Papers - Kawakami, Yutaka

Arbekacin

Kobayashi Y, Uchida H, Kawakami Y

Inter J Antimicrob Agent 5 ( 3 ) 227-230 1995.05

Research paper (scientific journal), Joint Work

Human tumor antigens recognized by T cells

Kawakami Y, Rosenberg SA

Immunologic. Res 16 ( 4 ) 313-339 1997.11

Research paper (scientific journal), Joint Work, Accepted

T細胞により認識されるヒトメラノーマ抗原の単離同定と免疫遺伝子治療への応用

Kawakami Yutaka

日本リンパ網内系会誌 37 ( 3 ) 137-144 1997.11

Research paper (scientific journal), Single Work

Immunobiology of melanoma antigens MART-1 and gp100 and their use in immunotherapy

Kawakami Y, Rosenberg SA

Int Rev Immunol 14 ( 2-3 ) 173-192 1997.04

Research paper (scientific journal), Joint Work

Melanosomal proteins as melanoma-specific immune targets

Sakai C, Kawakami Y, Law LW, Furumura M, Hearing VJ Jr

Melanoma Res 7 ( 2 ) 83-95 1997.04

Research paper (scientific journal), Joint Work, Accepted

癌の免疫遺伝子療法

Kawakami Yutaka

診断と治療 85 ( 4 ) 632-636 1997.04

Research paper (scientific journal), Single Work

遺伝子治療の臨床展開，米国の現状

Kawakami Yutaka

治療学 31 ( 4 ) 489-493 1997.04

Research paper (scientific journal), Single Work

Differential expression of MART-1 in primary and metastatic melanoma lesions

Kageshita T, Kawakami Y, Hirai S, Ono T

J Immunother 20 ( 6 ) 460-465 1997.11

Research paper (scientific journal), Joint Work, Accepted

The intronic region of an incompletely-spliced gp100 gene transcript encodes an epitope recognized by melanoma reactive tumor infiltrating lymphocytes

Robbins PF, El-Gamil M, Li YF, Fitzgerald EB, Kawakami Y, Rosenberg SA

J Immunol 159 ( 1 ) 303-308 1997.07

Research paper (scientific journal), Joint Work, Accepted

癌抗原遺伝子の単離同定と免疫遺伝子治療への応用

Kawakami Yutaka

現代医療 29   77-83 1997.08

Research paper (scientific journal), Single Work

T細胞が認識するヒト腫瘍抗原：同定と治療への応用

Kawakami Yutaka

臨床免疫 30 ( 1 ) 79-87 1998.01

Research paper (scientific journal), Single Work

Identification of a shared HLA-A*0201 restricted T cell epitope from the melanoma antigen tyrosinase related protein 2 (TRP2)

Parkhurst M, Fitzgerald E, Southwood S, Sette A, Rosenberg Sa, Kawakami Y

Cancer Research 58 ( 21 ) 4895-4901 1998.11

Research paper (scientific journal), Joint Work, Accepted

Immunotherapy using T cell defined tumor antigens for melanoma

Kawakami Y

Microbiology and Immunology 42 ( 12 ) 803-813 1998.12

Research paper (scientific journal), Single Work

Identification of new melanoma epitopes on melanosomal proteins recognized by tumor infiltrating T lymphocytes restricted by HLA-A1, -A2, and -A3 alleles

Kawakami Y, Robbins Pf, Wang X, Tupesis Jp, Parkhurst Mr, Kang X, Kazuyasu K, Appella E, Rosenberg Sa

Journal of Immunology 161 ( 12 ) 6985-6992 1998.12

Research paper (scientific journal), Joint Work, Accepted

T細胞認識癌抗原を用いた新しい癌の免疫療法

Kawakami Yutaka

日本内科学会雑誌 87 ( 12 ) 156-164 1998.12

Research paper (scientific journal), Single Work

がん患者にT細胞とB細胞の免疫応答を起こす腫瘍抗原分子の同定

Kawakami Yutaka

最新医学 53 ( 12 ) 2891-2897 1998.12

Research paper (scientific journal), Single Work

癌に対する免疫遺伝子治療

Kawakami Yutaka

Pharm Med 16 ( 2 ) 63-68 1998.02

Research paper (scientific journal), Single Work

Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma

Rosenberg S, Yang J, Schwartzentruber D, Hwu P, Marincola F, Topalian S, Restifo N, Dudley M, Schwarz S, Spiess P, Wunderlich J, Parkhurst M, Kawakami Y, Seipp C, Einhorn J, White D

Nature Med 4 ( 3 ) 321-321 1998.03

Research paper (scientific journal), Joint Work, Accepted

The use of melanosomal proteins in the immunotherapy of melanoma

Kawakami Y, Robbins Pf, Wang Rf, Parkhurst M, Kang X, Rosenberg SA

Journal of Immunotherapy 21 ( 4 ) 234-246 1998.07

Research paper (scientific journal), Joint Work, Accepted

T細胞認識ヒトメラノーマ抗原ペプチドを用いた癌の免疫療法

Kawakami Yutaka

Biotherapy 12 ( 5 ) 542-548 1998.05

Research paper (scientific journal), Single Work

T細胞認識腫瘍抗原の単離同定法

Ikeda Hideyuki, Kawakami Yutaka

細胞工学 17 ( 8 ) 1251-1253 1998.08

Research paper (scientific journal), Joint Work

T細胞によるがん細胞認識の分子機構

Kawakami Yutaka

細胞工学 17 ( 8 ) 1194-1199 1998.08

Research paper (scientific journal), Single Work

T細胞認識ヒトメラノーマ抗原の単離：T細胞エピトープ形成機序と免疫遺伝子治療への臨床応用

Kawakami Yutaka

細胞工学 17 ( 8 ) 1229-1238 1998.08

Research paper (scientific journal), Single Work

T細胞認識メラノーマ抗原を用いた新しい免疫療法の開発

Kawakami Yutaka

Biotherapy 12 ( 9 ) 1187-1194 1998.09

Research paper (scientific journal), Single Work

新しい癌の免疫療法の開発

Kawakami Yutaka

Molecular Medicine 35 ( 9 ) 1066-1068 1998.09

Research paper (scientific journal), Single Work

T細胞認識ヒトメラノーマ抗原を用いた免疫療法

Kawakami Yutaka

Molecular Medicine 35 ( 9 ) 1092-1099 1998.09

Research paper (scientific journal), Single Work

新しい癌抗原特異的免疫療法の開発に向けて

Kawakami Yutaka

Molecular Medicine 35 ( 9 ) 1100-1119 1998.09

Research paper (scientific journal), Single Work

メラノーマ腫瘍抗原と細胞治療

Kawakami Yutaka

血液腫瘍科 38 ( 1 ) 1-8 1999.01

Research paper (scientific journal), Single Work

遺伝子治療の現況

Sumimoto Hidetoshi, Kawakami Yutaka

肺癌の臨床    493-501 1999.11

Research paper (scientific journal), Joint Work

樹状細胞を用いた細胞療法-悪性黒色腫-

Kiniwa Yukiko，Kawakami Yutaka

最新医学 54   2674-2681 1999.11

Research paper (scientific journal), Joint Work

転移性脳腫瘍に対するin situ 癌ワクチン療法

Toda Masahiro, Iizuka Yukihiko, Kawase Takeshi, Kawakami Yutaka, Uemura Keiichi

神経免疫研究 12   181-185 1999.12

Research paper (scientific journal), Joint Work

脳内抗原提示細胞の単離とその解析

Toda Masahiro, Toba Youko, Suzuki Harumi, Koyasu Shigeo, Kawase Takeshi, Uemura Keiichi

神経免疫研究 12   29-33 1999.12

Research paper (scientific journal), Joint Work

造血器腫瘍における腫瘍抗原と免疫療法

Matsushita Maiko, Ikeda Hideyuki, Kawakami Yutaka

現代医療 31 ( 5 ) 1155-1160 1999.05

Research paper (scientific journal), Joint Work

皮膚悪性黒色腫に対する免疫療法

Kawakami Yutaka

Pharma Medica 17   49-56 1999.05

Research paper (scientific journal), Single Work

T細胞認識抗原を用いた癌の免疫療法開発におけるいくつかのポイント

Kawakami Yutaka

Biotherapy 13   429-433 1999.05

Research paper (scientific journal), Single Work

新しい癌ワクチン開発の進歩

Kawakami Yutaka

Cancer Frontier 1   27-38 1999.06

Research paper (scientific journal), Single Work

メラノーマ抗原ペプチドを用いた免疫療法

Kawakami Yutaka

医学のあゆみ 190   143-147 1999.07

Research paper (scientific journal), Single Work

悪性黒色腫抗原の単離と治療への応用

Kiniwa Yukiko，Kawakami Yutaka

血液・腫瘍科 39   13-20 1999.07

Research paper (scientific journal), Joint Work

癌に対する免疫制御法の進歩

Kawakami Yutaka

細胞工学 18   1379-1388 1999.09

Research paper (scientific journal), Single Work

造血器腫瘍におけるPRAME

Ikeda Hideyuki, Matsushita Maiko, Kawakami Yutaka

血液・腫瘍科 39 ( 1 ) 46-50 1999

Research paper (scientific journal), Joint Work

T cells that are autoreactive to β₂-glycoprtein I in patients with antiphospholipid syndrome and healthy individuals

Hattori Noriko, Kuwana Masataka, Kaburaki Junichi, Mimori Tsuneyo, Ikeda Yasuo, Kawakami Yutaka

Arthritis & Rheumatism 43 ( 1 ) 65-75 2000.01

Research paper (scientific journal), Joint Work

メラノーマの免疫療法

Kawakami Yutaka

現代医療 32   104-111 2000.01

Research paper (scientific journal), Single Work

Recognition of shared melanoma antigens in association with major HLA-A alleles by tumor infiltrating T lymphocytes from 123 patients with melanoma.

Kawakami Yutaka, Dang Nita, Wang Xiang, Tupesis Janis P, Robbins Paul F, Wang Rong-Fu, Wunderlich John R, Yannelli John R, Rosenberg Steven A

Journal of Immunotherapy 23 ( 1 ) 17-27 2000.01

Research paper (scientific journal), Joint Work

ヒト癌抗原の同定と樹状細胞を用いた免疫療法

Kawakami Yutaka

BIO Clinica 15   35-39 2000.01

Research paper (scientific journal), Single Work

腫瘍免疫とSEREX

Akada Masaaki，Kawakami Yutaka

癌治療と宿主 12 ( 4 ) 375-383 2000.10

Research paper (scientific journal), Joint Work

HLA class II alleles in Japanese patients with immune thrombocytopenic purpura. Associations with anti-platelet glycoprotein autoantibodies and responses to splenectomy

Kuwana Masataka, Kaburaki Junichi, Pandey JP, Murata Mitsuru, Kawakami Yutaka, Inoko Hidetoshi, Ikeda Yasuo

Tissue Antigens 56 ( 4 ) 337-343 2000.10

Research paper (scientific journal), Joint Work, Accepted

癌抗原遺伝子導入樹状細胞を用いた癌免疫療法

Sumimoto Hidetoshi, Kawakami Yutaka

Biotherapy 14 ( 12 ) 1197-1204 2000.12

Research paper (scientific journal), Joint Work

SEREX抗原によるグリオーマの血清診断

Toda Masahiro, Iizuka Yukihiko, Kawase Takeshi, Kawakami Yutaka

神経免疫研究 13   44-46 2000.12

Research paper (scientific journal), Joint Work

特発性血小板減少性紫斑病における病因的自己反応性T細胞の同定

Kuwana Masataka，Kawakami Yutaka，Ikeda Yasuo

日本臨床免疫学会会誌 23 ( 6 ) 555-557 2000.12

Research paper (scientific journal), Joint Work

メラノーマ抗原特異的免疫療法

Kawakami Yutaka

SkinCancer 15   208-216 2000.12

Research paper (scientific journal), Single Work

がん反応性T細胞の免疫モニタリング

Kawakami Yutaka

分子細胞治療 1   23-32 2000.02

Research paper (scientific journal), Single Work

癌ワクチンの可能性

Kawakami Yutaka

治療 82   1196-1199 2000.03

Research paper (scientific journal), Single Work

がんに対する免疫療法の可能性〜overview〜

Kawakami Yutaka

分子細胞治療 1 ( 3 ) 3-9 2000.04

Research paper (scientific journal), Single Work

ヒトメラノーマ抗原を標的とした免疫療法

Inodume Takashi，Kiniwa Yukiko，Kawakami Yutaka

血液・免疫・腫瘍 5 ( 2 ) 159-165 2000.04

Research paper (scientific journal), Joint Work

免疫遺伝子治療の現状

Kawakami Yutaka

BioTherapy 14 ( 4 ) 345-350 2000.04

Research paper (scientific journal), Single Work

Longitudinal analysis of autoantibody response to topoisomerase I in systemic sclerosis

Kuwana Masataka, Kaburaki Junichi, Mimori Tsuneyo, Kawakami Yutaka, Tojo Takeshi

Arthritis and Rheumatism 43 ( 5 ) 1074-1084 2000.05

Research paper (scientific journal), Joint Work

New cancer therapy by immunomanipulation: Development of immunotherapy for human melanoma as a model system

Kawakami Yutaka

Cornea 19 ( 3 Sppl ) S2-S6 2000.05

Research paper (scientific journal), Single Work

癌と免疫

Itou Kyougo，Kawakami Yutaka，Shiku Hiroshi

現代医療 32 ( 5 ) 1046-1066 2000.05

Research paper (scientific journal), Joint Work

白血病細胞抗原と免疫療法

Kawakami Yutaka，Matsushita Maiko

臨床医 26 ( 5 ) 676-677 2000.05

Research paper (scientific journal), Joint Work

癌抗原遺伝子導入樹状細胞を用いた癌免疫療法

Sumimoto Hidetoshi, Kawakami Yutaka

Surgery Frontier 7 ( 2 ) 166-169 2000.06

Research paper (scientific journal), Joint Work

ペプチド抗原によるCTL誘導

Matsushita Maiko，Kawakami Yutaka

Cancer Frontier 2 ( 1 ) 13-20 2000.07

Research paper (scientific journal), Joint Work

メラノーマと色素細胞に対するT細胞応答

Kawakami Yutaka

組織培養工学 26 ( 10 ) 18-22 2000.09

Research paper (scientific journal), Single Work

T cell immune responses against melanoma and melanocytes in cancer and autoimmunity

Kawakami Yutaka, Suzuki Yuriko, Shofuda Tomoko, Kiniwa Yukiko, Inozume Takashi, Dan Katsuaki, Sakurai Toshiharu, Fujita Tomonobu

Pigment Cell Research 13 ( Suppl 8 ) 163-169 2000

Research paper (scientific journal), Joint Work

Clinical significance of MART-1 and HLA-A2 expression and CD8+ T cell infiltration in melanocytic lesions in HLA-A2 phenotype patients

Kageshita Toshiro, Kawakami Yutaka, Ono Tomomichi

Journal of Dermatologicla Science 25 ( 1 ) 36-44 2001.01

Research paper (scientific journal), Joint Work

Graft-versus-leukemia(GVL)効果の免疫制御

Yamazaki Rie，Kawakami Yutaka

血液フロンティア 11 ( 11 ) 1399-1408 2001.10

Research paper (scientific journal), Joint Work

Tumor antigens isolated from a patient with vitiligo and T-cell-infiltrated melanoma

Kiniwa Yukiko, Fujita Tomonobu, Akada Masanori, Ito Keiichi, Shofuda Tomoko, Suzuki Yuriko, Yamamoto Akifumi, Saida Toshiaki, Kawakami Yutaka

Cancer Research 61 ( 21 ) 7900-7907 2001.11

Research paper (scientific journal), Joint Work

Induction of antigen-specific human CD4⁺ T cell anergy by peripheral blood DC2 precursors

Kuwana Masataka, Kaburaki Junichi, Wright Timothy M, Kawakami Yutaka, Ikeda Yasuo

European Journal of Immunology 31 ( 9 ) 2547-2557 2001.09

Research paper (scientific journal), Joint Work

免疫血液疾患研究の進歩と現状: 特発性血小板減少性紫斑病

Kuwana Masataka，Kawakami Yutaka，Ikeda Yasuo

臨床病理 49 ( 10 ) 992-995 2001.10

Research paper (scientific journal), Joint Work

Autoantibodies to the amino-terminal fragment of b-fodrin expressed in glandular epithelial cells in patients with Sjögren's syndrome

Kuwana Masataka, Okano Tetsuro, Ogawa Yoko, Kaburaki Junichi, Kawakami Yutaka

Journal of Immunology 167 ( 9 ) 5449-5456 2001.11

Research paper (scientific journal), Joint Work

メラノーマとメラノサイトに対する免疫応答の分子機構

Kawakami Yutaka

日本皮膚科学会雑誌 111 ( 12 ) 1701-1703 2001.11

Research paper (scientific journal), Single Work

グリオーマ抗原分子を用いた新しい診断法の開発

Toda Masahiro，Iizuka Yukihiko，Ueda Ryou，Yoshida Kazunari，Kawase Takeshi，Kawakami Yutaka

Neuro-Oncorogy 11 ( 2 ) 93-94 2001.12

Research paper (scientific journal), Joint Work

Isolation of a new melanoma antigen, MART-2, containing a mutated epitope recognized by autologous tumor infiltrating T lymphocytes

Kawakami Y, X Wang, Shofuda T, Sumimoto H, JP Tupesis, E Fitzgerald, SA Rosenberg

Journal of Immunology 166 ( 4 ) 2871-2877 2001.02

Research paper (scientific journal), Joint Work

Quantitative monitoring of PRAME gene for the detection of minimal residual disease in leukaemia

Matsushita Maiko, Ikeda Hideyuki, Kizaki Masahiro, Okamoto Shinichiro, Ogasawara Masahiro, Ikeda Yasuo, Kawakami Yutaka

British Journal of Haematology 112 ( 4 ) 916-926 2001.03

Research paper (scientific journal), Joint Work

Lack of evidence for an increased microchimerism in circulation of patients with Sjogrens syndrome

Toda Ikuko, Kuwana Masataka, Tsubota Kazuo, Kawakami Yutaka

Annals of Rheumatic Diseases 60 ( 3 ) 248-253 2001.03

Research paper (scientific journal), Joint Work

がんに対する分子標的としてのペプチド抗原

Kawakami Yutaka

最新医学 56 ( 3 ) 432-439 2001.03

Research paper (scientific journal), Single Work

Expression of the neural RNA-binding protein Musashi1 in human gliomas

Toda Masahiro, Iizuka Yukihiko, Yu Wang-Jie, Imai Takao, Ikeda Eiji, Yoshida Kazunari, Kawase Takeshi, Kawakami Yutaka, Okano Hideyuki, Uyemura Keiichi

Glia 34 ( 1 ) 1-7 2001.04

Research paper (scientific journal), Joint Work

メラノーマと樹状細胞

Inozume Takashi，Kawakami Yutaka

炎症と免疫5　Inflammation&Immunology 9 ( 3 ) 62-69 2001.04

Research paper (scientific journal), Joint Work

新しい癌の免疫療法開発

Kawakami Yutaka

実験医学 19 ( 6 ) 756-762 2001.04

Research paper (scientific journal), Single Work

新しい癌ワクチンの開発

Kawakami Yutaka

臨床と研究 78 ( 4 ) 695-700 2001.04

Research paper (scientific journal), Single Work

T細胞認識腫瘍抗原とその臨床応用

Kawakami Yutaka

medicina 38 ( 6 ) 1036-1038 2001.06

Research paper (scientific journal), Single Work

Immunodominant epitopes on glycoprotein IIb-IIIa recognized by autoreactive T cells in patients with immune thrombocytopenic purpura

Kuwana Masataka., Kaburaki Junichi, Kitasato Hidero, Kato Miyako, Kawai Shinichi, Kawakami Yutaka, Ikeda Yasuo

Blood 98 ( 1 ) 130-139 2001.07

Research paper (scientific journal), Joint Work

Heterogeneous immunogenetic background in Japanese adults with myasthenia gravis

Suzuki Shigeaki, Kuwana Masataka, Yasuoka Hidekata, Tanaka Kortaro, Fukuuchi Yasuo, Kawakami Yutaka

Journal of the Neurological Science 189 ( 1-2 ) 59-64 2001.08

Research paper (scientific journal), Joint Work

Heterogeneous immunogenetic background in Japanese adults with myasthenia gravis

Suzuki Sigeaki, Kuwana Masataka, Yasuoka Hidekata, Tanaka Koutaro, Fukuuchi Yasuo, Kawakami Yutaka

Journal of the Neurological Sciences 189 ( 1-2 ) 59-64 2001.08

Research paper (scientific journal), Joint Work, Accepted

Increase of oligodendrocyte progenitor cells after spinal cord injury

Ishii Ken, Toda Masahiro, Nakai Yoko, Asou Hiroaki, Watanabe Masahiko, Nakamura Masaya, Yato Yoshiyuki, Fujimura Yoshikazu, Kawakami Yutaka, Toyama Yoshiaki, Uyemura Keiichi

Journal of Neuroscience Research 65 ( 6 ) 500-507 2001.09

Research paper (scientific journal), Joint Work

メラノーマのワクチン療法

Kawakami Yutaka

がん治療最前線 1 ( 4 ) 10-13 2001.09

Research paper (scientific journal), Single Work

T細胞認識癌抗原を標的とした癌に対する免疫療法

Kawakami Yutaka

肝臓病学の進歩　第27回肝臓研究会別冊 27   31-36 2001.09

Research paper (scientific journal), Single Work

Autoreactive CD4+ T-cell clones to β2- glycoprotein I in patients with antiphospholipid syndrome: preferential recognition of the major phospholipid-binding site

Arai Takahide, Yoshida Kazue, Kaburaki Junichi, Inoko Hidetoshi, Ikeda Yasuo, Kawakami Yutaka, Kuwana Masataka

Blood 98 ( 6 ) 1889-1896 2001.09

Research paper (scientific journal), Joint Work

メラノーマに対する免疫遺伝子治療

Kawakami Yutaka

分子がん治療 2 ( 2 ) 131-137 2001

Research paper (scientific journal), Single Work

Dry eye assiciated with chronic graft-versus-host disease

Ogawa Yoko, Masataka Kuwana, Yamazaki Kazuto, Mashima Yukihiko, Okamoto Shinichiro, Tsubota Kazuo, Oguchi Yoshihisa, Yutaka Kawakami

Advances in experimental medicine and biology 506 ( Pt B ) 1041-1045 2002.10

Research paper (scientific journal), Joint Work

Identification of an immunodominant epitope on RNA polymerase III recognized by systemic sclerosis sera: application to enzyme-linked immunosorbent assay

Kuwana Masataka, Kimura Kyoko, Kawakami Yutaka

Arthritis rheumatism 46 ( 10 ) 2742-2747 2002.10

Research paper (scientific journal), Joint Work

Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells

Sumimoto Hidetoshi, Tsuji Takashi, Miyoshi Hiroyuki, Hagihara Masao, Takada-Yamazaki Rie, Okamoto Shinichiro, Ikeda Yasuo, Takahashi Tsuneo, Kawakami Yutaka

Journal of immunological methods 271 ( 1-2 ) 153-165 2002.12

Research paper (scientific journal), Joint Work

In Situ Cancer Vaccination with a Replication-Conditional HSV for the Treatment of Liver Metastasis of Colon Cancer

Endo Takashi, Toda Masahiro, Watanabe Msahiko, Iizuka Yukihiko, Kubota Tetsuo, Kitajima Masaki, Kawakami Yutaka

Cancer gene therapy 9   142-148 2002.02

Research paper (scientific journal), Joint Work

同種抗原を標的とした造血器腫瘍に対する免疫療法の開発

Matsushita Maiko，Yamazaki Rie，Fujita Tomonobu，Kawakami Yutaka

臨床血液 43 ( 2 ) 94-96 2002.02

Research paper (scientific journal), Joint Work

ミニ移植「Con」

Yamazaki Rie, Kawakami Yutaka

血液・免疫・腫瘍 7 ( 2 ) 94-98 2002.04

Research paper (scientific journal), Joint Work

Immuno-viral therapy of brain tumors by combination of viral therapy with cancer vaccination using a replication-conditional HSV

Toda Masahiro, Iizuka Yukihiko, Kawase Takeshi, Uemura Keiichi, Kawakami Yutaka

Cancer Gene Therapy 9 ( 4 ) 356-364 2002.04

Research paper (scientific journal), Joint Work

Spleen is a primary site for activation of platelet-reactive T and B cells in patients with immune thrombocytopenic purpura

Kuwana Masataka, Okazaki Yuka, Kaburaki Junichi, Kawakami Yutaka, Ikeda Yasuo

Journal of immunology 168 ( 7 ) 3675-3682 2002.04

Research paper (scientific journal), Joint Work

Restricted T-cell receptor β-chain usage by T cells autoreactive to β2-glycoprotein I in patients with antiphospholipid syndrome

Yoshida Kazue, Arai Takahide, Kaburaki Junichi, Ikeda Yasuo, Kawakami Yutaka, Kuwana Masataka

Blood 99 ( 7 ) 2499-2504 2002.04

Research paper (scientific journal), Joint Work

がん免疫療法の標的抗原

Ishikawa Toshiaki, Kawakami Yutaka

血液・腫瘍科 44 ( 5 ) 397-405 2002.05

Research paper (scientific journal), Joint Work

細胞傷害性T細胞による養子免疫療法

Sakurai Toshiharu, Kawakami Yutaka

メディカル・サイエンス・ダイジェスト 28 ( 7 ) 8-11 2002.06

Research paper (scientific journal), Joint Work

A simple and reliable behavioral analysis of locomotor function after spinal cord injury in mice

Mikami Yuji, Toda Masahiro, Watanabe Masahiko, Nakamura Masaya, Toyama Yoshiaki, Kawakami Yutaka

Journal of neurosurg 97 ( 1Suppl ) 142-147 2002.07

Research paper (scientific journal), Joint Work

SEREX法による癌抗原の同定

Okada Takaho, Kawakami Yutaka

血液・腫瘍科 45 ( 1 ) 1-7 2002.07

Research paper (scientific journal), Joint Work

Autoantibody to c-Mpl (thrombopoietin receptor) in systemic lupus erythematosus. - relationship to thrombocytopenia with megakaryocytic hypoplasia

Kuwana Masataka, Okazaki Yuka, Kajihara Mikio, Kaburaki Junichi, Miyazaki Hiroshi, Kawakami Yutaka, Ikeda Yasuo

Arthritis & rheumatism 46 ( 8 ) 2148-2159 2002.08

Research paper (scientific journal), Joint Work

Tumor antigens isolated from a patient with vitiligo and T-cell-infiltrated melanoma

Kawakami Yutaka, Kiniwa Yukiko

Cancer research 62 ( 16 ) 4836-4837 2002.08

Research paper (scientific journal), Joint Work

ヒト腫瘍抗原ペプチドの同定法と抗腫瘍免疫応答解析法

Fujita Tomonobu, Kawakami Yutaka

Biotherapy 16 ( 5 ) 441-448 2002.09

Research paper (scientific journal), Joint Work

Differences in autoantibody response to Th / To between systemic sclerosis and other autoimmune diseases

Kuwana Masataka, Kimura Kyoko, Hirakata Michito, Kawakami Yutaka, Ikeda Yasuo

Annals of the rheumatic diseases 61 ( 9 ) 842-846 2002.09

Research paper (scientific journal), Joint Work

DNAワクチン

Kawakami Yutaka

Surgery Frontier 9 ( 3 ) 65-67 2002.09

Research paper (scientific journal), Single Work

がんワクチンの開発と現況

Kawakami Yutaka

総合臨床 51 ( 9 ) 2552-2554 2002.09

Research paper (scientific journal), Single Work

Tumor-specific Immunological Recognition of Frameshift-mutated Peptidesan Colon Cancer with Microsatellite Instability.

Ishikawa Toshiaki, Fujita Tomonobu, Suzuki Yuriko, Okabe Satoshi, Yuasa Yasuhiko, Iwai Takehisa and Kawakami Yutaka.

Cancer Research 63 ( 17 ) 5564-55672 2003.09

Research paper (scientific journal), Single Work

Identification of Human Tumor Antigens Recognized by T-cells and Their Use for Immunotherapy.

kawakami Yutaka

International Journal of Hematology 77 ( 5 ) 427-434 2003.06

Research paper (scientific journal), Single Work

Suppression of autoreactive T-cell response to glycoprotein IIb-IIIa by blockade of CD40/CD154 interaction: implications for treatment of immune thrombocytopenic purpura

Kuwana Masataka, Kawakami Yutaka, Ikeda Yasuo

Blood 101 ( 2 ) 621-623 2003.01

Research paper (scientific journal), Joint Work

がんワクチン療法

Kawakami Yutaka

がん看護 8 ( 1 ) 39-39 2003.01

Research paper (scientific journal), Single Work

HLA class II alleles in systemic sclerosis patients with anti-RNA polymerase I/III antibody: associations with subunit reactivities.

'Kuwana Masataka, Janardan P. Pandey, Richard M. Silver, Kawakami Yutaka, Kaburaki Junichi'

The Journal of Rheumatology 30 ( 11 ) 2392-2397 2003.11

Research paper (scientific journal), Joint Work

Human circulating CD14^+^ monocytes as a source of progenitors that exhibit mesenchymal cell differentiation.

'Kuwana Masataka, Okazaki Yuka, Kodama Hiroaki, Izumi Keisuke, Yasuoka Hidekata, Ogawa Yoko, Kawakami Yutaka, Ikeda Yasuo'

Journal of Leukocyte Biology 74 ( 5 ) 833-845 2003.11

Research paper (scientific journal), Joint Work

A novel protein highly expressed in testis is overexpressed in systemic sclerosis fibroblasts and targeted by autoantibodies.

'Yasuoka Hidekata, Ihn hironobu, Thomas A.Medsger,Jr., Hirakata Michito, Kawakami Yutaka, Ikeda Yasuo, Kuwana Masataka'

The Journal of Immunology 171 ( 12 ) 6883-6890 2003.12

Research paper (scientific journal), Joint Work

樹状細胞を用いたがんの免疫細胞療法

Utagawa Masaru, Kawakami Yutaka

BioClinica 18 ( 2 ) 53(143)-57(147) 2003.02

Research paper (scientific journal), Joint Work

Preferentially Expressed Antigen of Melanoma (PRAME) in the development of diagnostic and therapeutic methods for hematological malignancies

Matsushita Maiko, Yamazaki Rie, Ikeda Hideyuki, Kawakami Yutaka

Leukemia and lymphoma 44 ( 3 ) 439-444 2003.03

Research paper (scientific journal), Joint Work

いまなぜ樹状細胞か

Kawakami Yutaka

臨床検査 47 ( 3 ) 233-234 2003.03

Research paper (scientific journal), Single Work

Genetic Reversion in an AML Cell Line from a Fanconi Anemia Patient withBiallelic Mutations in BRCA2.

'Ikeda Hideyuki, Matsushita Maiko, Waisfisz Quinten, Kinoshita Akitoshi, Oostra Anneke B, Nieuwint Aggie W M, Winter Johan P de, Hoatlin Maureen E, Kawai Yohko, Sasaki Masao S, D'Andrea Alan D , Kawakami Yutaka, Joenje Hans.'

Cancer Research 63 ( 10 ) 2688-2694 2003.05

Research paper (scientific journal), Joint Work, Accepted

Results of phase I clinical trail of immunotherapy with tumor lysate-pulsed monocyte-derived dendritic cells plus IL2 for stage IV malignant melanoma patients combined with low dose interleukin-2

'Nagayama Hitomi, Sato Katsuaki, Morishita Maiko, Uchiyama Kaoru, Inazawa Takeshi, Yamazaki Tomoko, Enomoto Makoto, Nakaoka Takashi, Nakamura Tetsuya, Maekawa Taira, Yamamoto Akifumi, Shimada Shinji, Saida Toshiaki, Kawakami Yutaka, Tani Kenzaburo, Asano Shigetaka, Takahashi Tsuneo A, and Yamashita Naohide.'

Melanoma Research 13 ( 13 ) 521-530 2003.10

Research paper (scientific journal), Joint Work, Accepted

Periductal area as the primary site for T-cell activation in lacrimal gland chronic graft-versus-host disease.

'Ogawa Yoko, Kuwana Masataka, Yamazaki Kazuto, Mashima Yukihiko, Yamada Masakazu, Okamoto Shinichiro, Oguchi Yoshihisa, Kawakami Yutaka'

Investigative Ophthalmology & Visual Science 44 ( 5 ) 1888-1896 2003.05

Research paper (scientific journal), Joint Work

悪性腫瘍に対する細胞免疫療法

河上裕

臨床血液 44 ( 6 ) 358-367 2003.06

Research paper (scientific journal), Single Work

癌に対する新しい免疫療法の開発：ポストゲノム時代の医療

河上裕

JCLS    39087 2003.06

Research paper (scientific journal), Single Work

A role of autoantibody-mediated platelet destruction in thrombocytopenia in patients with cirrhosis.

'Kajihara Mikio, Kato Shinzo, Okazaki Yuka, Kawakami Yutaka, Ishii Miromasa, Ikeda Yasuo, Kuwana Masataka'

Hepatology 37 ( 6 ) 1267-1276 2003.06

Research paper (scientific journal), Joint Work

癌のワクチン療法（特集：癌の免疫学—癌免疫療法の理論と臨床—）

河上裕

呼吸 22 ( 12 ) 1207-1212 2003

Research paper (scientific journal), Single Work

腫瘍に対する免疫応答

河上裕

キーワードで理解する免疫学イラストマップ    186-193 2003

Research paper (scientific journal), Single Work

腫瘍免疫療法

河上裕

現代医学 35 ( 7 ) 111-117 2003

Research paper (scientific journal), Single Work

細胞免疫療法　④医学・医療の進歩を世界へ向けて『治療の最前線』Ｓ−1造血幹細胞移植から細胞療法へ）

河上裕

第26回日本医学会総会会誌2003福岡 1 2003

Research paper (conference, symposium, etc.), Single Work

Human melanoma antigens recognized by CD8+ T cells, in Tumor Antigens Recognized by T cells and Antibodies eds

Kawakami Yutaka

Tumor immunology series 3   47-74 2003

Research paper (scientific journal), Single Work

Expression of a transcriptional factor, SOX6, in human gliomas.

Ueda Ryo, Yoshida Kazunari, Kawakami Yutaka, Kawase Takeshi, and Toda Masahiro.

Brain Tumor Pathology. 21 ( 1 ) 35-38 2004.02

Research paper (scientific journal), Single Work

Defective vasculogenesis in systemic sclerosis.

Kuwana Masataka, Okazaki Yuka, Yasuoka Hidetaka, Kawakami Yutaka, Ikeda Yasuo.

Lancet 364 ( 9493 ) 603-610 2004.08

Research paper (scientific journal), Single Work

Identification of an epigenetically silenced gene, RFX1,in human glioma cells using restriction landmark genomic scanning.

Ohashi Yohei, Ueda Masakazu, Kawase Takeshi, Kawakami Yutaka, Toda Masahiro.

Oncogene 23 ( 47 ) 7772-7779 2004.10

Research paper (scientific journal), Single Work

新規腫瘍抗原同定法

'木村康子, 河上裕'

血液・免疫・腫瘍 9 ( 1 ) 41-44 2004.01

Research paper (scientific journal), Joint Work

Autoreactive CD8＋Cytotoxic T Lymphocytes to Major Histocompatibility Complex Class I Chain-Related Gene A in Patients With Behcet’s Disease.

Yasuoka Hidetaka, Okazaki Yuka, Kawakami Yutaka, Hirakata Michito, Inoko Hidetoshi, Ikeda Yasuo and Kuwana Masataka.

ARTHRITIS&RHEUMATISM 50 ( 11 ) 3658-3662 2004.11

Research paper (scientific journal), Single Work

樹状細胞腫瘍内投与による免疫療法

河上裕

医学のあゆみ 208 ( 7 ) 634 2004.02

Research paper (scientific journal), Single Work

癌免疫療法の開発：メラノーマ研究からのレッスン

河上裕

日本臨床免疫学会会誌 27 ( 2 ) 87-98 2004.03

Research paper (scientific journal), Single Work

メラノーマに対する免疫療法における最近の展開

河上裕

Skin Cancer 19 ( 1 ) 25-33 2004.03

Research paper (scientific journal), Single Work

Single nucleotide polymorphisms of the inflammatory cytokine genes in adults with chronic immune thrombocytopenic purpura.

'Satoh Takashi, Janardan P. Pandey, Okazaki Yuka, Yasuoka Hidekata, Kawakami Yutaka, Ikeda Yasuo, Kuwana Masataka'

British Journal of Haematology 124 ( 6 ) 796-801 2004.05

Research paper (scientific journal), Joint Work

Development of Immunotherapy for Pancreatic Cancer.

'Kawakami Yutaka, Okada Takaho, Akada Masanori'

Pancreas 28 ( 3 ) 320-325 2004.04

Research paper (scientific journal), Joint Work

癌抗原:SEREX法による癌抗原の単離

'岡田勉, 河上裕'

Biotherapy 18 ( 3 ) 205-211 2004.05

Research paper (scientific journal), Joint Work

Identification of bladder cancer antigens recoginized by IgG antibodies of a patient with metastatic bladder cancer.

Ito Keiichi, Fujita Tomonobu, Akada Masanori, Kiniwa Yukiko, Tsukamoto Makoto,Yamamoto Aiko, Matsuzaki Yuriko, Matsushita Maiko, Asano Takako, Nakamura Jun, Tachibana Masaaki, Hayakawa Masamichi, Ikeda Hideyuki, Murai Masaru and Kawakami Yutaka.

International Journal of Cancer 108 ( 5 ) 712-724 2004.02

Research paper (scientific journal), Joint Work

　がん抗原ペプチドを標的とした治療

河上裕

現代医療 36 ( 7 ) 185-190 2004

Research paper (scientific journal), Single Work

Inhibition of growth and invasive ability of melanoma by inactivation of mutated BRAF with lentivirus-mediated RNA interference.

Sumimoto Hidetoshi, Miyagishi Makoto, Miyoshi Hiroyuki, Yamagata Shizuko, Shimizu Ayako, Taira Kazunari, and Kawakami Yutaka

Oncogene 23 ( 36 ) 6031-6039 2004.08

Research paper (scientific journal), Single Work

Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells.

Miyagishi Makoto, Sumimoto Hidetoshi, Miyoshi Hiroyuki, Kawakami Yutaka and Taira Kazunari.

Journal of Gene Medicine 6 ( 7 ) 715-723 2004.07

Research paper (scientific journal), Single Work

Identification of human tumor antigens and its implication for diagnosis and treatment of cancer.

Kawakami Yutaka, Fujita Tomonobu, Matsuzaki Yuriko, Sakurai Toshiharu, Tsukamoto Makoto, Toda Masahiro, and Sumimoto Hideroshi.

Cancer Science 95 ( 10 ) 784-791 2004.10

Research paper (scientific journal), Single Work

Involvement of overexpressed wild-type BRAF in the growth of malignant melanoma cell lines.

Tanami H, Imoto I, Hirasawa A, Yuki Y, Sonoda I, Inoue J, Yasui K, Misawa-Furihata A, Kawakami Y, Inazawa J.

Oncogene 23 ( 54 ) 8796-8804 2004.11

Research paper (scientific journal), Single Work

Systematic identification of human melanoma antigens using serial analysis of gene expression (SAGE).

Matsuzaki Yuriko, Hashimoto Shin-ichi, Fujita Tomonobu, Suzuki Takuji, Sakurai Toshiharu, Matsushima Kouji, Kawakami Yutaka.

Journal Immunother 28 ( 1 ) 39374 2005.01

Research paper (scientific journal), Single Work

Immune responses to DNA mismatch repair enzymes hMSH2 and hPMS1 in patients with pancreatic cancer, dermatomyositis and polymyositis.

Okada Takaho，Noji Shinobu, Goto Yasufumi, Iwata Takashi, Fujita Tomonobu, Okada Tsutomu, Matsuzaki Yuriko, Kuwana Masataka, Hirakata M, Horii A, Matsuno S, Sunamura M and Kawakami Yutaka

International Journal of Cancer 116 ( 6 ) 925-933 2005.10

Research paper (scientific journal), Joint Work

Carbonic anhydrase-Ⅱis a tumor vessel endothelium-associated antigen targeted by dendritic cell therapy.

Yoshiura K, Nakaoka T, Nishishita T, Sato K, Yamamoto A, Shimada S, Saida T, Kawakami Y, Takahashi T, Fukuda H, Imajoh-Ohmi S, Oyaizu N, and Yamashita N

Clinical Cancer Research 11 ( 22 ) 8201-8207 2005.11

Research paper (scientific journal), Joint Work

Donor fibroblast chimerism in the pathogenic fibrotic lesion of human chronic graft-versus-host disease. Invest

Ogawa Y, Kodama H, Kameyama K, Yamazaki K, Yasuoka H, Okamoto S, Inoko H, Kawakami Y, and Kuwana M.

Invest Ophthalmol 46 ( 12 ) 4519-4527 2005.12

Research paper (scientific journal), Joint Work

Gene therapy for human small cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference.

Sumimoto Hidetoshi, Yamagata Sizuko, Shimizu Ayako, Miyoshi Hiroyuki, Mizuguchi Makoto, Hayakawa T, Miyagishi Makoto, Taira Kazunari and Kawakami Yutaka.

Gene Therapy 12 ( 1 ) 95-100 2005.01

Research paper (scientific journal), Single Work

Novel melanoma antigen, FCRL/FREB, identified by cDNA profile comparison using DNA chip are immunogenic in multiple melanoma patients.

Inozume Takashi, Matsuzaki Yuriko, Kurihara Sachiko, Fujita Tomonobu, Yamamoto Akifumi, Aburatani Hiroyuki, Shimada Shinji and Kawakami Yutaka .

International Journal of Cancer 114 ( 2 ) 283-290 2005.03

Research paper (scientific journal), Single Work

Genomic alterations in primary cutaneous melanomas detected by　metaphase comparative genomic hybridization with laser capture or manual microdissection: 6p gains may predict poor outcome

Namiki Takeshi, Yanagawa Shigeru, Izumo Toshiyuki, Ishikawa Masashi, Tachibana Masayoshi, Kawakami Yutaka, Yokozeki Hiroo, Nishioka Kiyoshi, Kaneko Yasuhiko

Cancer Genet Cytogenet 157 ( 1 ) 1-11 2005.02

Research paper (scientific journal), Joint Work

Lack of circulating autoantibodies to bone morphogenetic protein receptor-II or activin receptor-like kinase 1 in mixed connective tissue disease patients with pulmonary arterial hypertension

Satoh Takashi, Kimura K, Okano Y, Hirakata M, Kawakami Yutaka, and Kuwana Masataka

Rheumatology 44 ( 2 ) 192-196 2005.02

Research paper (scientific journal), Joint Work

Binding of β2-glycoprotein I to anionic phospholipids facilitates processing and presentation of a cryptic epitope that activates pathogenic autoreactive T cells

Kuwana Masataka, Matsuura E, Kobayashi Kazuko, Okazaki Yuka, Kaburaki J, Ikeda Yasuo and Kawakami Yutaka

Blood 105 ( 4 ) 1552-1557 2005.02

Research paper (scientific journal), Joint Work

Immunological detection of altered signaling molecules involved in melanoma development.

Kawakami Yutaka, Sumimoto Hidetoshi, Fujita Tomonobu, Matsuzaki Yuriko

Cancer Metastasis and Rev 24 ( 2 ) 357-366 2005.01

Research paper (scientific journal), Joint Work

Frequent immune responses to a cancer/testis antigen,CAGE,in patients with microsatellite instability positive endometrial cancer

Iwata Takashi , Fujita Tomonobu, Hirao Nobumaru, Matsuzaki Yuriko, Okada Tsutomu, Mochimaru Hiroshi, Susumu N, Matsumoto E, Sugano K, Yamashita N, Nozawa S, and Kawakami Yutaka

Clinical Cancer Res. 11 ( 10 ) 3949-3957 2005.05

Research paper (scientific journal), Joint Work

Evaluation of Cytomegalovirus-specific T cell reconstitution in patients after various allogeneic hematopoietic stem cell transplantation using interferon-γ-enzyme-linked immunospot and HLA tetramer assays with an immunodominant T cell epitope.

Ohnishi Mutsuko, Sakurai Toshiharu, Heike Y, Yamasaki Rie, Kanda Y, Takaue Y, Mizoguchi H and Kawakami Yutaka

British Journal of Heamatology 131 ( 4 ) 472-479 2005.11

Research paper (scientific journal), Joint Work

Nobel autoantibodies to a voltage-gated potassium channel Kv1.4 in a severe form of myasthenia gravis.

Suzuki Shigeaki, Satoh Takashi, Yasuoka Hidekata, Hamaguchi Yasuhito, Tanaka Kortaro, Kawakami Yutaka, Suzuki Norihiro, Kuwana Masataka

Journal of Neuroimmunology 170 ( 1-2 ) 141-149 2005.12

Research paper (scientific journal), Joint Work

Cardiomyogenic Potential of Mesenchymal Progenitors Derived from Human Circulation CD14+ Monocytes

Kodama H, Inoue T, Watanabe R, Yasuoka H, Kawakami Y, Ogawa S, Ikeda Y, Mikoshiba K, Kuwana M

Stem Cells and Development 14 ( 6 ) 676-686 2005.12

Research paper (scientific journal), Joint Work

A novel cancer testis antigen frequently expressed in pancreatic, lung and endometrial cancers

Okada T, Akada M, Fujita T, Iwata T, Goto Y, Kido K, Okada T, Matsuzaki Y, Kobayashi K, Matsuno S, Sunamura M, Kawakami Y

Clinical Cancer Research 12 ( 1 ) 191-197 2006.01

Research paper (scientific journal), Joint Work

Possible involvement of allogeneic antigens recognized by donor-derived CD4+ cytotoxic T cells in selective GVL effects after stem cell transplantation of patients with haematological malignancy.

Matsushita M, Yamazaki R, Ikeda H, Mori T, Sumimoto H ,Fujita T, Okamoto S, Ikeda Y and Kawakami Y.

British journal of Heamatology 132 ( 1 ) 56-65 2006.01

Research paper (scientific journal), Joint Work

Identification of a antigen, GARC-1, using cytotoxic T lymphocytes induced by HSV cancer vaccine

Iizuka Y, Kojima H, Kobata T, Kawase T, Kawakami Y and Toda M

International journal of Cancer 118 ( 4 ) 942-949 2006.02

Research paper (scientific journal), Joint Work

Autoantidoby to CD40 ligand in systemic lupus erythematosus: association with thrombocytopenia but not thromboembolism

M.Nakamura, Y. Tanaka, T. Satoh, M. Kawai, M. Hirakata, J.Kaburaki, Y. Kawakami, Y. Ikeda and M. Kuwana

Reumatology 45 ( 2 ) 150-156 2006.02

Research paper (scientific journal), Joint Work

A new melanoma antigen FABP7 involved in proliferation and invasion is a potential target for immunotherapy and molecular target therapy.

Goto Y, Matsuzaki Y, Kurihara S, Shimizu A, Okada T, Yamamoto K, Murata H, Takata M, Aburatani H, Hoon D, Saida T, Kawakami Y.

Cancer Res. 66 ( 8 ) 4443-4449 2006.04

Research paper (scientific journal), Joint Work

Increase in Circulating Endothelial Precursors by Atorvastain in Patients With Systemic Sclerosis.

Kuwana M, Kaburaki J, Okazaki Y, Yasuoka H, Kawakami Y, Ikeda Y,

Arthritis & Pheumatism. 54 ( 6 ) 1946-1951 2006.06

Research paper (scientific journal), Joint Work

The BRAF-MAPK signaling pathway is essential for cancer immune evasion in human melanoma cells.

Sumimoto H, Imabayashi F, Iwata T, Kawakami Y.

J Exp Med. 203 ( 7 ) 1651-1656 2006.07

Research paper (scientific journal), Joint Work

Effective inhibition of cell growth and invasion of melanoma by combined suppression of BRAF (V599E) and Skp2 with lentiviral RNAi.

Sumimoto Hidetoshi, Hirata K, Yamagata S, Miyoshi H, Miyagishi M, Taira K and Kawakami Yutaka

International Journal of Cancer 118 ( 2 ) 472-476 2006.01

Research paper (scientific journal), Joint Work

Neurogenic potential of Progenitors derived from human circulating CD14+ monocytes.

Kodama H, Inoue T, Watanabe R, Yasutomi D, Kawakami Y, Ogawa S, Mikoshiba K, Ikeda Y, Kuwana M.

Immunology and Cell Biology. 84 ( 2 ) 209-217 2006.04

Research paper (scientific journal), Joint Work

Frequent immune response to a melanocyte-specific protein KU-MEL-1 in patients with Vogt-Koyanagi-Harada disease.

Ohtani S, Sakurai T, Yamamoto K, Fujita T, Matsuzaki Y, Goto Y, Ando Y, Suzuki S, Usui M, Takeuchi M, Kawakami Y .

Br J Ophthalmol. 90 ( 6 ) 773-777 2006.06

Research paper (scientific journal), Joint Work

Analysis of the tumor reactivity of tumor-infiltrating lymphocytes in a metastatic melanoma lesion that lost MHC class I expression after anti-PD-1 therapy.

Inozume T, Yaguchi T, Ariyasu R, Togashi Y, Ohnuma T, Honobe A, Nishikawa H, Kawakami Y, Kawamura T.

J Invest Dermatol. pii: S0022-202X(19)30023-5 2019.01

Research paper (scientific journal), Joint Work

A defined commensal consortium induces CD8 T cells and anti-cancer immunity.

Tanoue T, Morita S, Plichta DR, Skelly AN, Suda W, Sugiura Y, Narushima S, Vlamakis H, Motoo I, Sugita K, Shiota A, Takeshita K, Yasuma K, Riethmacher D, Kaisho T, Norman JM, Mucida D, Suematsu M, Yaguchi T, Bucci V, Inoue T, Kawakami Y, Olle B, Roberts B, Hattori M, Xavier RJ, Atarashi K, Honda K.

Nature. 565 ( 7741 ) 600 - 605 2019.01

Research paper (scientific journal), Joint Work

Novel elucidation and treatment of pancreatic chronic graft-versus-host disease in mice.

Mukai S, Ogawa Y, Urano F, Kawakami Y, Tsubota K.

R Soc Open Sci. 5 ( 10 ) 181067 2018.10

Research paper (scientific journal), Joint Work

Therapeutic potential of tranilast for the treatment of chronic graft-versus-host disease in mice.

Mukai S, Ogawa Y, Saya H, Kawakami Y, Tsubota K.

PLOS ONE. 13 ( 10 ) e0203742 2018.10

Research paper (scientific journal), Joint Work

Inhibition of Vascular Adhesion Protein-1 for Treatment of Graft-Versus-Host Disease in Mice.

Mukai S, Ogawa Y, Kawakami Y, Mashima Y, Tsubota K.

FASEB J. 32 ( 8 ) 4085 - 4095 2018.08

Research paper (scientific journal), Joint Work

Carboplatin and epirubicin combination therapy for advanced malignant epithelial skin tumors: retrospective study of six patients.

Nakaymura Y, Tanase K, Hirai I, Kawakami Y, Funakoshi T

The Journal of Dermatology 45 ( 7 ) 874 - 875 2018.07

Research paper (scientific journal), Joint Work

Pilot study of WT1 peptide-pulsed dendritic cell vaccination with docetaxel in esophageal cancer.

Matsuda T,Takeuchi H, Sakurai T, Mayanagi S, Booka E, Fujita T, Higuchi H, Taguchi J, Hamamoto Y, Takaishi H, Kawakubo H, Okamoto M, Sunamura M, Kawakami Y, Kitagawa Y.

oncology letters. 16 ( 1 ) 1348 - 1356 2018.07

Research paper (scientific journal), Joint Work

Current status of immunotherapy against gastrointestinal cancers and its biomarkers: Perspective for precision immunotherapy.

Hazama S, Tamada K, Yamaguchi Y, Kawakami Y, Nagano H.

Annals of Gastroenterological Surgery 2 ( 4 ) 289 - 303 2018.06

Research paper (scientific journal), Joint Work

Evaluation of the appropriate surgical margin for pigmented basal cell carcinoma according to the risk factors for recurrence: a single-institute retrospective study in Japan.

Nakamura Y, Tanese K, Hirai I, Kameyama K, Kawakami Y, Amagai M, Funakoshi T.

J Eur Acad Dermatol Venereol. [Epub ahead of print] 2018.05

Research paper (scientific journal), Joint Work

Tumor associated macrophages support the growth of FGF9-induced lung adenocarcinoma by multiple mechanisms.

Ahmed Hegab, Mari Ozaki, Kagawa Shizuko, Junko Hamamoto, Hiroyuki Yasuda, Katsuhiko Naoki, Kenzo Soejima, Yongjun Yin, Tomonari Kinoshita, Tomonori Yaguchi, Yutaka Kawakami, David Orntiz, and Tomoko Betsuyaku.

Lung Cancer. 119   25 - 35 2018.05

Research paper (scientific journal), Joint Work

IgG response to MHC class I epitope peptides is a quantitative predictive biomarker in the early course of treatment of colorectal cancer using therapeutic peptides.

Kanekiyo S, Hazama S, Takenouchi H, Nakajima M, Shindo Y, Matsui H, Tokumitsu Y, Tomochika S, Tsunedomi R, Tokuhisa Y, Iida M, Sakamoto K, Suzuki N, Takeda S, Yamamoto S, Yoshino S, Okuno K, Udaka K, Kawakami Y, Matsueda S, Ito K, Nagano H.

Oncol Rep. 39 ( 5 ) 2385 - 2392 2018.05

Research paper (scientific journal), Joint Work

IgG response to MHC class I epitope peptides is a quantitative predictive biomarker in the early course of treatment of colorectal cancer using therapeutic peptides.

Kanekiyo S, Hazama S, Takenouchi H, Nakajima M, Shindo Y, Matsui H, Tokumitsu Y, Tomochika S, Tsunedomi R, Tokuhisa Y, Iida M, Sakamoto K, Suzuki N, Takeda S, Yamamoto S, Yoshino S, Okuno K, Udaka K, Kawakami Y, Matsueda S, Ito K, Nagano H.

Oncol Rep. 39 ( 5 ) 2385 - 2392 2018.05

Research paper (scientific journal), Joint Work

International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study.

Franck Pagès, Bernhard Mlecnik, Florence Marliot, Gabriela Bindea, Fang-Shu Ou, Carlo Bifulco, Alessandro Lugli, Inti Zlobec, Tilman T. Rau, Martin D. Berger, Iris D. Nagtegaal, Elisa Vink-Börger, Arndt Hartmann, Carol Geppert, Julie Kolwelter, Susanne Merkel, Robert Grützmann, Marc Van den Eynd, Anne Jouret-Mourin, Alex Kartheuser, Daniel Léonard, Christophe Remue, Michael H. A. Roehrl, Prashant Bavi, Pamela S. Ohashi, Julia Y. Wang, Linh T.Nguyen, SeongJun Han, Heather L. MacGregor, Sara Hafezi-Bakhtiari, Bradly G. Wouters, Giuseppe V. Masucci, Emilia K. Andersson, Eva Zavadova, Michal Vocka, Jan Spacek, Lubos Petruzelka, Bohuslav Konopasek, Pavel Dundr, Helena Skalova, Kristyna Nemejcova, Gerardo Botti, Fabiana Tatangelo, Paolo Delrio, Gennaro Cilberto, Michele Maio, Luigi Laghi, Fabio Grizzi, Tessa Fredriksen, Bénédicte Buttard, Mihaela Angelova, Angela Vasaturo, Pauline Maby, Sarah E. Church, Helen K. Angell, Lucie Lafontaine, Daniela Bruni, Carine El Sissy, Nacilla Haicheur, Amos Kirilovsky, Anne Berger, Christine Lagorce, Jeffrey P. Meyers, Christopher Paustian, Carmen Ballesteros-Merino, Jeroen Dijkstra, Carlijn van de Water, Shannon van Lent- van Vliet, Nikki Knijn, Ana-Maria Mușină, Dragos-Viorel Scripcariu, Boryana Popivanova, Mingli Xu, Tomonobu Fujita, Shoichi Hazama, Nobuaki Suzuki, Hiroaki Nagano, Kiyotaka Okuno, Toshihiko Torigoe, Noriyuki Sato, Tomohisa Furuhata, Ichiro Takemasa, Kyogo Itoh, Prabhu S. Patel, Hemangini H. Vora, Birva Shah, ayendrakumar B. Patel, Kruti N. Rajvik, Shashank J. Pandya, Shilin N. Shukla, Yili Wang, Guanjun Zhang, Yutaka Kawakami, Francesco M. Marincola, Paolo A. Ascierto, Daniel J. Sargent, Bernard A. Fox, Jérôme Galon.

Lancet. 391 ( 10135 ) 2128 - 2139 2018.05

Research paper (scientific journal), Joint Work

Evaluation of the appropriate surgical margin for pigmented basal cell carcinoma according to the risk factors for recurrence: a single-institute retrospective study in Japan.

Nakamura Y, Tanese K, Hirai I, Kameyama K, Kawakami Y, Amagai M, Funakoshi T.

J Eur Acad Dermatol Venereol. [Epub ahead of print] 2018.05

Research paper (scientific journal), Joint Work

A pilot study of peptide vaccines for VEGF receptor 1 and 2 in patients with recurrent/progressive high grade glioma.

Shibao S, Ueda R, Saito K, Kikuchi R, Nagashima H, Kojima A, Kagami H, Sasaki H, Noji S, Kawakami Y, Yoshida K, Toda M.

Oncotarget. 9 ( 30 ) 21569 - 21579 2018.04

Research paper (scientific journal), Joint Work

Association between programmed cell death ligand-1 expression and extracranial metastasis in intracranial solitary fibrous tumor/hemangiopericytoma.

Kamamoto D, Ohara K, Kitamura Y, Yoshida K, Kawakami Y, Sasaki H.

Journal of neuro-oncology 139 ( 2 ) 251 - 259 2018.04

Research paper (scientific journal), Joint Work

Generation of human immunosuppressive myeloid cell populations in human interleukin-6 transgenic NOG mice.

Hanazawa A, Ito R, Katano I, Kawai K, Goto M, Suemizu H, Kawakami Y, Ito M, Takahashi T.

Frontiers in Immunology. 9 ( 152 )  2018.02

Research paper (scientific journal), Joint Work

Ocular Surface and Tear Film Characteristics in a Sclerodermatous Chronic Graft-Versus-Host Disease Mouse Model.

He J, Yamane M, Shibata S, Fukui M, Shimizu E, Yano T, Mukai S, Kawakami Y, Li S, Tsubota K, Ogawa Y.

Cornea. 37 ( 4 ) 486 - 494 2018.01

Research paper (scientific journal), Joint Work

Functional Role of Lacrimal Gland Fibroblasts in a Mouse Model of Chronic Graft-Versus-Host Disease.

Yamane M, Ogawa Y, Mukai S, Yaguchi S, Kamijuku H, Inaba T, Asai K, Morikawa S, Kawakami Y, Shimmura S, Tsubota K.

Cornea 37 ( 1 ) 102 - 108 2018.01

Research paper (scientific journal), Joint Work

Inovolbement of local renin-angiotensin system in immunosuppression of tumor microenvironment.

Nakamura K, Yaguchi T, Ohmura G, Kobayashi A, Kawamura N, Iwata T, Kiniwa Y, Okuyama R, Kawakami Y.

Cancer Science. 109 ( 1 ) 54 - 64 2018.01

Research paper (scientific journal), Joint Work

Long-term maintenance of peripheral blood derived human NK cells in a novel human IL-15- transgenic NOG mouse.

Katano I, Nishime C, Ito R, Kamisako T, Mizusawa T, Ka Y, Ogura T, Suemizu H, Kawakami Y, Ito M, Takahashi T.

Scientific Reports. 7 ( 1 ) 17230 2017.12

Research paper (scientific journal), Joint Work

Determination of poor prognostic immune features of tumor microenvironment in nonsmoking patients with lung adenocarcinoma.

Kinoshita T, Kudo-Saito C, Muramatsu R, Fujita T, Saito M, Nagumo H, Sakurai T, Noji S, Takahata E, Yaguchi T, Tsukamoto N, Hayashi Y, Kaseda K, Kamiyama I, Ohtsuka T, Tomizawa K, Shimoji M, Mitsudomi T, Asamura H, Kawakami Y.

European Journal of Cancer. 86   15 - 27 2017.11

Research paper (scientific journal), Joint Work

Role of the autonomic nervous system in the tumor micro-environment and its therapeutic potential.

Xu Z, Shioda S, Jinushi M, Kawakami Y, Ohtaki H, Wang S, Zhao X, Liu Y, Zhou D, Guo Y.

Current pharmaceutical design 23 ( 11 ) 1687 - 1692 2017.11

Research paper (scientific journal), Joint Work

Human PBMC transferred murine MHC class I/II deficient NOG mice enable long-term evaluation of human immune responses.

Yaguchi T, Kobayashi A, Inozume T, Morii K, Nagumo H, Nishio H, Iwata T, Ka Y, Katano I, Ito R, Ito M, Kawakami Y.

Cellular & Molecular Immunology 14   1-10 2017.11

Research paper (scientific journal), Joint Work

Reduced Expression of VAMP8 in Lacrimal Gland Affected by Chronic Graft-versus-Host Disease.

Fukui M, Ogawa Y, Mukai S, Kamoi M, Asato T, Kawakami Y, Tsubota K.

Journal of Ophthalmology 2017   1639012 2017.09

Research paper (scientific journal), Joint Work

CXorf48 is a potential therapeutic target for achieving treatment-free remission in CML patients.

Matsushita M, Ozawa K, Suzuki T, Nakamura M, Nakano N, Kanchi S, Ichikawa D, Matsuki E, Sakurai M, Karigane D, Kasahara H, Tsukamoto N, Shimizu T, Mori T, Nakajima H, Okamoto S, Kawakami Y, Hattori Y.

Blood Cancer Journal 7   e601 2017.09

Research paper (scientific journal), Joint Work

Peptide-pulsed dendritic cell vaccine in combination with carboplatin and paclitaxel chemotherapy for stage IV melanoma.

Fukuda K, Funakoshi T, Sakurai T, Nakamura Y, Mori M, Tanese K, Tanikawa A, Taguchi J, Fujita T, Okamoto M, Amagai M, Kawakami Y.

Melanoma Research. 27 ( 4 ) 326 - 334 2017.08

Research paper (scientific journal), Joint Work

miR-196b, miR-378a and miR-486 are predictive biomarkers for the efficacy of vaccine treatment in colorectal cancer

Shindo Yoshitaro, Hazama Shoichi, Nakamura Yusuke, Inoue Yuka, Kanekiyo Shinsuke, Suzuki Nobuaki, Takenouchi Hiroko, Tsunedomi Ryouichi, Nakajima Masao, Ueno Tomio, Takeda Shigeru, Yoshino Shigefumi, Okuno Kiyotaka, Fujita Yusuke, Hamamoto Yoshihiko, Kawakami Yutaka, Oka Masaaki, Nagano Hiroaki

Oncology Letters 14 ( 2 ) 1355 - 1362 2017.08

Research paper (scientific journal),  ISSN  1792-1074

　View Summary

<p>MicroRNAs (miRNAs/miRs) regulate the levels of transcripts and serve a critical function in the regulation of tumor microenvironments. Therefore, miRNA levels in cancer tissues are thought to be potential biomarkers for immunotherapy. From a phase I trial of a vaccine treatment using 5 human leukocyte antigen (HLA)-A* 2402-restricted peptides (registration no. UMIN000004948), colorectal cancer (CRC) tissues were obtained from 8 patients and normal colorectal tissues from 5 patients via surgery. From a phase II trial using the same peptides (registration no. UMIN000001791), CRC tissues were obtained from 16 patients from the HLA-A* 2402-matched group and 10 patients from the HLA-A* 2402-unmatched group. These tissues were used for miRNA microarray analysis. As the first step, cancer tissues from the phase I study were used and 10 candidate miRNAs were selected by comparing the miRNA expression between two groups one with improved prognosis and the other with poor prognosis. The miRNAs were subsequently validated using the cases enrolled in the phase II study. Significantly improved prognoses were identified in 16 patients in the HLA-A* 2402-matched group with high expression of miR-196b-5p and low expression of miR-378a-3p and miR-486-5p. There was no difference in prognosis in the 10 patients in the HLA-A* 2402-unmatched group. Therefore, high miR-196b expression and low miR-378a-3p and miR-486-5p expression were indicated as useful biomarkers for prediction of the efficacy of vaccine treatment for patients with metastatic CRC. In a planned phase III study, expression levels of these 3 miRNAs (miR-196b-5p, miR-378a-3p and miR-486-5p) may be useful biomarkers for assessing patients who are likely to have an improved outcome following vaccination.</p>

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Autophagy-dependent regulation of tumor metastasis by myeloid cells.

Jinushi M, Morita T, Xu Z, Kinoshita I, Dosaka-Akita H, Yagita H, Kawakami Y.

PLOS ONE. 12 ( 7 ) e0179357 2017.07

Research paper (scientific journal), Joint Work

MIF: functions in brain and glioblastoma.

Ohta Shigeki, Kawakami Yutaka, Okano Hideyuki

Oncotarget 8 ( 29 ) 46706 - 46707 2017.06

Research paper (scientific journal), Joint Work,  ISSN  1949-2553

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Lack of XAGE-1b and NY-ESO-1 in metastatic lymph nodes may predict the potential survival of stage III melanoma patients

Mori Mariko, Funakoshi Takeru, Kameyama Kaori, Kawakami Yutaka, Sato Eiichi, Nakayama Eiichi, Amagai Masayuki, Tanese Keiji

Journal of Dermatology 44 ( 6 ) 671 - 680 2017.06

Research paper (scientific journal), Joint Work,  ISSN  0385-2407

　View Summary

<p>The cancer-testis antigens (CTA) are a large family of tumor-associated antigens expressed by a variety of cancer cells and primitive germ cells of the adult testis and placenta. These tumor-restricted expressing patterns suggest that CTA would be ideal targets for tumor-specific immunotherapy. XAGE-1 is a CTA that was originally identified by computer-based screening, and four transcription variants, XAGE-1a, -1b, -1c and -1d, have been characterized to date. Although the presence of XAGE-1 transcripts has been reported in various cancers, the expression of XAGE-1b in melanoma has not been fully characterized. In this study, we performed immunohistochemical staining of XAGE-1b together with NY-ESO-1, a well-known CTA, in 113 melanoma samples obtained from 84 patients and evaluated their expression in tumor cells. The effects of expression on tumor progression and patient prognosis were analyzed. Both XAGE-1b and NY-ESO-1 were expressed at high levels in lymph node metastasis and skin metastasis samples compared with the primary site (P &lt; 0.01 in XAGE-1b and P &lt; 0.05 in NY-ESO-1). In a subgroup analysis of 22 patients with stage III lymph node metastasis, overall survival was significantly higher in the XAGE-1b and NY-ESO-1 double-negative group than in the other groups (P &lt; 0.05). These results suggest that lack of XAGE-1b and NY-ESO-1 expression could have a positive influence on clinical outcome in patients with melanoma.</p>

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A randomized phase II study comparing nivolumab with carboplatin-pemetrexed for patients with EGFR mutation-positive nonsquamous non-small cell lung cancer who acquire resistance to tyrosine kinase inhibitors not due to a secondary T790M mutation

Hayashi H, Chiba Y, Sakai K, Fujita T, Yoshioka H, Sakai D, Kitagawa C, Naito T, Takeda K, Okamoto I, Mitsudomi T, Kawakami Y, Nishio K, Nakamura S, Yamamoto N, Nakagawa K.

Clinical Lung Cancer. 18   719 - 723 2017.05

Research paper (scientific journal), Joint Work

Inter-patient and Intra-tumor Heterogeneity in the Sensitivity to Tumor-targeted Immunity in Colorectal Cancer.

Miyauchi T, Yaguchi T, Kawakami Y.

Jpn J Clin Immunol. 40 ( 1 ) 54 - 59 2017.05

Research paper (scientific journal), Joint Work

Prospects for personalized combination immunotherapy for solid tumors based on adoptive cell therapies and immune checkpoint blockade therapies.

Kato D, Yaguchi T, Iwata T, Morii K, Nakagawa T, Nishimura R, Kawakami Y.

Jpn J Clin Immunol. 40 ( 1 ) 68 - 77 2017.05

Research paper (scientific journal), Joint Work

Novel Treatment of Chronic Graft-Versus-Host Disease in Mice Using the ER Stress Reducer 4-Phenylbutyric Acid

Mukai Shin, Ogawa Yoko, Urano Fumihiko, Kudo-Saito Chie, Kawakami Yutaka, Tsubota Kazuo

Scientific Reports 7 2017.02

Research paper (scientific journal), Joint Work,  ISSN  2045-2322

　View Summary

<p>Chronic graft-versus-host disease (cGVHD) is a notorious complication of allogeneic hematopoietic stem cell transplantation and causes disabling systemic inflammation and fibrosis. In this novel study, we focused on a relationship between endoplasmic reticulum (ER) stress and cGVHD, and aimed to create effective treatment of cGVHD. A series of experiments were conducted using a mouse model of cGVHD. Our data suggested (1) that ER stress was elevated in organs affected by cGVHD and (2) that 4-phenylbutyric acid (PBA) could reduce cGVHD-induced ER stress and thereby alleviate systemic inflammation and fibrosis. Because fibroblasts are thought to be implicated in cGVHD-elicited fibrosis and because macrophages are reported to play a role in the development of cGVHD, we investigated cGVHD-triggered ER stress in fibroblasts and macrophages. Our investigation demonstrated (1) that indicators for ER stress and activation markers for fibroblasts were elevated in cGVHD-affected lacrimal gland fibroblasts and (2) that they could be reduced by PBA. Our work also indicated that splenic macrophages from PBA-dosed mice exhibited the lower levels of ER stress and M2 macrophage markers than those from cGVHD-affected mice. Collectively, this study suggests that the reduction of ER stress utilizing PBA can be a clinically translatable method to treat systemic cGVHD.</p>

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Predictive biomarkers for the efficacy of peptide vaccine treatment: based on the results of a phase II study on advanced pancreatic cancer.

Shindo Y, Hazama S, Suzuki N, Iguchi H, Uesugi K, Tanaka H, Aruga A, Hatori T, Ishizaki H, Umeda Y, Fujiwara T, Ikemoto T, Shimada M, Yoshimatsu K, Takenouchi H, Matsui H, Kanekiyo S, Iida M, Koki Y, Arima H, Furukawa H, Ueno T, Yoshino S, Fujita T, Kawakami Y, Nakamura Y, Oka M, Nagano H.

Journal of Experimental & Clinical Cnacer Research 36 ( 1 ) 36 2017.02

Research paper (scientific journal), Joint Work

Functional analysis of KIF20A, a potential immunotherapeutic target for glioma

Saito Katsuya, Ohta Shigeki, Kawakami Yutaka, Yoshida Kazunari, Toda Masahiro

Journal of Neuro-Oncology 132 ( 1 ) 63 - 74 2017.01

Research paper (scientific journal), Joint Work,  ISSN  0167-594X

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<p>Kinesin family member 20A (KIF20A), an ideal cancer-testis antigen, was reported to be a promising immunotherapeutic target for pancreatic cancers. Clinical trials of KIF20A peptide vaccine immunotherapy have been conducted against pancreatic cancers. To demonstrate the efficacy of KIF20A as a candidate molecular target for gliomas, we analyzed the expression and function of KIF20A in gliomas. Western blot and quantitative PCR analyses showed that KIF20A expression in glioma cell lines and glioma tissues was high compared with that found in a normal brain. KIF20A immunostaining of glioma cells and glioma tissues demonstrated that KIF20A was involved in spindle formation and cytokinesis, and that KIF20A was highly expressed, especially in glioma cells undergoing mitosis. In silico analysis of a cancer microarray database revealed that KIF20A was highly expressed in gliomas depending on the pathological grade, and glioma patients with higher expression of KIF20A showed poorer prognosis. Down-regulating KIF20A reduced cell proliferation in glioma cells due to the failure of cytokinesis and generation of binucleated cells. Additionally, KIF20A inhibition induced significant apoptosis in SF126 glioma cells. Taken together, KIF20A is a tumor-associated antigen involved in the glioma cell growth and cell survival, suggesting that KIF20A is an oncoantigen of gliomas. Thus, KIF20A is a candidate novel immunotherapeutic target for gliomas.</p>

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Progression of Human Renal Cell Carcinoma via Inhibition of RhoA-ROCK Axis by PARG1.

Miyazaki J, Ito K, Fujita T, Matsuzaki Y, Asano T, Hayakawa M, Asano T, Kawakami Y.

Transl Oncol. 10 ( 2 ) 142 - 152 2017.01

Research paper (scientific journal), Joint Work

CHD7 promotes proliferation of neural stem cells mediated by MIF

Ohta Shigeki, Yaguchi Tomonori, Okuno Hironobu, Chneiweiss Hervé, Kawakami Yutaka, Okano Hideyuki

Molecular Brain 9 ( 1 ) 1 - 12 2016.12

Research paper (scientific journal), Joint Work,  ISSN  1756-6606

　View Summary

<p>Macrophage migration inhibitory factor (MIF) plays an important role in supporting the proliferation and/or survival of murine neural stem/progenitor cells (NSPCs); however, the downstream effectors of this factor remain unknown. Here, we show that MIF increases the expression of Pax6 and Chd7 in NSPCs in vitro. During neural development, the chromatin remodeling factor Chd7 (chromatin helicase-DNA-binding protein 7) is expressed in the ventricular zone of the telencephalon of mouse brain at embryonic day 14.5, as well as in cultured NSPCs. Retroviral overexpression of Pax6 in NSPCs increased Chd7 gene expression. Lentivirally-expressed Chd7 shRNA suppressed cell proliferation and neurosphere formation, and inhibited neurogenesis in vitro, while decreasing gene expression of Hes5 and N-myc. In addition, CHD7 overexpression increased cell proliferation in human embryonic stem cell-derived NSPCs (ES-NSPCs). In Chd7 mutant fetal mouse brains, there were fewer intermediate progenitor cells (IPCs) compared to wildtype littermates, indicating that Chd7 contributes to neurogenesis in the early developmental mouse brain. Furthermore, in silico database analysis showed that, among members of the CHD family, CHD7 is highly expressed in human gliomas. Interestingly, high levels of CHD7 gene expression in human glioma initiating cells (GICs) compared to normal astrocytes were revealed and gene silencing of CHD7 decreased GIC proliferation. Collectively, our data demonstrate that CHD7 is an important factor in the proliferation and stemness maintenance of NSPCs, and CHD7 is a promising therapeutic target for the treatment of gliomas.</p>

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Prognostic value of tumor-infiltrating lymphocytes differs depending on histological type and smoking habit in completely resected non-small-cell lung cancer.

Kinoshita T, Muramatsu R, Fujita T, Nagumo H, Sakurai T, Noji S, Takahata E, Yaguchi T, Tsukamoto N, Kudo-Saito C, Hayashi Y, Kamiyama I, Ohtsuka T, Asamura H, Kawakami Y.　

Annals Of Oncology. 27 ( 11 ) 2117 - 2123 2016.11

Research paper (scientific journal), Joint Work

Targeting ALCAM in the cryo-treated tumour microenvironment successfully induces systemic anti-tumour immunity

Kudo-Saito Chie, Fuwa Takafumi, Kawakami Yutaka

European Journal of Cancer 62   54 - 61 2016.07

Research paper (scientific journal), Joint Work,  ISSN  0959-8049

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<p>Cryoablative treatment has been widely used for treating cancer. However, the therapeutic efficacies are still controversial. The molecular mechanisms of the cryo-induced immune responses, particularly underlying the ineffectiveness, remain to be fully elucidated. In this study, we identified a new molecular mechanism involved in the cryo failure. We used cryo-ineffective metastatic tumour models that murine melanoma B16-F10 cells were subcutaneously and intravenously implanted into C57BL/6 mice. When the subcutaneous tumours were treated cryoablation on day 7 after tumour implantation, cells expressing activated leucocyte cell adhesion molecule (ALCAM/CD166) were significantly expanded not only locally in the treated tumours but also systemically in spleen and bone marrow of the mice. The cryo-induced ALCAM⁺ cells including CD45^- mesenchymal stem/stromal cells, CD11b⁺Gr1⁺ myeloid-derived suppressor cells, and CD4⁺Foxp3⁺ regulatory T cells significantly suppressed interferon γ production and cytotoxicity of tumour-specific CD8⁺ T cells via ALCAM expressed in these cells. This suggests that systemic expansion of the ALCAM⁺ cells negatively switches host-immune directivity to the tumour-supportive mode. Intratumoural injection with anti-ALCAM blocking monoclonal antibody (mAb) following the cryo treatment systemically induced tumour-specific CD8⁺ T cells with higher cytotoxic activities, resulting in suppression of tumour growth and metastasis in the cryo-resistant tumour models. These suggest that expansion of ALCAM⁺ cells is a determinant of limiting the cryo efficacy. Further combination with an immune checkpoint inhibitor anti-CTLA4 mAb optimized the anti-tumour efficacy of the dual-combination therapy. Targeting ALCAM may be a promising strategy for overcoming the cryo ineffectiveness leading to the better practical use of cryoablation in clinical treatment of cancer.</p>

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MIF maintains the tumorigenic capacity of brain tumor-initiating cells by directly inhibiting p53

Fukaya Raita, Ohta Shigeki, Yaguchi Tomonori, Matsuzaki Yumi, Sugihara Eiji, Okano Hideyuki, Saya Hideyuki, Kawakami Yutaka, Kawase Takeshi, Yoshida Kazunari, Toda Masahiro

Cancer Research 76 ( 9 ) 2813 - 2823 2016.05

Research paper (scientific journal), Joint Work,  ISSN  0008-5472

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<p>Tumor-initiating cells thought to drive brain cancer are embedded in a complex heterogeneous histology. In this study, we isolated primary cells from 21 human brain tumor specimens to establish cell lines with high tumorigenic potential and to identify the molecules enabling this capability. The morphology, sphereforming ability upon expansion, and differentiation potential of all cell lines were indistinguishable in vitro. However, testing for tumorigenicity revealed two distinct cell types, brain tumor-initiating cells (BTIC) and non-BTIC. We found that macrophage migration inhibitory factor (MIF) was highly expressed in BTIC compared with non-BTIC. MIF bound directly to both wild-type and mutant p53 but regulated p53-dependent cell growth by different mechanisms, depending on glioma cell line and p53 status. MIF physically interacted with wild-type p53 in the nucleus and inhibited its transcription-dependent functions. In contrast, MIF bound to mutant p53 in the cytoplasm and abrogated transcription-independent induction of apoptosis. Furthermore, MIF knockdown inhibited BTIC-induced tumor formation in a mouse xenograft model, leading to increased overall survival. Collectively, our findings suggest that MIF regulates BTIC function through direct, intracellular inhibition of p53, shedding light on the molecular mechanisms underlying the tumorigenicity of certain malignant brain cells.</p>

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MHC-compatible bone marrow stromal/stem cells trigger fibrosis by activating host T cells in a scleroderma mouse model

Ogawa Yoko, Morikawa Satoru, Okano Hideyuki, Mabuchi Yo, Suzuki Sadafumi, Yaguchi Tomonori, Sato Yukio, Mukai Shin, Yaguchi Saori, Inaba Takaaki, Okamoto Shinichiro, Kawakami Yutaka, Tsubota Kazuo, Matsuzaki Yumi, Shimmura Shigeto

eLife 5   e09394 2016.01

Research paper (scientific journal), Joint Work

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<p>Fibrosis of organs is observed in systemic autoimmune disease. Using a scleroderma mouse, we show that transplantation of MHC compatible, minor antigen mismatched bone marrow stromal/stem cells (BMSCs) play a role in the pathogenesis of fibrosis. Removal of donor BMSCs rescued mice from disease. Freshly isolated PDGFRα⁺ Sca-1⁺ BMSCs expressed MHC class II following transplantation and activated host T cells. A decrease in FOXP3⁺ CD25⁺ Treg population was observed. T cells proliferated and secreted IL-6 when stimulated with mismatched BMSCs in vitro. Donor T cells were not involved in fibrosis because transplanting T cell-deficient RAG2 knock out mice bone marrow still caused disease. Once initially triggered by mismatched BMSCs, the autoimmune phenotype was not donor BMSC dependent as the phenotype was observed after effector T cells were adoptively transferred into naïve syngeneic mice. Our data suggest that minor antigen mismatched BMSCs trigger systemic fibrosis in this autoimmune scleroderma model.</p>

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Melanoma cells control antimelanoma CTL responses via interaction between TIGIT and CD155 in the effector phase

Inozume Takashi, Yaguchi Tomonori, Furuta Junpei, Harada Kazutoshi, Kawakami Yutaka, Shimada Shinji

Journal of Investigative Dermatology 136 ( 1 ) 255 - 263 2016.01

Research paper (scientific journal), Joint Work,  ISSN  0022-202X

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<p>Recently, T-cell immunoreceptor with Ig and ITIM domains (TIGIT) was reported as a candidate for novel immune checkpoints. However, the impact of TIGIT on melanoma-specific cytotoxic T lymphocytes in the effector phase remains unclear. In this study, we demonstrated that melanoma cells control antimelanoma cytotoxic T lymphocyte responses via the TIGIT-CD155 interaction in the effector phase. TIGIT is an inhibitory receptor expressed on T cells, and CD155 is one of the cognate ligands expressed on the tumor cells or antigen presenting cells. First, we confirmed that CD155 was constitutively expressed on melanoma cells. We then demonstrated that CD155 on melanoma cells suppressed cytokine release from melanoma-specific cytotoxic T lymphocytes via interaction with TIGIT. Overexpression of CD155 enhanced and its downregulation attenuated the suppressive effect. This suggested that antimelanoma cytotoxic T lymphocyte responses are controlled not only by an imbalance in CD226 (an activating molecule that binds to CD155) and TIGIT expression on T cells but also by the expression levels of CD155 on melanoma cells. In addition, the co-blockade of TIGIT and PD-1 signals synergistically elicited a response of tumor-infiltrating lymphocytes on autologous melanoma cells. These results suggest that the CD155-TIGIT interaction should be blocked for enhancement of antimelanoma immune responses.</p>

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Identification of novel HLA-A*24:02-restricted epitope derived from a homeobox protein expressed in hematological malignancies.

Matsushita Maiko, Otsuka Yohei, Tsutsumida Naoya, Tanaka Chiaki, Uchiumi Akane, Ozawa Koji, Suzuki Takuma, Ichikawa Daiju, Aburatani Hiroyuki, Okamoto Shinichiro, Kawakami Yutaka, Hattori Yutaka

PLoS One 11 ( 1 ) e0146371 2016.01

Research paper (scientific journal), Joint Work

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<p>The homeobox protein, PEPP2 (RHOXF2), has been suggested as a cancer/testis (CT) antigen based on its expression pattern. However, the peptide epitope of PEPP2 that is recognized by cytotoxic T cells (CTLs) is unknown. In this study, we revealed that PEPP2 gene was highly expressed in myeloid leukemia cells and some other hematological malignancies. This gene was also expressed in leukemic stem-like cells. We next identified the first reported epitope peptide (PEPP2^271-279). The CTLs induced by PEPP2^271-279 recognized PEPP2-positive target cells in an HLA-A∗24:02-restricted manner. We also found that a demethylating agent, 5-aza-2′-deoxycytidine, could enhance PEPP2 expression in leukemia cells but not in blood mononuclear cells from healthy donors. The cytotoxic activity of anti-PEPP2 CTL against leukemic cells treated with 5-aza-2′-deoxycytidine was higher than that directed against untreated cells. These results suggest a clinical rationale that combined treatment with this novel antigen-specific immunotherapy together with demethylating agents might be effective in therapy-resistant myeloid leukemia patients.</p>

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Cancer-induced heterogeneous immunosuppressive tumor microenvironments and their personalized modulation

Yaguchi Tomonori, Kawakami Yutaka

International Immunology 28 ( 8 ) 393 - 399 2016

Research paper (scientific journal), Joint Work,  ISSN  0953-8178

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<p>Although recent cancer immunotherapy strategies, including immune-checkpoint blockade (i.e. blocking PD-1, PD-L1 or CTLA-4), have shown durable clinical effects in some (but not all) patients with various advanced cancers, further understanding of human immunopathology, particularly in tumor microenvironments, is essential to improve this type of therapy. The major hurdle for immunotherapy is the immunosuppression that is found in cancer patients. There are two types of immunosuppression: one is induced by gene alterations in cancer; the other is local adaptive immunosuppression, triggered by tumor-specific T cells in tumors. The former is caused by multiple mechanisms via various immunosuppressive molecules and via cells triggered by gene alterations, including activated oncogenes, in cancer cells. The various immunosuppressive mechanisms involve signaling cascades that vary among cancer types, subsets within cancer types and individual cancers. Therefore, personalized immune-interventions are necessary to appropriately target oncogene-induced signaling that modulates anti-cancer immune responses, on the basis of genetic and immunological analysis of each patient. Further understanding of human cancer immunopathology may lead to real improvement of current cancer immunotherapies.</p>

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2015 Guidance on cancer immunotherapy development in early-phase clinical studies

Arato Teruyo, Yamaguchi Yoshiyuki, Daimon Takashi, Heike Yuji, Ishii Ken, Kobiyama Kouji, Itho Kyogo, Kageyama Shinichi, Shiku Hiroshi, Miyahara Yoshihiro, Ikeda Hiroaki, Harada Naozumi, Muraoka Daisuke, Kawakami Yutaka, Nakayama Eiichi, Ozawa Keiya, Sato Noriyuki, Takeuchi Masahiro, Tani Kenzaburo, Tamada Koji, Ueda Ryuzo, Yamanaka Takeharu, Yamaue Hiroki, Yasukawa Masaki, Iguchi Toyotaka, Terashima Takeshi, Amakasu Kohei, Kitano Shigehisa, Nagata Yasuhiro, Noguchi Masanori, Asano Takeo, Asonuma Motohiro, Kakimi Kazuhiro, Takesako Kazutoh, Tanaka Masanori, Yamada Akira, Aoshi Taiki, Kuroda Etsushi, Yamazaki Naoya, Kuchiba Aya, Tanaka Shiro, Michimae Hirofumi, Yamada Tomomi, Hanada Kenichi, Okada Hideho, Takebe Naoko, Hirano Naoto, Okumura Satoshi, Shibatsuji Masayoshi, Sato Daisaku, Yamaguchi Teruhide

Cancer Science 106 ( 12 ) 1761 - 1771 2015.12

Research paper (scientific journal), Joint Work,  ISSN  1347-9032

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<p>The development of cancer immunotherapies is progressing rapidly with a variety of technological approaches. They consist of "cancer vaccines", which are based on the idea of vaccination, "effector cell therapy", classified as passive immunotherapy, and "inhibition of immunosuppression", which intends to break immunological tolerance to autoantigens or immunosuppressive environments characterizing antitumor immune responses. Recent reports showing clinical evidence of efficacy of immune checkpoint inhibitors and adoptive immunotherapies with tumor-infiltrating lymphocytes and tumor-specific receptor gene-modified T cells indicate the beginning of a new era for cancer immunotherapy. This guidance summarizes ideas that will be helpful to those who plan to develop cancer immunotherapy. The aims of this guidance are to discuss and offer important points in early phase clinical studies of innovative cancer immunotherapy, with future progress in this field, and to contribute to the effective development of cancer immunotherapy aligned with the scope of regulatory science. This guidance covers cancer vaccines, effector cell therapy, and inhibition of immunosuppression, including immune checkpoint inhibitors.</p>

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Cancer immunotherapy by immuno-checkpoint blockade

Kawakami Yutaka

[Rinshō ketsueki] The Japanese journal of clinical hematology 56 ( 10 ) 2186 - 2194 2015.10

ISSN  0485-1439

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<p>As cancer immunotherapies utilizing anti-tumor T-cell responses, immuno-checkpoint blockade and adoptive T-cell immunotherapy have recently achieved durable responses even in advanced cancer patients with metastases. Administration of antibodies on the T-cell surface, CTLA-4 and PD-1 (or PD-1 ligand PD-L1), resulted in tumor regression of not only melanoma and renal cell cancer which were known to be relatively sensitive to immunotherapy, but also various malignancies including lung, bladder, ovarian, gastric, and head and neck cancers, as well as hematological malignancies such as Hodgkin and B-cell malignant lymphomas. These findings have changed the status of immunotherapy in the development of cancer treatments. Currently, development of combinations employing cancer immunotherapy with immuno-checkpoint blockade, as well as personalized cancer immunotherapy based on the evaluation of pretreatment immune status, are in progress. </p>

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NUAK2 amplification coupled with PTEN deficiency promotes melanoma development via CDK Activation

Namiki Takeshi, Yaguchi Tomonori, Nakamura Kenta, Valencia Julio C., Coelho Sergio G., Yin Lanlan, Kawaguchi Masakazu, Vieira Wilfred D., Kaneko Yasuhiko, Tanemura Atsushi, Katayama Ichiro, Yokozeki Hiroo, Kawakami Yutaka, Hearing Vincent J.

Cancer Research 75 ( 13 ) 2708 - 2715 2015.07

Research paper (scientific journal), Joint Work,  ISSN  0008-5472

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<p>The AMPK-related kinase NUAK2 has been implicated in melanoma growth and survival outcomes, but its therapeutic utility has yet to be confirmed. In this study, we show how its genetic amplification in PTEN-deficient melanomas may rationalize the use of CDK2 inhibitors as a therapeutic strategy. Analysis of array-CGH data revealed that PTEN deficiency is coupled tightly with genomic amplification encompassing the NUAK2 locus, a finding strengthened by immunohistochemical evidence that phospho-Akt overexpression was correlated with NUAK2 expression in clinical specimens of acral melanoma. Functional studies in melanoma cells showed that inactivation of the PI3K pathway upregulatedp21 expression and reduced the number of cells in S phase. NUAK2 silencing and inactivation of the PI3K pathway efficiently controlled CDK2 expression, whereas CDK2 inactivation specifically abrogated the growth of NUAK2-amplified and PTEN-deficient melanoma cells. Immunohistochemical analyses confirmed an association of CDK2 expression with NUAK2 amplification and p-Akt expression in melanomas. Finally, pharmacologic inhibition of CDK2 was sufficient to suppress the growth of NUAK2-amplified and PTEN-deficient melanoma cells in vitro and in vivo. Overall, our results show how CDK2 blockade may offer a promising therapy for genetically defined melanomas, where NUAK2 is amplified and PTEN is deleted.</p>

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Synergistic antiproliferative effect of imatinib and adriamycin in platelet-derived growth factor receptor-expressing osteosarcoma cells

Yamaguchi Sayaka I., Ueki Arisa, Sugihara Eiji, Onishi Nobuyuki, Yaguchi Tomonori, Kawakami Yutaka, Horiuchi Keisuke, Morioka Hideo, Matsumoto Morio, Nakamura Masaya, Muto Akihiro, Toyama Yoshiaki, Saya Hideyuki, Shimizu Takatsune

Cancer Science 106 ( 7 ) 875 - 882 2015.07

Research paper (scientific journal), Joint Work,  ISSN  1347-9032

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<p>Osteosarcoma (OS) is the most frequent primary solid malignant tumor of bone. Its prognosis remains poor in the substantial proportion of patients who do not respond to chemotherapy and novel therapeutic options are therefore needed. We previously established a mouse model that mimics the aggressive behavior of human OS. Enzyme-linked immunosorbent assay-based screening of such mouse tumor lysates identified platelet-derived growth factor-BB (PDGF-BB) as an abundant soluble factor, the gene for which was expressed dominantly in surrounding non-malignant cells of the tumor, whereas that for the cognate receptor (PDGF receptor β) was highly expressed in OS cells. Platelet-derived growth factor-BB induced activation of both MEK-ERK and phosphatidylinositol 3-kinase-protein kinase B signaling pathways and promoted survival in OS cells deprived of serum, and these effects were blocked by the PDGF receptor inhibitor imatinib. However, these actions of PDGF-BB and imatinib were mostly masked in the presence of serum. Whereas imatinib alone did not manifest an antitumor effect in mice harboring OS tumors, combined treatment with imatinib and adriamycin exerted a synergistic antiproliferative effect on OS cells in vivo. These results suggest that treatment of OS with imatinib is effective only when cell survival is dependent on PDGF signaling or when imatinib is combined with another therapeutic intervention that renders the tumor cells susceptible to imatinib action, such as by inducing cellular stress.</p>

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Phase I pilot study of Wilms tumor gene 1 peptide-pulsed dendritic cell vaccination combined with gemcitabine in pancreatic cancer

Mayanagi Shuhei, Kitago Minoru, Sakurai Toshiharu, Matsuda Tatsuo, Fujita Tomonobu, Higuchi Hajime, Taguchi Junichi, Takeuchi Hiroya, Itano Osamu, Aiura Koichi, Hamamoto Yasuo, Takaishi Hiromasa, Okamoto Masato, Sunamura Makoto, Kawakami Yutaka, Kitagawa Yuko

Cancer Science 106 ( 4 ) 397 - 406 2015.04

Research paper (scientific journal), Joint Work,  ISSN  1347-9032

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<p>This study aimed to evaluate the feasibility of and immune response to Wilms tumor gene 1 (WT1) peptide-pulsed dendritic cell vaccination combined with gemcitabine (DCGEM) as a first-line therapy among patients with advanced pancreatic cancer. Ten HLA-A*2402 patients were treated with WT1 peptide-pulsed DC vaccination (1 × 10&lt;sup&gt;7&lt;/sup&gt; cells) on days 8 and 22 and gemcitabine (1000 mg/m&lt;sup&gt;2&lt;/sup&gt;) on days 1, 8 and 15. Induction of a WT1-specific immune response was evaluated using the delayed-type hypersensitivity (DTH) skin test, interferon-γ enzyme-linked immunospot and HLA tetramer assays, along with assays for various immunological factors. DCGEM was well-tolerated, and the relative dose intensity of gemcitabine was 87%. Disease control associated with a low neutrophil/lymphocyte ratio was observed in all three patients with DTH positivity; it was also correlated with a low percentage of granulocytic myeloid derived suppressor cells in the pretreatment peripheral blood (P = 0.017). Patients with liver metastases and high levels of inflammatory markers such as C-reactive protein and interleukin-8 (IL-8) showed poor survival even though a WT1-specific immune response was induced in them. WT1 peptide-pulsed DCGEM is feasible and effective for inducing anti-tumor T-cell responses. Our results support future investigations for pancreatic cancer patients with non-liver metastases and favorable immunological conditions. This trial was registered with the University hospital Medical Information Network (UMIN) Clinical Trials Registry (http://www.umin.ac.jp/ctr/ number: UMIN-000004855).</p>

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Aberrant myosin 1b expression promotes cell migration and lymph node metastasis of HNSCC

Ohmura Gaku, Tsujikawa Takahiro, Yaguchi Tomonori, Kawamura Naoshi, Mikami Shuji, Sugiyama Juri, Nakamura Kenta, Kobayashi Asuka, Iwata Takashi, Nakano Hiroshi, Shimada Taketoshi, Hisa Yasuo, Kawakami Yutaka

Molecular Cancer Research 13 ( 4 ) 721 - 731 2015.04

Research paper (scientific journal), Joint Work,  ISSN  1541-7786

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<p>Lymph node metastasis is the major clinicopathologic feature associated with poor prognosis in patients with head and neck squamous cell carcinoma (HNSCC). Here, web-based bioinformatics meta-analysis was performed to elucidate the molecular mechanism of lymph node metastasis of human HNSCC. Preferential upregulation of Myosin 1b (MYO1B) transcript in HNSCC datasets was identified. Myo1b mRNA was highly expressed in human HNSCC cells and patient tissue specimens compared with their normal counterparts as shown by quantitative PCR (qPCR) analyses. Immunohistochemistry (IHC)-detected Myo1b expression was significantly correlated with lymph node metastases in patients with oral cancer of the tongue. HNSCC with high expression of Myo1b and chemokine receptor 4 (CCR4), another metastasis-associated molecule, was strongly associated with lymph node metastasis. RNA interference (RNAi) of Myo1b in HNSCC cells, SAS and HSC4, significantly inhibited migratory and invasive abilities through decreased large protrusion formation of cell membranes. Finally, Myo1b knockdown in SAS cells significantly inhibited in vivo cervical lymph nodemetastases in a cervical lymph node metastatic mouse model system. Implications: Myo1b is functionally involved in lymph node metastasis of human HNSCC through enhanced cancer cell motility and is an attractive target for new diagnostic and therapeutic strategies for patients with HNSCC. Mol Cancer Res; 13(4); 721-31.</p>

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Predominant development of mature and functional human NK cells in a novel human IL-2 - Producing transgenic NOG mouse

Katano Ikumi, Takahashi Takeshi, Ito Ryoji, Kamisako Tsutomu, Mizusawa Takuma, Ka Yuyo, Ogura Tomoyuki, Suemizu Hiroshi, Kawakami Yutaka, Ito Mamoru

Journal of Immunology 194 ( 7 ) 3513 - 3525 2015.04

Research paper (scientific journal), Joint Work,  ISSN  0022-1767

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<p>We generated a severe immunodeficient NOD/Shi-scid-IL-2Rγ&lt;sup&gt;null&lt;/sup&gt; (NOG) mouse substrain expressing the transgenic human IL-2 gene (NOG-IL-2 Tg). Upon transfer of human cord blood-derived hematopoietic stem cells (HSCs), CD3&lt;sup&gt;-&lt;/sup&gt;CD56&lt;sup&gt;high&lt;/sup&gt;CD16&lt;sup&gt;+/-&lt;/sup&gt; cells developed unexpectedly, predominantly in the NOG-IL-2 Tg (hu-HSC NOG-IL-2 Tg). These cells expressed various NK receptors, including NKp30, NKp44, NKp46, NKG2D, and CD94, as well as a diverse set of killer cell Ig-like receptor molecules at levels comparable to normal human NK cells from the peripheral blood, which is evidence of their maturity. They produced levels of granzyme A as high as in human peripheral blood-derived NK cells, and a considerable amount of perforin protein was detected in the plasma. Human NK cells in hu-HSC NOG-IL-2 Tg produced IFN-γ upon stimulation, and IL-2, IL-15, or IL-12 treatment augmented the in vitro cytotoxicity. Inoculation of K562 leukemia cells into hu-HSC NOG-IL-2 Tg caused complete rejection of the tumor cells, whereas inoculation into hu-HSC NOG fully reconstituted with human B, T, and some NK cells did not. Moreover, when a CCR4&lt;sup&gt;+&lt;/sup&gt; Hodgkin's lymphoma cell line was inoculated s.c. into hu-HSC NOG-IL-2 Tg, the tumor growth was significantly suppressed by treatment with a therapeutic humanized anti-CCR4 Ab (mogamulizumab), suggesting that the human NK cells in the mice exerted active Ab-dependent cellular cytotoxicity in vivo. Taken together, these data suggest that the new NOG-IL-2 Tg strain is a unique model that can be used to investigate the biological and pathological functions of human NK cells in vivo.</p>

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Cytokine profile in cervical mucosa of Japanese patients with cervical intraepithelial neoplasia

Iwata Takashi, Fujii Takuma, Morii Kenji, Saito Miyuki, Sugiyama Juri, Nishio Hiroshi, Morisada Tohru, Tanaka Kyoko, Yaguchi Tomonori, Kawakami Yutaka, Aoki Daisuke

International Journal of Clinical Oncology 20 ( 1 ) 126 - 133 2015.02

Research paper (scientific journal), Joint Work,  ISSN  1341-9625

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<p>BACKGROUND: Immune responses in the uterine cervix are considered to play an important role in persistent human papillomavirus (HPV) infection and carcinogenesis, but many aspects of the mechanism are still unclear. The goal of this study was to measure cytokines to analyze immune responses in patients with cervical intraepithelial neoplasia (CIN).</p><p>MATERIALS AND METHODS: The levels of 17 cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, G-CSF, GM-CSF, INF-γ, MCP-1, MIP-1β, and TNFα) in cervical mucus were simultaneously measured using a multiplex immunoassay in 52 high-grade squamous intraepithelial lesion (HSIL) cases and overproduction of IL-1β, IL-8, and MIP-1β was identified. The levels of these 3 cytokines were measured in 130 patients with or without CIN lesions using enzyme-linked immunosorbent assay. The associations of the cytokine levels with the cytology, infecting HPV type, and status of cigarette smoking were investigated.</p><p>RESULTS: IL-1β and IL-8 levels were associated with the cytology, and these levels were higher in HSIL cases than in NILM (negative for intraepithelial lesion and malignancy) and LSIL (low-grade squamous intraepithelial lesion) cases (P = 0.005, P = 0.001, respectively). The MIP-1β level was significantly lower in smokers (P = 0.018) and high-risk (HR)-HPV-infected patients (P = 0.021).</p><p>CONCLUSIONS: Enhanced expression of IL-1β and IL-8 indicates that Th2 inflammatory responses become stronger in the local uterine cervical region with the progression of CIN lesions, and a decrease in the MIP-1β level may be advantageous for immunoescape of HPV. Cigarette smoking may further facilitate persistent HPV infection.</p>

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Prognostic significance of interleukin-8 and CD163-positive cell-infiltration in tumor tissues in patients with oral squamous cell carcinoma

Fujita Yohei, Okamoto Masato, Goda Hiroyuki, Tano Tomoyuki, Nakashiro Koh Ichi, Sugita Atsuro, Fujita Tomonobu, Koido Shigeo, Homma Sadamu, Kawakami Yutaka, Hamakawa Hiroyuki

PLoS One 9 ( 12 ) e110378 2014.12

Research paper (scientific journal), Joint Work

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<p>Purpose: We investigated whether serum interleukin (IL)-8 reflects the tumor microenvironment and has prognostic value in patients with oral squamous cell carcinoma (OSCC). Experimental Design: Fifty OSCC patients who received radical resection of their tumor(s) were enrolled. Preoperative sera were measured for IL-8 by ELISA. Expression of IL-8 and the infiltration of immune cells in tumor tissues were analyzed by an immunohistochemical staining of surgical specimens. Results: We found that disease-free survival (DFS) was significantly longer in the Stage I/II OSCC patients with low serum IL-8 levels compared to those with high levels (p=0.001). The tumor expression of IL-8, i.e., IL-8(T) and the density of CD163-positive cells in the tumor invasive front, i.e., CD163(IF) were correlated with the serum IL-8 level (p =0.033 and p=0.038, respectively), and they were associated with poor clinical outcome (p= 0.007 and p=0.002, respectively, in DFS) in all patients. A multivariate analysis revealed that N status, IL-8(T) and CD163(IF) significantly affected the DFS of the patients. Further analysis suggested that combination of N status with serum IL-8, IL-8(T) or CD163(IF) may be a new criterion for discriminating between OSCC patients at high and low risk for tumor relapse. Interestingly, the in vitro experiments demonstrated that IL-8 enhanced generation of CD163-positive M2 macrophages from peripheral blood monocytes, and that the cells produced IL-10. Conclusions: These findings indicate that IL-8 may be involved in poor clinical outcomes via generation of CD163-positive M2 macrophages, and that these factors in addition to N status may have prognostic value in patients with resectable OSCSS.</p>

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Functional analysis of a novel glioma antigen, EFTUD1

Saito Katsuya, Iizuka Yukihiko, Ohta Shigeki, Takahashi Satoshi, Nakamura Kenta, Saya Hideyuki, Yoshida Kazunari, Kawakami Yutaka, Toda Masahiro

Neuro-Oncology 16 ( 12 ) 1618 - 1629 2014.12

Research paper (scientific journal), Joint Work,  ISSN  1522-8517

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<p>BACKGROUND: A cDNA library made from 2 glioma cell lines, U87MG and T98G, was screened by serological identification of antigens by recombinant cDNA expression (SEREX) using serum from a glioblastoma patient. Elongation factor Tu GTP binding domain containing protein 1 (EFTUD1), which is required for ribosome biogenesis, was identified. A cancer microarray database showed overexpression of EFTUD1 in gliomas, suggesting that EFTUD1 is a candidate molecular target for gliomas.</p><p>METHODS: EFTUD1 expression in glioma cell lines and glioma tissue was assessed by Western blot, quantitative PCR, and immunohistochemistry. The effect on ribosome biogenesis, cell growth, cell cycle, and induction of apoptosis and autophagy in glioma cells during the downregulation of EFTUD1 was investigated. To reveal the role of autophagy, the autophagy-blocker, chloroquine (CQ), was used in glioma cells downregulating EFTUD1. The effect of combining CQ with EFTUD1 inhibition in glioma cells was analyzed.</p><p>RESULTS: EFTUD1 expression in glioma cell lines and tissue was higher than in normal brain tissue. Downregulating EFTUD1 induced G1 cell-cycle arrest and apoptosis, leading to reduced glioma cell proliferation. The mechanism underlying this antitumor effect was impaired ribosome biogenesis via EFTUD1 inhibition. Additionally, protective autophagy was induced by glioma cells as an adaptive response to EFTUD1 inhibition. The antitumor effect induced by the combined treatment was significantly higher than that of either EFTUD1 inhibition or CQ alone.</p><p>CONCLUSION: These results suggest that EFTUD1 represents a novel therapeutic target and that the combination of EFTUD1 inhibition with autophagy blockade may be effective in the treatment of gliomas.</p>

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Predictive biomarkers for the outcome of vaccination of five therapeutic epitope peptides for colorectal cancer

Hazama Shoichi, Takenouchi Hiroko, Tsunedomi Ryouichi, Iida Michihisa, Suzuki Nobuaki, Iizuka Norio, Inoue Yuka, Sakamoto Kazuhiko, Nakao Mitsuhiro, Shindo Yoshitaro, Kanekiyo Shinsuke, Tokumitsu Yukio, Yoshimura Kiyoshi, Maeda Noriko, Maeda Kazunari, Maeda Yoshinari, Matsui Hiroto, Yoshino Shigefumi, Nakamura Yusuke, Fujita Yusuke, Hamamoto Yoshihiko, Okamoto Masato, Fujita Tomonobu, Kawakami Yutaka, Oka Masaaki

Anticancer Research 34 ( 8 ) 4201 - 4206 2014.08

Research paper (scientific journal), Joint Work,  ISSN  0250-7005

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<p>Background: We previously reported a phase I study of a cancer vaccine using five novel HLA-A∗2402-restricted peptides, and demonstrated the safety and the promising potential of our five-peptide cocktail for advanced colorectal cancer. The objective of this analysis was to investigate predictive biomarkers for the prior selection of patients who are likely to have clinical benefit from such therapy. Patients and Methods: Seventeen patients with colorectal cancer who were treated with the five peptides underwent a complete blood count, serum chemistry tests and enzyme-linked ImmunoSpot assay before the treatment as predictive markers of high reactivity to the peptides. Results: Interleukin-6 level was a significant predictor for overall survival of patients treated with the peptide cocktail (p=0.017). A high neutrophil/lymphocyte ratio was likely to have some association with the poor induction of peptide-specific immune reaction. Conclusion: Interleukin-6 level might be a good predictive biomarker for clinical benefit of patients treated with this peptide vaccine.</p>

Absence of recognition of common melanocytic antigens by T cells isolated from the cerebrospinal fluid of a Vogt-Koyanagi-Harada patient

Abad Sébastien, Wieërs Grégoire, Colau Didier, Wildmann Claude, Delair Emmanuelle, Dhote Robin, Brézin Antoine P., Kawakami Yukata, Coulie Pierre G., van der Bruggen Pierre

Molecular Vision 20   956 - 969 2014.07

ISSN  1090-0535

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<p>Purpose: Vogt-Koyanagi-Harada (VKH) syndrome is an autoimmune disease characterized by inaugural uveomeningitidis and hearing loss and at late stages a depigmentation in eyes and skin. Melanocytes are the cells common to the four affected tissues, namely eye, brain, inner ear, and skin. Melanocytes are therefore considered as the source of self-antigens. The melanocytic proteins tyrosinase-related protein-1 (TRP1), TRP2, tyrosinase, and gp100 have been proposed as the proteins targeted by autoreactive T cells from VKH patients bearing human leukocyte antigen (HLA)-DRB1*04:05, the HLA allele classically associated with VKH disease. The objective of this work was to determine the antigens recognized by a large number of potentially autoreactive CD4 T lymphocytes obtained from the cerebrospinal fluid of one VKH patient who did not express HLA-DRB1*04:05. Methods: T cells were isolated from the cerebrospinal fluid of a newly diagnosed HLA-DRB1*14:01,*15:03;-DPB1*01:01,*04:02 patient in the acute phase of the VKH disease and cloned by limiting dilution. Each of the 107 T cell clones, of which 90% were CD4+, was tested for its ability to secrete cytokines upon contact with autologous antigen-presenting cells loaded with either of the melanocytic proteins TRP1, TRP2, tyrosinase, gp100, Melan-A and KU-MEL-1. The sensitivity of our recombinant bacteria-based approach was validated with a CD4 T cell clone with known antigen specificity. The ability of each of the 107 clones to secrete cytokines upon nonspecific stimulation was verified. Results: None of the 107 T cell clones was able to secrete tumor necrosis factor-α, interferon-γ, interleukin (IL)-5, or IL-17 upon contact with autologous B cells loaded with any of the six common melanocytic proteins. Nine clones secreted high-level IL-17 upon stimulation with beads coated with antibodies. Conclusions: The self-antigens that triggered the VKH disease in this patient probably derive from proteins other than the six melanocytic proteins mentioned above. Further study of antigens that are recognized by potential autoreactive T cells from VKH patients is likely to benefit from testing a broader set of melanocytic proteins.</p>

NOD-Rag2^null IL-2Rγ^null mice an alternative to NOG mice for generation of humanized mice.

Katano Ikumi, Ito Ryoji, Kamisako Tsutomu, Eto Tomoo, Ogura Tomoyuki, Kawai Kenji, Suemizu Hiroshi, Takahashi Takeshi, Kawakami Yutaka, Ito Mamoru

Experimental Animals 63 ( 3 ) 321 - 330 2014.07

Research paper (scientific journal), Joint Work,  ISSN  1341-1357

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<p>We have developed NOD-Rag2^null IL-2Rγ^null (NR2G) mice similar to NOD-scid IL-2Rγ^null (NOG) mice that are known as an excellent host to generate humanized mice. To evaluate the usefulness of NR2G mice as a host for humanized mice, the engraftment rates and differentiation of human cells after human hematopoietic stem cell (HSC) transplantation were compared among NR2G, NOG, and NOD-scid mice. For this purpose, the appropriate irradiation doses to expand the niche for human stem cells in the bone marrow were first determined. As a result, 8 and 2.5 Gy in adult, and 4 and 1 Gy in newborn NR2G and NOG mice, respectively, were found to be appropriate. Next, 5 × 10⁴ human umbilical cord blood CD34⁺ cells were intravenously inoculated into irradiated adult or newborn of the immunodeficient mice. These HSC transplantation experiments demonstrated that both NR2G and NOG mice showed high engraftment rates compared with NOD-scid mice, although NOG mice showed a slightly higher engraftment rate than that for NR2G mice. However, no difference was found in the human cell populations differentiated from HSCs between NR2G and NOG mice. The HSC transplantation experiments to adults and newborns of two immunodeficient mice also revealed that the HSC transplantation into newborn mice resulted in higher engraftment rate than those into adults. These results showed that NR2G mice could be used as an alternative host to NOG mice to generate humanized mice.</p>

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Immunosuppression through constitutively activated NF-κB signalling in human ovarian cancer and its reversal by an NF-κB inhibitor

Nishio H., Yaguchi T., Sugiyama J., Sumimoto H., Umezawa K., Iwata T., Susumu N., Fujii T., Kawamura N., Kobayashi A., Park J., Aoki D., Kawakami Y.

British Journal of Cancer 110 ( 12 ) 2965 - 2974 2014.06

ISSN  0007-0920

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<p>Background:Although T-cell immunity is thought to be involved in the prognosis of epithelial ovarian cancer (EOC) patients, immunosuppressive conditions hamper antitumour immune responses. Thus, their mechanisms and overcoming strategies need to be investigated.Methods:The role of NF-κB in human EOC cells and macrophages was evaluated by in vitro production of immunosuppressive IL-6 and IL-8 by EOC cells and in vivo analysis of immune responses in nude mice implanted with human EOC cells using an NF-κB inhibitor DHMEQ.Results:In EOC patients, increased plasma IL-6, IL-8, and arginase were observed. The NF-κB inhibitor DHMEQ inhibited the production of IL-6 and IL-8 by EOC cell lines. Immunosuppression of human DCs and macrophages by culture supernatant of EOC cells was reversed with the pretreatment of DHMEQ. Administration of DHMEQ to nude mice implanted with human EOC resulted in the restoration of T-cell stimulatory activity of murine DCs along with the reduction of tumour accumulation and arginase expression of MDSCs. Nuclear factor-κB inhibition in tumour-bearing mice also enhanced antitumour effects of transferred murine naive T cells.Conclusions:NF-κB is involved in the immunosuppression induced by human EOC, and its inhibitor may restore antitumour immune responses, indicating that NF-κB is an attractive target for EOC treatment.</p>

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A phase ΙI study of five peptides combination with oxaliplatin-based chemotherapy as a first-line therapy for advanced colorectal cancer (FXV study)

Hazama Shoichi, Nakamura Yusuke, Tanaka Hiroaki, Hirakawa Kosei, Tahara Ko, Shimizu Ryoichi, Ozasa Hiroaki, Etoh Ryuichi, Sugiura Fumiaki, Okuno Kiyotaka, Furuya Takumi, Nishimura Taku, Sakata Koichiro, Yoshimatsu Kazuhiko, Takenouchi Hiroko, Tsunedomi Ryouichi, Inoue Yuka, Kanekiyo Shinsuke, Shindo Yoshitaro, Suzuki Nobuaki, Yoshino Shigefumi, Shinozaki Hirokazu, Kamiya Akira, Furukawa Hiroyuki, Yamanaka Takeharu, Fujita Tomonobu, Kawakami Yutaka, Oka Masaaki

Journal of Translational Medicine 12 ( 1 )  2014.04

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<p>Background: We previously conducted a phase I trial for advanced colorectal cancer (CRC) using five HLA-A*2402-restricted peptides, three derived from oncoantigens and two from vascular endothelial growth factor (VEGF) receptors, and confirmed safety and immunological responses. To evaluate clinical benefits of cancer vaccination treatment, we conducted a phase II trial using the same peptides in combination with oxaliplatin-based chemotherapy as a first-line therapy.Methods: The primary objective of the study was the response rates (RR). Progression free survival (PFS), overall survival (OS), and immunological parameters were evaluated as secondary objective. The planned sample size was more than 40 patients for both HLA2402-matched and -unmatched groups. All patients received a cocktail of five peptides (3 mg each) mixed with 1.5 ml of IFA which was subcutaneously administered weekly for the first 12 weeks followed by biweekly administration. Presence or absence of the HLA-A*2402 genotype were used for classification of patients into two groups.Results: Between February 2009 and November 2012, ninety-six chemotherapy naïve CRC patients were enrolled under the masking of their HLA-A status. Ninety-three patients received mFOLFOX6 and three received XELOX. Bevacizumab was added in five patients. RR was 62.0% and 60.9% in the HLA-A*2402-matched and -unmatched groups, respectively (p = 0.910). The median OS was 20.7 months in the HLA-A*2402-matched group and 24.0 months in the unmatched group (log-rank, p = 0.489). In subgroup with a neutrophil/lymphocyte ratio (NLR) of &lt; 3.0, patients in the HLA-matched group did not survive significantly longer than those in the unmatched group (log-rank, p = 0.289) but showed a delayed response.Conclusions: Although no significance was observed for planned statistical efficacy endpoints, a delayed response was observed in subgroup with a NLR of &lt; 3.0. Biomarkers such as NLR might be useful for selecting patients with a better treatment outcome by the vaccination.Trial registration: Trial registration: UMIN000001791.</p>

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Induction of immunoregulatory CD271⁺ cells by metastatic tumor cells that express human endogenous retrovirus H

Kudo-Saito Chie, Yura Masahiro, Yamamoto Ryusuke, Kawakami Yutaka

Cancer Research 74 ( 5 ) 1361 - 1370 2014.03

ISSN  0008-5472

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<p>Human endogenous retroviruses (HERV) are associated with many diseases such as autoimmune diseases and cancer. Although the frequent expression of a variety of HERVs in tumor cells has been demonstrated, their functional contributions in cancer are as yet unclear. Intriguingly, HERVs and other retroviruses include an immunosuppressive domain in their transmembrane envelope proteins, but its mechanism of action and cancer relevance are obscure. In this study, we demonstrate that the human endogenous retrovirus HERV-H has a critical role in tumor metastasis and immune escape. We found that expression of herv-h mRNA was elevated in metastatic tumor cells undergoing epithelial-to-mesenchymal transition (EMT) and in primary tumor tissues from advanced colon cancer. The immunosuppressive peptide H17 derived from HERV-H was sufficient to induce EMT in tumor cells that expressed low levels of HERV-H, and it amplified this event within the tumor microenvironment. H17 also stimulated CCL19 expression in tumor cells, which in turn recruited and expanded a population of pluripotent immunoregulatory CD271⁺ cells, which included mesenchymal stem cells and myeloid-derived suppressor cells. In tumor tissues from patients with advanced colon cancer, we confirmed that CD271 ⁺ cells were increased in HERV-H⁺CCL19⁺ tumor tissues. Notably, RNAi-mediated change of HERVH or CCL19, or depletion of CD271⁺ cells, improved immune responses in vitro and in vivo accompanied by tumor regression. Together, our results argued that HERV-H is a critical determinant of immune escape in cancer, suggesting its candidacy as a promising therapeutic target to treat patients with advanced cancer.</p>

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TGF-β1 in tumor microenvironments induces immunosuppression in the tumors and sentinel lymph nodes and promotes tumor progression

Nakamura Shoko, Yaguchi Tomonori, Kawamura Naoshi, Kobayashi Asuka, Sakurai Toshiharu, Higuchi Hajime, Takaishi Hiromasa, Hibi Toshifumi, Kawakami Yutaka

Journal of Immunotherapy 37 ( 2 ) 63 - 72 2014.02

ISSN  1524-9557

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<p>In cancer patients, sentinel lymph nodes (SLNs) are crucial in the induction of antitumor T cells. However, in many cases, SLNs and tumors appear to be in immunosuppressive condition through mechanisms yet to be elucidated. In this study, the role of tumor-derived TGF-β1 in the generation of immunosuppressive microenvironments of tumors and SLNs was investigated. Murine colorectal carcinoma CT26 transduced with TGF-β1 cDNA (CT26-TGF-β1) showed enhanced tumor growth compared with mock-transduced CT26 (CT26-Mock) when implanted in syngeneic Balb/c mice, even though CT26-TGF-β1 shows slower growth in vitro. This enhanced growth was not observed when implanted in immunodeficient mice, suggesting that TGF-β1 enhanced tumor growth by suppressing antitumor T-cell responses. Analysis of immune cells in CT26-TGF-β1-implanted mice revealed impairment of dendritic cells (DCs), decrease of CD8 T cells, and increase of MDSCs and Tregs in the tumors. Similarly, the SLNs of these mice showed an increase of MDSCs, Tregs, and PD-L1 DCs, and decrease of T-cell stimulatory activity of DCs accompanied by decreased CD80 expression and TNF-α production. In addition, induction of tumor antigen-specific T cells from SLNs of the CT26-TGF-β1-implanted mice was significantly reduced. These results demonstrate that overproduction of TGF-β1 is critical for the generation of immunosuppressive microenvironments in both tumors and SLNs, which may result in suppression of spontaneous antitumor CD8 T-cell responses. Therefore, TGF-β1 is an attractive target for restoration of immunosuppressive condition in cancer patients.</p>

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Roles of signaling pathways in cancer cells and immune cells in generation of immunosuppressive tumor-associated microenvironments

Kawakami Yutaka, Yaguchi Tomonori, Sumimoto Hidetoshi, Kudo-Saito Chie, Tsukamoto Nobuo, Iwata-Kajihara Tomoko, Nakamura Shoko, Nishio Hiroshi, Satomi Ryosuke, Kobayashi Asuka, Tanaka Mayuri, Park Jeong Hoon, Kamijuku Hajime, Tsujikawa Takahiro, Kawamura Naoshi

  307 - 323 2014.01

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<p>Cancer cells trigger multiple immunosuppressive cascades and generate immunosuppressive tumor-associated microenvironments including tumor and sentinel lymph nodes. Constitutive activation of various signaling pathways (e.g., MAPK, STAT3, NF-κB, β-catenin) in human cancer cells was found to trigger the multiple immunosuppressive cascades through the production of immunosuppressive cytokines, such as TGF-β, IL-10, IL-6, and VEGF, and induction of immunosuppressive immune cells, such as regulatory T cells, tolerogenic dendritic cells, and myeloid derived suppressor cells. Some of these cancer-derived cytokines impair various immune cells through activation of their signaling molecules such as STAT3 and NF-κB. Inhibitors for these activated signals could inhibit the multiple immunosuppressive cascades by acting on both cancer cells and immune cells. Since common signaling mechanisms are often utilized for some of the hallmarks of cancer (e.g., cell proliferation/survival, invasion/metastasis, and immunosuppression), targeting these common signaling pathways may be an attractive strategy for cancer therapy, including immunotherapy.</p>

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Towards the introduction of the 'Immunoscore' in the classification of malignant tumours

Galon Jérôme, Mlecnik Bernhard, Bindea Gabriela, Angell Helen K., Berger Anne, Lagorce Christine, Lugli Alessandro, Zlobec Inti, Hartmann Arndt, Bifulco Carlo, Nagtegaal Iris D., Palmqvist Richard, Masucci Giuseppe V., Botti Gerardo, Tatangelo Fabiana, Delrio Paolo, Maio Michele, Laghi Luigi, Grizzi Fabio, Asslaber Martin, D'Arrigo Corrado, Vidal-Vanaclocha Fernando, Zavadova Eva, Chouchane Lotfi, Ohashi Pamela S., Hafezi-Bakhtiari Sara, Wouters Bradly G., Roehrl Michael, Nguyen Linh, Kawakami Yutaka, Hazama Shoichi, Okuno Kiyotaka, Ogino Shuji, Gibbs Peter, Waring Paul, Sato Noriyuki, Torigoe Toshihiko, Itoh Kyogo, Patel Prabhu S., Shukla Shilin N., Wang Yili, Kopetz Scott, Sinicrope Frank A., Scripcariu Viorel, Ascierto Paolo A., Marincola Francesco M., Fox Bernard A., Pagès Franck

Journal of Pathology 232 ( 2 ) 199 - 209 2014.01

ISSN  0022-3417

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<p>The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression-based stratification. These parameters rely on tumour-cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named 'Immunoscore' has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).</p>

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Targeting FSTL1 prevents tumor bone metastasis and consequent immune dysfunction

Kudo-Saito Chie, Fuwa Takafumi, Murakami Kouichi, Kawakami Yutaka

Cancer Research 73 ( 20 ) 6185 - 6193 2013.10

ISSN  0008-5472

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<p>Bone metastasis greatly deteriorates the quality of life in patients with cancer. Although mechanisms have been widely investigated, the relationship between cancer bone metastasis and antitumor immunity in the host has been much less studied. Here, we report a novel mechanism of bone metastasis mediated by FSTL1, a follistatinlike glycoprotein secreted by Snail⁺ tumor cells, which metastasize frequently to bone. We found that FSTL1 plays a dual role in bone metastasis-in one way by mediating tumor cell invasion and bone tropism but also in a second way by expanding a population of pluripotent mesenchymal stem-like CD45 ALCAM cells derived from bone marrow. CD45^- ALCAM ⁺ cells induced bone metastasis de novo, but they also generated CD8^low T cells with weak CTL activity in the periphery, which also promoted bone metastasis in an indirect manner. RNA interferencemediated attenuation of FSTL1 in tumor cells prevented bone metastasis along with the parallel increase in ALCAM⁺ cells and CD8^low T cells. These effects were accompanied by heightened antitumor immune responses in vitro and in vivo. In clinical specimens of advanced breast cancer, ALCAM⁺ cells increased with FSTL1 positivity in tumor tissues, but not in adjacent normal tissues, consistent with a causal connection between these molecules. Our findings define FSTL1 as an attractive candidate therapeutic target to prevent or treat bone metastasis, which remains a major challenge in patients with cancer.</p>

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Sox6 Up-Regulation by Macrophage Migration Inhibitory Factor Promotes Survival and Maintenance of Mouse Neural Stem/Progenitor Cells

Ohta Shigeki, Misawa Aya, Lefebvre Véronique, Okano Hideyuki, Kawakami Yutaka, Toda Masahiro

PLoS One 8 ( 9 )  2013.09

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<p>Macrophage migration inhibitory factor (MIF) has important roles in supporting the proliferation and/or survival of murine neural stem/progenitor cells (NSPCs), but downstream effectors remain unknown. We show here that MIF robustly increases the expression of Sox6 in NSPCs in vitro. During neural development, Sox6 is expressed in the ventricular zone of the ganglionic eminence (GE) of mouse brains at embryonic day 14.5 (E14.5), cultured NSPCs from E14.5 GE, and NSPCs in the subventricular zone (SVZ) around the lateral ventricle (LV) of the adult mouse forebrain. Retroviral overexpression of Sox6 in NSPCs increases the number of primary and secondary neurospheres and inhibits cell differentiation. This effect is accompanied with increased expression of Hes1 and Bcl-2 and Akt phosphorylation, thus suggesting a role for Sox6 in promoting cell survival and/or self-renewal ability. Constitutive activation of the transcription factor Stat3 results in up-regulation of Sox6 expression and chromatin immunoprecipitation analysis showed that MIF increases Stat3 binding to the Sox6 promoter in NSPCs, indicating that Stat3 stimulates Sox6 expression downstream of MIF. Finally, the ability of MIF to increase the number of primary and secondary neurospheres is inhibited by Sox6 gene silencing. Collectively, our data identify Sox6 as an important downstream effector of MIF signaling in stemness maintenance of NSPCs.</p>

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Immunochemoradiotherapy for patients with oral squamous cell carcinoma

Tano Tomoyuki, Okamoto Masato, Kan Shin, Bando Takashi, Goda Hiroyuki, Nakashiro Koh Ichi, Shimodaira Shigetaka, Koido Shigeo, Homma Sadamu, Fujita Tomonobu, Sato Mitsunobu, Yamashita Naomi, Hamakawa Hiroyuki, Kawakami Yutaka

Neoplasia (United States) 15 ( 7 ) 805 - 814 2013.07

ISSN  1522-8002

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<p>Eighty-one patients with oral squamous cell carcinoma (OSCC) received oral fluoropyrimidine UFT and radiotherapy (RT) with or without an immunotherapeutic agent OK-432. Both overall survival and progression-free survival of patients who received RT + UFT + OK-432 were significantly longer than those of patients who received RT + UFT (P =.0075 and P =.0175, respectively). Clinical response was also more favorable in RT + UFT + OK-432 group than in RT + UFT group (P =.0066). Next, in vitro experiments were conducted to examine the effect of 5-fluorouracil (5-FU) and X-ray irradiation in OK-432-induced immunity. Human peripheral blood mononuclear cells stimulated with OK-432 produced helper T cell 1 (Th1)-type cytokines as well as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), which are produced by Th2 and regulatory T cells (Tregs), respectively, and are inhibitory in antitumor immunity. OK-432-induced IL-10 and TGF-β but not Th1 cytokines were significantly inhibited by 5-FU and/or X-ray. 5-FU and X-ray also inhibited the expression of mRNAs for GATA-3 and Foxp3, which are transcription factors for Th2 and Tregs, respectively, but not for T-bet, a transcription factor for Th1. In addition, 5-FU and X-ray decreased the expression of mRNAs for suppressor of cytokine signaling 1 (SOCS1) and SOCS3. Antisense oligonucleotides for SOCS1 and SOCS3 markedly reduced OK-432-induced IL-10 and TGF-β. This is the first report clearly demonstrating that OK-432-based immunotherapy significantly enhanced the therapeutic effects of chemoradiotherapy in patients with OSCC as well as elucidating the mechanism of the synergistic effect of immunochemoradiotherapy in which 5-FU and radiation enhanced OK-432-induced Th1 response mediated by the inhibition of SOCS1 and SOCS3 gene expression.</p>

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Autocrine and paracrine loops between cancer cells and macrophages promote lymph node metastasis via CCR4/CCL22 in head and neck squamous cell carcinoma

Tsujikawa Takahiro, Yaguchi Tomonori, Ohmura Gaku, Ohta Shigeki, Kobayashi Asuka, Kawamura Naoshi, Fujita Tomonobu, Nakano Hiroshi, Shimada Taketoshi, Takahashi Takeshi, Nakao Ryuta, Yanagisawa Akio, Hisa Yasuo, Kawakami Yutaka

International Journal of Cancer 132 ( 12 ) 2755 - 2766 2013.06

ISSN  0020-7136

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<p>Lymph node metastasis is a poor prognostic factor for patients with head and neck squamous cell carcinoma (HNSCC). However, its molecular mechanism has not yet been fully understood. In our study, we investigated the expression of CCR4 and its ligand CCL22 in the HNSCC tumor microenvironment and found that the CCR4/CCL22 axis was involved in lymph node metastasis of HNSCC. CCR4 was expressed in 20 of 31 (64.5%) human tongue cancer tissues, and its expression was significantly correlated with lymph node metastasis (p &lt; 0.01) and lymphatic invasion (p &lt; 0.05). CCR4 was expressed in three of five human HNSCC cell lines tested. CCR4⁺ HNSCC cells, but not CCR4^- cells, showed enhanced migration toward CCL22, indicating that functional CCR4 was expressed in HNSCC cell lines. CCL22 was also expressed in cancer cells (48.4% of tongue cancer tissues) or CD206⁺ M2-like macrophages infiltrated in tumors and draining lymph nodes. CCL22 produced by cancer cells or CD206^high M2-like macrophages increased the cell motility of CCR4⁺ HNSCC cells in vitro in an autocrine or paracrine manner. In the mouse SCCVII in vivo model, CCR4⁺ cancer cells, but not CCR4 ^- cells, metastasized to lymph nodes which contained CCL22 producing M2-like macrophages. These results demonstrate that lymph node metastasis of CCR4⁺ HNSCC is promoted by CCL22 in an autocrine or M2-like macrophage-dependent paracrine manner. Therefore, the CCR4/CCL22 axis may be an attractive target for the development of diagnostic and therapeutic strategies for patients with HNSCC.</p>

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Angiotensin II Type 1 Receptor Antagonist Attenuates Lacrimal Gland, Lung, and Liver Fibrosis in a Murine Model of Chronic Graft-Versus-Host Disease

Yaguchi Saori, Ogawa Yoko, Shimmura Shigeto, Kawakita Tetsuya, Hatou Shin, Satofuka Shingo, Nakamura Shigeru, Imada Toshihiro, Miyashita Hideyuki, Yoshida Satoru, Yaguchi Tomonori, Ozawa Yoko, Mori Takehiko, Okamoto Shinichiro, Kawakami Yutaka, Ishida Susumu, Tsubota Kazuo

PLoS One 8 ( 6 )  2013.06

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<p>Chronic graft-versus-host disease (cGVHD), a serious complication following allogeneic HSCT (hematopoietic stem cell transplantation), is characterized by systemic fibrosis. The tissue renin-angiotensin system (RAS) is involved in the fibrotic pathogenesis, and an angiotensin II type 1 receptor (AT1R) antagonist can attenuate fibrosis. Tissue RAS is present in the lacrimal gland, lung, and liver, and is known to be involved in the fibrotic pathogenesis of the lung and liver. This study aimed to determine whether RAS is involved in fibrotic pathogenesis in the lacrimal gland and to assess the effect of an AT1R antagonist on preventing lacrimal gland, lung, and liver fibrosis in cGVHD model mice. We used the B10.D2→BALB/c (H-2^d) MHC-compatible, multiple minor histocompatibility antigen-mismatched model, which reflects clinical and pathological symptoms of human cGVHD. First, we examined the localization and expression of RAS components in the lacrimal glands using immunohistochemistry and quantitative real-time polymerase chain reaction (PCR). Next, we administered an AT1R antagonist (valsartan; 10 mg/kg) or angiotensin II type 2 receptor (AT2R) antagonist (PD123319; 10 mg/kg) intraperitoneally into cGVHD model mice and assessed the fibrotic change in the lacrimal gland, lung, and liver. We demonstrated that fibroblasts expressed angiotensin II, AT1R, and AT2R, and that the mRNA expression of angiotensinogen was greater in the lacrimal glands of cGVHD model mice than in controls generated by syngeneic-HSCT. The inhibition experiment revealed that fibrosis of the lacrimal gland, lung, and liver was suppressed in mice treated with the AT1R antagonist, but not the AT2R antagonist. We conclude that RAS is involved in fibrotic pathogenesis in the lacrimal gland and that AT1R antagonist has a therapeutic effect on lacrimal gland, lung, and liver fibrosis in cGVHD model mice. Our findings point to AT1R antagonist as a possible target for therapeutic intervention in cGVHD.</p>

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Cancer-induced immunosuppressive cascades and their reversal by molecular-targeted therapy

Kawakami Yutaka, Yaguchi Tomonori, Sumimoto Hidetoshi, Kudo-Saito Chie, Tsukamoto Nobuo, Iwata-Kajihara Tomoko, Nakamura Shoko, Nishio Hiroshi, Satomi Ryosuke, Kobayashi Asuka, Tanaka Mayuri, Park Jeong Hoon, Kamijuku Hajime, Tsujikawa Takahiro, Kawamura Naoshi

Annals of the New York Academy of Sciences 1284 ( 1 ) 80 - 86 2013.05

ISSN  0077-8923

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<p>Immunological status in tumor tissues varies among patients. Infiltration of memory-type CD8⁺ T cells into tumors correlates with prognosis of patients with various cancers. However, the mechanism of the differential CD8⁺ T cell infiltration has not been well investigated. In general, tumor-associated microenvironments, including tumor and sentinel lymph nodes, are under immunosuppressive conditions such that the immune system is not able to eliminate cancer cells without immune-activating interventions. Constitutive activation of various signaling pathways in human cancer cells triggers multiple immunosuppressive cascades that involve various cytokines, chemokines, and immunosuppressive cells. Signaling pathway inhibitors could inhibit these immunosuppressive cascades by acting on either cancer or immune cells, or both. In addition, common signaling mechanisms are often utilized for multiple hallmarks of cancer (e.g., cell proliferation/survival, invasion/metastasis, and immunosuppression). Therefore, targeting these common signaling pathways may be an attractive strategy for cancer therapy including immunotherapy.</p>

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CCL2 is critical for immunosuppression to promote cancer metastasis

Kudo-Saito Chie, Shirako Hiromi, Ohike Misa, Tsukamoto Nobuo, Kawakami Yutaka

Clinical and Experimental Metastasis 30 ( 4 ) 393 - 405 2013.04

ISSN  0262-0898

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<p>We previously found that cancer metastasis is accelerated by immunosuppression during Snail-induced epithelial-to-mesenchymal transition (EMT). However, the molecular mechanism still remained unclear. Here, we demonstrate that CCL2 is a critical determinant for both tumor metastasis and immunosuppression induced by Snail⁺ tumor cells. CCL2 is significantly upregulated in various human tumor cells accompanied by Snail expression induced by snail transduction or TGFβ treatment. The Snail ⁺ tumor-derived CCL2 amplifies EMT events in other cells including Snail^- tumor cells and epithelial cells within tumor microenvironment. CCL2 secondarily induces Lipocalin 2 (LCN2) in the Snail ⁺ tumor cells in an autocrine manner. CCL2 and LCN2 cooperatively generate immunoregulatory dendritic cells (DCreg) having suppressive activity accompanied by lowered expression of costimulatory molecules such as HLA-DR but increased expression of immunosuppressive molecules such as PD-L1 in human PBMCs. The CCL2/LCN2-induced DCreg cells subsequently induce immunosuppressive CD4⁺FOXP3⁺ Treg cells, and finally impair tumor-specific CTL induction. In murine established tumor model, however, CCL2 blockade utilizing the specific siRNA or neutralizing mAb significantly inhibits Snail⁺ tumor growth and metastasis following systemic induction of anti-tumor immune responses in host. These results suggest that CCL2 is more than a chemoattractant factor that is the significant effector molecule responsible for immune evasion of Snail⁺ tumor cells. CCL2 would be an attractive target for treatment to eliminate cancer cells via amelioration of tumor metastasis and immunosuppression.</p>

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Suppression of myeloid cell leukemia-1 (Mcl-1) enhances chemotherapy-associated apoptosis in gastric cancer cells

Akagi Hideko, Higuchi Hajime, Sumimoto Hidetoshi, Igarashi Toru, Kabashima Ayano, Mizuguchi Hiroyuki, Izumiya Motoko, Sakai Gen, Adachi Masayuki, Funakoshi Shinsuke, Nakamura Shoko, Hamamoto Yasuo, Kanai Takanori, Takaishi Hiromasa, Kawakami Yutaka, Hibi Toshifumi

Gastric Cancer 16 ( 1 ) 100 - 110 2013.01

ISSN  1436-3291

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<p>Background: Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein that regulates apoptosis sensitivity in a variety of cell types. Here we evaluate the roles of Mcl-1 in chemotherapy-associated apoptosis in gastric cancer cells. In addition, our study examined whether Mcl-1 contributed to apoptosis resistance in so-called cancer stem cell (CSC)-like populations in gastric cancer. Methods: Seven gastric cancer cell lines were used. The expression of Mcl-1 was assessed by either real-time polymerase chain reaction or Western blot analysis. Apoptosis was quantitated by morphological observation and caspase activity measurement. Adenovirus-mediated RNA interference (RNAi) technology was used to knockdown the expression of Mcl-1. The release of cytochrome c was evaluated by subcellular fractionation and immunoblot analysis. To identify and isolate the CSC-like populations, we used the CSC-associated cell surface marker CD44 and flow cytometry. Results: Six out of the 7 gastric cancer cell lines overexpressed Mcl-1 protein. These Mcl-1-expressing cell lines were relatively resistant to chemotherapeutic agents such as 5-fluorouracil (5-FU) and cisplatin (CDDP). Depletion of Mcl-1 protein by RNAi technology effectively sensitized the cells to anticancer drug-induced mitochondrial cytochrome c release, caspase activation, and apoptosis. In addition, vast amounts of Mcl-1 mRNA were expressed in CD44-positive CSC-like cells. Mcl-1 suppression enhanced the apoptosis in CD44-positive cells to a level equivalent to that in CD44-negative cells, suggesting that Mcl-1 mediates chemotherapy resistance in CSC-like populations. Conclusion: These results suggest that Mcl-1 mediates the resistance to apoptosis in gastric cancer cells by blocking the mitochondrial pathway of cell death. Mcl-1 depletion appears to be an attractive strategy to overcome chemotherapy resistance in gastric cancer cells.</p>

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Expression and localization of aging markers in lacrimal gland of chronic graft-versus-host disease.

Kawai Masataka, Ogawa Yoko, Shimmura Shigeto, Ohta Shigeki, Suzuki Takanori, Kawamura Naoshi, Kuwana Masataka, Kawakami Yutaka, Tsubota Kazuo

Scientific Reports 3   2455 2013

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<p>Aging is commonly defined as the accumulation of diverse deleterious changes in cells and tissues with advancing age. To investigate whether aging changes are involved in the lacrimal glands of chronic graft-versus-host disease (cGVHD) model mice, we obtained the specimens from cGVHD model mice, untreated aged and young mice, and examined by histopathology, and immunoblotting. Oxidative stress markers, 8-OHdG, 4-HNE, and hexonoyl lesion (HEL), and other aging markers, p16 and p38, were used to assess the samples. The infiltrating mononuclear cells and endothelia of capillaries in the cGVHD and aged mice expressed the oxidative stress markers and other aging markers, but not in the young mice. Histological changes and the expression of aging markers in the samples from cGVHD mice exhibited similar features to those in aging mice. These results suggest that changes that typically appear with advanced age occur earlier in the lives of mice with lacrimal gland cGVHD.</p>

Prognostic impact of expression of Bcl-2 and Bax genes in circulating immune cells derived from patients with head and neck carcinoma

Tano Tomoyuki, Okamoto Masato, Kan Shin, Nakashiro Koh Ichi, Shimodaira Shigetaka, Koido Shigeo, Homma Sadamu, Sato Mitsunobu, Fujita Tomonobu, Kawakami Yutaka, Hamakawa Hiroyuki

Neoplasia (United States) 15 ( 3 ) 305 - 314 2013

ISSN  1522-8002

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<p>Antitumor functions of the host immune system are frequently compromised in patients with malignancies. In the current study, we evaluated the relationship between expression ratio of mRNAs for the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax (the Bcl-2/Bax ratio) in peripheral blood mononuclear cells and clinical outcomes in patients with head and neck carcinomas. The overall survival (OS) time of patients with Bcl-2/Bax ratios ≥ 1.2 tended to be longer than that of patients with Bcl-2/Bax ratios &lt; 1.2 but not significantly so (P =.084, n = 61). Disease-free survival (DFS) of patients with Bcl-2/Bax ratios ≥ 1.2 was statistically significantly longer than that of patients with Bcl-2/Bax ratios &lt; 1.2 (P =.001, n = 76). All of the patients whose Bcl-2/Bax ratio is ≥ 2.0 were alive after 36 months and survived without any evidence of disease for 24 months (Bcl-2/Bax ≥ 2.0 versus Bcl-2/Bax &lt; 2.0; P =.035, n = 61 in OS, P &lt;.001, n = 76 in DFS, respectively). In 56 patients who received immunochemoradiotherapy using UFT and OK-432 in combination with radiotherapy, a statistically significant relationship between the Bcl-2/Bax ratio and the therapeutic effect estimated using Response Evaluation Criteria in Solid Tumors was observed, as well as a relation with interferon-γ (IFN-γ) induction in response to the therapy [P =.002 in complete response versus partial response + stable disease; P =.046 in IFN-γ(+) versus IFN-γ(-)]. In addition, there were significant correlations of the Bcl-2/Bax ratio with both the absolute number of CD4⁺ T cells and the rate of CD4⁺ T cell and natural killer cell activity. These findings strongly suggest that the balance of expression of Bcl-2 and Bax genes in circulating immune cells has a high prognostic value in head and neck cancer patients.</p>

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Generation of human melanocytes from induced pluripotent stem cells

Ohta Shigeki, Imaizumi Yoichi, Akamatsu Wado, Okano Hideyuki, Kawakami Yutaka

  193 - 215 2013

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<p>The discovery of human induced pluripotent stem cells (iPSCs) has provided a model system for studying early events during human development. Developmentally melanocytes originate from migratory neural crest cells that emerge from the neural plate during embryogenesis after a complex process of differentiation, proliferation, and migration out of the neural tube along defined pathways. In the adult, human melanocytes are located in the basal layer of the epidermis, hair follicles, uvea, inner ear, and meninges. In the epidermis, melanocytes produce melanin pigment that gives color to the skin as well as providing protection from ultraviolet light damage. In addition, melanocytes transfer melanin pigment to hair matrix keratinocytes during each hair cycle to maintain hair pigmentation. Characterization of mouse melanocyte stem cells (MELSCs) is more complete than for humans. MELSCs are located in the bulge region of hair follicles, where hair follicle stem cells (HFSCs) also reside. Recently, it has been demonstrated that HFSCs provide a functional nice for MELSCs. According to current cancer stem cell theory, melanomas are considered to evolve from MELSCs, although the exact mechanism remains to be elucidated fully. In humans, importantly, the lack of more specific markers of MELSCs, current understanding of the molecular regulations of melanocyte development remains incomplete. Recently, the generation of melanocytes from iPSCs has lead to some clarification of human melanocyte development in vitro. Utilization of iPSC-derived melanocytes may prove invaluable in further study of human melanocytic development and novel therapies for patients suffering with pigmentation disorders and melanoma.</p>

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Improvement of cancer immunotherapy by combining molecular targeted therapy

Kawakami Yutaka, Yaguchi Tomonori, Sumimoto Hidetoshi, Kudo-Saito Chie, Iwata-Kajihara Tomoko, Nakamura Shoko, Tsujikawa Takahiro, Park Jeong Hoon, Popivanova Boryana K., Miyazaki Junichiro, Kawamura Naoshi

Frontiers in Oncology 3 MAY 2013

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<p>In human cancer cells, a constitutive activation of MAPK, STAT3, β-catenin, and various other signaling pathways triggers multiple immunosuppressive cascades. These cascades result in the production of immunosuppressive molecules (e.g., TGF-β, IL-10, IL-6, VEGF, and CCL2) and induction of immunosuppressive immune cells (e.g., regulatory T cells, tolerogenic dendritic cells, and myeloid-derived suppressor cells). Consequently, immunosuppressive conditions are formed in tumor-associated microenvironments, including the tumor and sentinel lymph nodes. Some of these cancer-derived cytokines and chemokines impair immune cells and render them immunosuppressive via the activation of signaling molecules, such as STAT3, in the immune cells. Thus, administration of signal inhibitors may inhibit the multiple immunosuppressive cascades by acting simultaneously on both cancer and immune cells at the key regulatory points in the cancer-immune network. Since common signaling pathways are involved in manifestation of several hallmarks of cancer, including cancer cell proliferation/survival, invasion/metastasis, and immunosuppression, targeting these shared signaling pathways in combination with immunotherapy may be a promising strategy for cancer treatment.</p>

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Pivotal roles of T-helper 17-related cytokines, IL-17, IL-22, and IL-23, in inflammatory diseases

Qu Ning, Xu Mingli, Mizoguchi Izuru, Furusawa Jun Ichi, Kaneko Kotaro, Watanabe Kazunori, Mizuguchi Junichiro, Itoh Masahiro, Kawakami Yutaka, Yoshimoto Takayuki

Clinical and Developmental Immunology 2013 2013

ISSN  1740-2522

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<p>T-helper 17 (Th17) cells are characterized by producing interleukin-17 (IL-17, also called IL-17A), IL-17F, IL-21, and IL-22 and potentially TNF-α and IL-6 upon certain stimulation. IL-23, which promotes Th17 cell development, as well as IL-17 and IL-22 produced by the Th17 cells plays essential roles in various inflammatory diseases, such as experimental autoimmune encephalomyelitis, rheumatoid arthritis, colitis, and Concanavalin A-induced hepatitis. In this review, we summarize the characteristics of the functional role of Th17 cells, with particular focus on the Th17 cell-related cytokines such as IL-17, IL-22, and IL-23, in mouse models and human inflammatory diseases.</p>

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[Immunotherapy targeting cancer stem cells].

Kawakami Yutaka, Matsushita Maiko, Ueda Ryo, Tsukamoto Nobuo, Ohta Shigeki

Nihon rinsho. Japanese journal of clinical medicine 70 ( 12 ) 2142 - 2146 2012.12

ISSN  0047-1852

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<p>Cancer stem cells are relatively resistant to chemotherapy, and cause relapse of cancer. Thus, various strategies to eliminate cancer stem cells have recently been exploited. One of them is immunotherapy. To develop the immunotherapy targeting cancer stem cells, tumor antigens expressed in cancer stem cells have been identified, and their use in the immunotherapy is expected. However, cancer stem cells may have an immunosuppressive ability. Therefore, blockade of the immunosuppressive mechanisms of cancer stem cells may also be required for development of effective immunotherapies against cancer stem cells.</p>

[Molecular targets for development of cancer immunotherapy].

Kawakami Yutaka

Nihon rinsho. Japanese journal of clinical medicine 70 Suppl 8   144 - 148 2012.11

ISSN  0047-1852

Cancer classification using the Immunoscore

Galon Jérôme, Pagès Franck, Marincola Francesco M., Angell Helen K., Thurin Magdalena, Lugli Alessandro, Zlobec Inti, Berger Anne, Bifulco Carlo, Botti Gerardo, Tatangelo Fabiana, Britten Cedrik M., Kreiter Sebastian, Chouchane Lotfi, Delrio Paolo, Arndt Hartmann, Asslaber Martin, Maio Michele, Masucci Giuseppe V., Mihm Martin, Vidal-Vanaclocha Fernando, Allison James P., Gnjatic Sacha, Hakansson Leif, Huber Christoph, Singh-Jasuja Harpreet, Ottensmeier Christian, Zwierzina Heinz, Laghi Luigi, Grizzi Fabio, Ohashi Pamela S., Shaw Patricia A., Clarke Blaise A., Wouters Bradly G., Kawakami Yutaka, Hazama Shoichi, Okuno Kiyotaka, Wang Ena, O'Donnell-Tormey Jill, Lagorce Christine, Pawelec Graham, Nishimura Michael I., Hawkins Robert, Lapointe Réjean, Lundqvist Andreas, Khleif Samir N., Ogino Shuji, Gibbs Peter, Waring Paul, Sato Noriyuki, Torigoe Toshihiko, Itoh Kyogo, Patel Prabhu S., Shukla Shilin N., Palmqvist Richard, Nagtegaal Iris D., Wang Yili, D'Arrigo Corrado, Kopetz Scott, Sinicrope Frank A., Trinchieri Giorgio, Gajewski Thomas F., Ascierto Paolo A., Fox Bernard A.

Journal of Translational Medicine 10 ( 1 )  2012.10

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<p>Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Recent literature has alluded to the importance of the host immune system in controlling tumor progression. Thus, evidence supports the notion to include immunological biomarkers, implemented as a tool for the prediction of prognosis and response to therapy. Accumulating data, collected from large cohorts of human cancers, has demonstrated the impact of immune-classification, which has a prognostic value that may add to the significance of the AJCC/UICC TNM-classification. It is therefore imperative to begin to incorporate the 'Immunoscore' into traditional classification, thus providing an essential prognostic and potentially predictive tool. Introduction of this parameter as a biomarker to classify cancers, as part of routine diagnostic and prognostic assessment of tumors, will facilitate clinical decision-making including rational stratification of patient treatment. Equally, the inherent complexity of quantitative immunohistochemistry, in conjunction with protocol variation across laboratories, analysis of different immune cell types, inconsistent region selection criteria, and variable ways to quantify immune infiltration, all underline the urgent requirement to reach assay harmonization. In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. This review represents a follow-up of the announcement of this initiative, and of the J Transl Med. editorial from January 2012. Immunophenotyping of tumors may provide crucial novel prognostic information. The results of this international validation may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).</p>

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Macrophage migration inhibitory factor (MIF) promotes cell survival and proliferation of neural stem/progenitor cells

Ohta Shigeki, Misawa Aya, Fukaya Raita, Inoue Satoshi, Kanemura Yonehiro, Okano Hideyuki, Kawakami Yutaka, Toda Masahiro

Development (Cambridge) 139 ( 19 )  2012.10

ISSN  0950-1991

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<p>In a previous study, we showed that murine dendritic cells (DCs) can increase the number of neural stem/progenitor cells (NSPCs) in vitro and in vivo. In the present study, we identified macrophage migration inhibitory factor (MIF) as a novel factor that can support the proliferation and/or survival of NSPCs in vitro. MIF is secreted by DCs and NSPCs, and its function in the normal brain remains largely unknown. It was previously shown that in macrophages, MIF binds to a CD74-CD44 complex. In the present study, we observed the expression of MIF receptors in mouse ganglionic-eminence-derived neurospheres using flow cytometry in vitro. We also found CD74 expression in the ganglionic eminence of E14 mouse brains, suggesting that MIF plays a physiological role in vivo. MIF increased the number of primary and secondary neurospheres. By contrast, retrovirally expressed MIF shRNA and MIF inhibitor (ISO-1) suppressed primary and secondary neurosphere formation, as well as cell proliferation. In the neurospheres, MIF knockdown by shRNA increased caspase 3/7 activity, and MIF increased the phosphorylation of Akt, Erk, AMPK and Stat3 (Ser727), as well as expression of Hes3 and Egfr, the products of which are known to support cell survival, proliferation and/or maintenance of NSPCs. MIF also acted as a chemoattractant for NSPCs. These results show that MIF can induce NSPC proliferation and maintenance by multiple signaling pathways acting synergistically, and it may be a potential therapeutic factor, capable of activating NSPC, for the treatment of degenerative brain disorders.</p>

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Accumulation of Secretory Vesicles in the Lacrimal Gland Epithelia Is Related to Non-Sjögren's Type Dry Eye in Visual Display Terminal Users

Kamoi Mizuka, Ogawa Yoko, Nakamura Shigeru, Dogru Murat, Nagai Toshihiro, Obata Hiroto, Ito Masataka, Kaido Minako, Kawakita Tetsuya, Okada Yasunori, Kawakami Yutaka, Shimmura Shigeto, Tsubota Kazuo

PLoS One 7 ( 9 )  2012.09

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<p>Previous observations in a rat model of a non-Sjögren's syndrome (non-SS) type of dry eye seen in users of visual display terminals (VDT) indicated that secretory vesicle (SV) accumulation in the lacrimal gland epithelia contributes to the condition. Here, to examine this possibility in humans, we compared the lacrimal gland histology and percent SV area in the cytoplasm of acinar epithelial cells using light microscopy and transmission electron microscopy, in patients with VDT work-related non-SS dry-eye (VDT group), SS-induced dry-eye, and autopsied normal controls. In addition, the VAMP8 (vesicle-associated membrane protein 8, an exocrine-pathway molecule) and Rab3D (mature vesicle marker) were histochemically examined in lacrimal gland tissue sections. The lacrimal gland acini were larger in the VDT group than in the SS group, and the percent SV area was significantly higher in the VDT group than in the normal controls (P = 0.021) or SS group (P = 0.004). Immunostaining revealed abnormal distributions of VAMP8 in the VDT and SS groups. Rab3D was more strongly expressed in the cytoplasm of acinar epithelial cells in the VDT group than in that of normal controls. The duration of VDT use was significantly longer in the VDT group than in the other groups. These findings suggest that excessive SV accumulation in the acinar epithelia may contribute to the reduced tear secretion in VDT users.</p>

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Immune suppression and resistance mediated by constitutive activation of Wnt/-β-catenin signaling in human melanoma cells

Yaguchi Tomonori, Goto Yasufumi, Kido Kenji, Mochimaru Hiroshi, Sakurai Toshiharu, Tsukamoto Nobuo, Kudo-Saito Chie, Fujita Tomonobu, Sumimoto Hidetoshi, Kawakami Yutaka

Journal of Immunology 189 ( 5 ) 2110 - 2117 2012.09

ISSN  0022-1767

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<p>Cancer-induced immunosuppression is a major problem reducing antitumor effects of immunotherapies, but its molecular mechanism has not been well understood. We evaluated immunosuppressive roles of activated Wnt/β-catenin pathways in human melanoma for dendritic cells (DCs) and CTLs. IL-10 expression was associated with β-catenin accumulation in human melanoma cell lines and tissues and was induced by direct β-catenin/TCF binding to the IL-10 promoter. Culture supernatants from β-catenin-accumulated melanoma have activities to impair DC maturation and to induce possible regulatory DCs. Those immunosuppressive culture supernatant activities were reduced by knocking down β-catenin in melanoma cells, partly owing to downregulation of IL-10. Murine splenic and tumor-infiltrating DCs obtained from nude mice implanted with human mutant β-catenin-overexpressed melanoma cells had less ability to activate T cells than did DCs from mice with control melanoma cells, showing in vivo suppression of DCs by activated Wnt/β-catenin signaling in human melanoma. This in vivo DC suppression was restored by the administration of a β-catenin inhibitor, PKF115-584. β-catenin-overexpressed melanoma inhibited IFN-γ production by melanoma-specific CTLs in an IL-10-independent manner and is more resistant to CTL lysis in vitro and in vivo. These results indicate that Wnt/β-catenin pathways in human melanoma may be involved in immunosuppression and immunoresistance in both induction and effector phases of antitumor immunoresponses partly through IL-10 production, and they may be attractive targets for restoring immunocompetence in patients with Wnt/β-catenin-activated melanoma.</p>

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Transplantation of side population cells restores the function of damaged exocrine glands through clusterin

Mishima Kenji, Inoue Hiroko, Nishiyama Tatsuaki, Mabuchi Yo, Amano Yusuke, Ide Fumio, Matsui Makoto, Yamada Hiroyuki, Yamamoto Gou, Tanaka Junichi, Yasuhara Rika, Sakurai Takashi, Lee Masaichi Chang Il, Chiba Kan, Sumimoto Hidetoshi, Kawakami Yutaka, Matsuzaki Yumi, Tsubota Kazuo, Saito Ichiro

Stem Cells 30 ( 9 ) 1925 - 1937 2012.09

ISSN  1066-5099

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<p>Stem cell-based therapy has been proposed as a promising strategy for regenerating tissues lost through incurable diseases. Side population (SP) cells have been identified as putative stem cells in various organs. To examine therapeutic potential of SP cells in hypofunction of exocrine glands, SP cells isolated from mouse exocrine glands, namely, lacrimal and salivary glands, were transplanted into mice with irradiation-induced hypofunction of the respective glands. The secretions from both glands in the recipient mice were restored within 2 months of transplantation, although the transplanted cells were only sparsely distributed and produced no outgrowths. Consistent with this, most SP cells were shown to be CD31-positive endothelial-like cells. In addition, we clarified that endothelial cell-derived clusterin, a secretory protein, was an essential factor for SP cell-mediated recovery of the hypofunctioning glands because SP cells isolated from salivary glands of clusterin-deficient mice had no therapeutic potential, whereas lentiviral transduction of clusterin restored the hypofunction. In vitro and in vivo studies showed that clusterin had an ability to directly inhibit oxidative stress and oxidative stress-induced cell damage. Thus, endothelial cell-derived clusterin possibly inhibit oxidative stress-induced hypofunction of these glands.</p>

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Cancer-testis antigen BORIS is a novel prognostic marker for patients with esophageal cancer

Okabayashi Koji, Fujita Tomonobu, Miyazaki Junichiro, Okada Tsutomu, Iwata Takashi, Hirao Nobumaru, Noji Shinobu, Tsukamoto Nobuo, Goshima Naoki, Hasegawa Hirotoshi, Takeuchi Hiroya, Ueda Masakazu, Kitagawa Yuko, Kawakami Yutaka

Cancer Science 103 ( 9 ) 1617 - 1624 2012.09

ISSN  1347-9032

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<p>Esophageal squamous cell cancer (ESCC) is one of the most common lethal tumors in the world, and development of new diagnostic and therapeutic methods is needed. In this study, cancer-testis antigen, BORIS, was isolated by functional cDNA expression cloning using screening technique with serum IgG Abs from ESCC patients. BORIS was previously reported to show cancer-testis antigen like expression, but its immunogenicity has remained unclear in cancer patients. BORIS was considered to be an immunogenic antigen capable of inducing IgG Abs in patients with various cancers, including four of 11 ESCC patients. Immunohistochemical study showed that the BORIS protein was expressed in 28 of 50 (56%) ESCC tissues. The BORIS expression was significantly associated with lymph node metastasis in ESCC patients with pT1 disease (P = 0.036). Furthermore, the patients with BORIS-positive tumors had a poor overall survival (5-year survival rate: BORIS-negative 70.0% vs BORIS-positive 29.9%, log-rank P = 0.028) in Kaplan-Meier survival analysis and log-rank test. Multivariate Cox proportional hazard model demonstrated that BORIS expression was an independent poor prognostic factor (hazard ratio = 4.158 [95% confidence interval 1.494-11.57], P = 0.006). Downregulation of BORIS with specific siRNAs resulted in decreased cell proliferation and invasion ability of ESCC cell lines. BORIS may be a useful biomarker for prognostic diagnosis of ESCC patients and a potential target for treatment including by BORIS-specific immunotherapy and molecular target therapy.</p>

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Presence and physiologic function of the renin-angiotensin system in mouse lacrimal gland

Yaguchi Saori, Ogawa Yoko, Shimmura Shigeto, Hatou Shin, Nakamura Shigeru, Inaba Takaaki, Imada Toshihiro, Ozawa Yoko, Kawakami Yutaka, Ishida Susumu, Tsubota Kazuo

Investigative Ophthalmology and Visual Science 53 ( 9 ) 5416 - 5425 2012.08

ISSN  0146-0404

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<p>PURPOSE. To investigate the expression, localization, and physiologic function of renin-angiotensin system (RAS) components in the mouse lacrimal gland. METHODS. Lacrimal glands and cultured lacrimal gland fibroblasts from wild-type (WT) BALB/c (H-2^d) mice were used. Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry were used to determine the expression and localization of the RAS components, prorenin/renin, angiotensin-converting enzyme (ACE), angiotensin II, angiotensin II type 1 receptor (AT1R), and angiotensin II type 2 receptor (AT2R) in the normal mouse lacrimal gland. To examine the change in tear secretion, mice received ARB (AT1R blocker) or AT2R antagonist. Tear secretion was assessed by cotton thread test before and after drug administration. RESULTS. The mRNAs coding for angiotensinogen, prorenin, ACE, and both AT1R and AT2R were found in normal lacrimal gland tissue and cultured lacrimal gland fibroblasts. Prorenin/renin and ACE were identified in myoepithelial cells around ducts and acini and in blood vessels. Angiotensin II, AT1R, and AT2R were observed in the ducts and interstitial fibroblasts. AT1R and AT2R were also localized in blood vessels. All the cultured lacrimal gland fibroblasts expressed angiotensin II, AT1R, and AT2R. Tear secretion increased in mice that received ARB. CONCLUSIONS. The results are consistent with the hypothesis that a tissue-specific RAS is present in the lacrimal gland, and suggest that fibroblasts are one of the cell types playing a role in the tissue RAS. Tissue RAS might be involved in tissue function of regulating tear secretion in the lacrimal gland.</p>

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Growth inhibition and apoptosis by an active component of OK-432, a streptococcal agent, via Toll-like receptor 4 in human head and neck cancer cell lines

Tano Tomoyuki, Okamoto Masato, Kan Shin, Nakashiro Koh Ichi, Shimodaira Shigetaka, Yamashita Naomi, Kawakami Yutaka, Hamakawa Hiroyuki

Oral Oncology 48 ( 8 ) 678 - 685 2012.08

ISSN  1368-8375

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<p>Toll-like receptor 4 (TLR4) plays a significant role in cancer therapy as receptors of bacteria-derived immunotherapeutic agents such as OK-432, a streptococcal immunotherapeutic agent. In addition, recent reports demonstrated that TLRs, including TLR4, are also expressed in cancer cells as well as in immunocompetent cells. It is a problem in cancer therapy that the immunoadjuvant may activate survival signals such as nuclear factor (NF)-κB or mitogen-activated protein kinases (MAPKs) in cancer cells via TLRs. In the current study, we investigated responsiveness of human head and neck cancer cell lines against TLR4 ligands, OK-PSA, an active component of OK-432, and a lipopolysaccharide (LPS). Stimulation with LPS or OK-PSA resulted in the activation of NF-κB in these cell lines expressing TLR4 and MD-2 that is a significant coreceptor for TLR4 signaling. Interestingly, OK-PSA induced cell-growth inhibition, while LPS enhanced the proliferation of the cancer cells. OK-PSA induced NF-κB activation more slowly than that induced by LPS. In addition, phosphorylation of p38 MAPK by OK-PSA was only slight compared with that by LPS. OK-PSA also induced apoptosis of the cancer cells mediated by the activation of caspase 1, 3 and 8 in a p53-independent manner. These findings strongly suggest that active components of OK-432 may elicit anti-cancer effects via enhancing host immunity as well as via directly inducing the growth inhibition and apoptosis of head and neck cancer cells through TLR4 signal.</p>

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[Biomarkers for cancer immunotherapy].

Kawakami Yutaka, Kawase Yoshie, Ohizumi Azusa, Noji Shinobu, Sakurai Toshiharu, Fujita Tomonobu

Nihon rinsho. Japanese journal of clinical medicine 70 ( 5 ) 759 - 766 2012.05

ISSN  0047-1852

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<p>Immune-biomarkers and -assays are important for development of cancer immunotherapy to select the patients who are expected to respond to immunotherapy before or early after immunotherapy, to monitor immune induction following immunotherapy, and to evaluate anti-tumor effects early after immunotherapy. Comprehensive immune-evaluation including positive and negative immune responses against cancer cells and identification of blood biomarkers which reflect immune-conditions in tumor microenvironment are required although direct evaluation of tumor tissues can be possible for some patients. Importance of immune responses has recently been recognized even for standard cancer treatments including chemotherapy and molecular target therapy. Therefore, immunological biomarkers may be useful for any cancer treatment.</p>

Mucosal microvilli in dry eye patients with chronic GVHD

Tatematsu Y., Ogawa Y., Shimmura S., Dogru M., Yaguchi S., Nagai T., Yamazaki K., Kameyama K., Okamoto S., Kawakami Y., Tsubota K.

Bone Marrow Transplantation 47 ( 3 ) 416 - 425 2012.03

ISSN  0268-3369

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<p>The ocular surface is a frequent target tissue of mucosal chronic GVHD (cGVHD). We investigated the histopathological features of the conjunctival microvilli in patients with cGVHD. Conjunctival tissue specimens from patients with cGVHD or Sjögren's syndrome (SS) or from healthy individuals were examined by light microscopy and EM, impression cytology, and immunohistochemistry. The cGVHD conjunctivae showed significantly more metaplasia and fewer goblet cells than the SS and normal conjunctivae. Abundant CD8 ⁺ T cells infiltrated the basal epithelia in the cGVHD conjunctiva. The microvilli per standard epithelial unit and the secretory vesicles were counted by analyzing electron micrographs. The mean number of mucosal microvilli was significantly lower in the cGVHD than that in the SS or normal specimens, and the microvilli were significantly shorter, with a smaller height-width ratio. The mean number of secretory vesicles was also significantly lower, and the membrane-spanning mucin thinner, in the cGVHD compared with the SS and normal specimens. Thus, the conjunctival mucosal microvilli of cGVHD patients were significantly different in number and morphology from those of SS and normal subjects. These may be important factors affecting the stability of the tear-film layer and its contribution to cGVHD-related dry eye.</p>

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Fibroblast growth factor-2 is an important factor that maintains cellular immaturity and contributes to aggressiveness of osteosarcoma

Shimizu Takatsune, Ishikawa Tomoki, Iwai Sayaka, Ueki Arisa, Sugihara Eiji, Onishi Nobuyuki, Kuninaka Shinji, Miyamoto Takeshi, Toyama Yoshiaki, Ijiri Hiroshi, Mori Hajime, Matsuzaki Yumi, Yaguchi Tomonori, Nishio Hiroshi, Kawakami Yutaka, Ikeda Yasuo, Saya Hideyuki

Molecular Cancer Research 10 ( 3 ) 454 - 468 2012.03

ISSN  1541-7786

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<p>Osteosarcoma is the most frequent, nonhematopoietic, primary malignant tumor of bone. Histopathologically, osteosarcoma is characterized by complex mixtures of different cell types with bone formation. The role of environmental factors in the formation of such a complicated tissue structure as osteosarcoma remains to be elucidated. Here, a newly established murine osteosarcoma model was used to clarify the roles of environmental factors such as fibroblast growth factor-2 (Fgf2) or leukemia-inhibitory factor (Lif) in the maintenance of osteosarcoma cells in an immature state. These factors were highly expressed in tumor environmental stromal cells, rather than in osteosarcoma cells, and they potently suppressed osteogenic differentiation of osteosarcoma cells in vitro and in vivo. Further investigation revealed that the hyperactivation of extracellular signal-regulated kinase (Erk)1/2 induced by these factors affected in the process of osteosarcoma differentiation. In addition, Fgf2 enhanced both proliferation and migratory activity of osteosarcoma cells and modulated the sensitivity of cells to an anticancer drug. The results of the present study suggest that the histology of osteosarcoma tumors which consist of immature tumor cells and pathologic bone formations could be generated dependent on the distribution of such environmental factors. The combined blockade of the signaling pathways of several growth factors, including Fgf2, might be useful in controlling the aggressiveness of osteosarcoma.</p>

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Downregulation of KIF23 suppresses glioma proliferation

Takahashi Satoshi, Fusaki Noemi, Ohta Shigeki, Iwahori Yoshihiro, Iizuka Yukihiko, Inagawa Kohei, Kawakami Yutaka, Yoshida Kazunari, Toda Masahiro

Journal of Neuro-Oncology 106 ( 3 ) 519 - 529 2012.02

ISSN  0167-594X

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<p>To identify therapeutic molecular targets for glioma, we performed modified serological identification of antigens by recombinant complementary DNA (cDNA) expression cloning using sera from a mouse glioma model. Two clones, kinesin family member 23 (Kif23) and structural maintenance of chromosomes 4 (Smc4), were identified as antigens through immunological reaction with sera from mice harboring synergic GL261 mouse glioma and intratumoral inoculation with a mutant herpes simplex virus. The human Kif23 homolog KIF23 is a nuclear protein that localizes to the interzone of mitotic spindles, acting as a plus-end-directed motor enzyme that moves antiparallel microtubules in vitro. Expression analysis revealed a higher level of KIF23 expression in glioma tissues than in normal brain tissue. The introduction of small interfering RNA (siRNA) targeting KIF23 into two different glioma cell lines, U87MG and SF126, downregulated KIF23 expression, which significantly suppressed glioma cell proliferation in vitro. KIF23 siRNA-treated glioma cells exhibited larger cell bodies with two or more nuclei compared with control cells. In vivo analysis using mouse xenograft showed that KIF23 siRNA/DNA chimeratreated tumors were significantly smaller than tumors treated with control siRNA/DNA chimera. Taken together, our results indicate that downregulation of KIF23 decreases proliferation of glioma cells and that KIF23 may be a novel therapeutic target in malignant glioma.</p>

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Human tumor antigens recognized by T cells and their implications for cancer immunotherapy

Ueda Ryo, Yaguchi Tomonori, Kawakami Yutaka

  335 - 345 2012.01

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<p>Recent clinical trials of immunotherapies indicate that tumor reactive autologous T cells are able to regress even advanced, large tumors in melanoma patients. For example, the adoptive transfer of CD8+ cytotoxic T lymphocytes (CTL) specifically targeted for identified tumor antigens following lymphodepletive treatment, such as fludarabine/cyclophosphamide administration and total body irradiation, led to objective tumor responses in more than 70% of patients with melanoma (Dudley et al. 2008). Immunological analyses on these tumor tissues demonstrated that administered T cells may eliminate tumor cells through direct killing and cytokine secretion. Therefore, CD8+ CTLs that recognize MHC class I positive cancer cells are important for in vivo tumor rejection.</p>

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Defining the critical hurdles in cancer immunotherapy

Fox Bernard A., Schendel Dolores J., Butterfield Lisa H., Aamdal Steinar, Allison James P., Ascierto Paolo Antonio, Atkins Michael B., Bartunkova Jirina, Bergmann Lothar, Berinstein Neil, Bonorino Cristina C., Borden Ernest, Bramson Jonathan L., Britten Cedrik M., Cao Xuetao, Carson William E., Chang Alfred E., Characiejus Dainius, Raja Choudhury A., Coukos George, de Gruijl Tanja, Dillman Robert O., Dolstra Harry, Dranoff Glenn, Durrant Lindy G., Finke James H., Galon Jerome, Gollob Jared A., Gouttefangeas Cécile, Grizzi Fabio, Guida Michele, Håkansson Leif, Hege Kristen, Herberman Ronald B., Stephen Hodi F., Hoos Axel, Huber Christoph, Hwu Patrick, Imai Kohzoh, Jaffee Elizabeth M., Janetzki Sylvia, June Carl H., Kalinski Pawel, Kaufman Howard L., Kawakami Koji, Kawakami Yutaka, Keilholtz Ulrich, Khleif Samir N., Kiessling Rolf, Kotlan Beatrix, Kroemer Guido, Lapointe Rejean, Levitsky Hyam I., Lotze Michael T., Maccalli Cristina, Maio Michele, Marschner Jens Peter, Mastrangelo Michael J., Masucci Giuseppe, Melero Ignacio, Melief Cornelius, Murphy William J., Nelson Brad, Nicolini Andrea, Nishimura Michael I., Odunsi Kunle, Ohashi Pamela S., O'Donnell-Tormey Jill, Old Lloyd J., Ottensmeier Christian, Papamichail Michael, Parmiani Giorgio, Pawelec Graham, Proietti Enrico, Qin Shukui, Rees Robert, Ribas Antoni, Ridolfi Ruggero, Ritter Gerd, Rivoltini Licia, Romero Pedro J., Salem Mohamed L., Scheper Rik J., Seliger Barbara, Sharma Padmanee, Shiku Hiroshi, Singh-Jasuja Harpreet, Song Wenru, Straten Per Thor, Tahara Hideaki, Tian Zhigang, van der Burg Sjoerd H., von Hoegen Paul, Wang Ena, Welters Marij J P, Winter Hauke, Withington Tara, Wolchok Jedd D., Xiao Weihua, Zitvogel Laurence, Zwierzina Heinz, Marincola Francesco M., Gajewski Thomas F., Wigginton Jon M., Disis Mary L.

Journal of Translational Medicine 9 ( 1 )  2011.12

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<p>Scientific discoveries that provide strong evidence of antitumor effects in preclinical models often encounter significant delays before being tested in patients with cancer. While some of these delays have a scientific basis, others do not. We need to do better. Innovative strategies need to move into early stage clinical trials as quickly as it is safe, and if successful, these therapies should efficiently obtain regulatory approval and widespread clinical application. In late 2009 and 2010 the Society for Immunotherapy of Cancer (SITC), convened an "Immunotherapy Summit" with representatives from immunotherapy organizations representing Europe, Japan, China and North America to discuss collaborations to improve development and delivery of cancer immunotherapy. One of the concepts raised by SITC and defined as critical by all parties was the need to identify hurdles that impede effective translation of cancer immunotherapy. With consensus on these hurdles, international working groups could be developed to make recommendations vetted by the participating organizations. These recommendations could then be considered by regulatory bodies, governmental and private funding agencies, pharmaceutical companies and academic institutions to facilitate changes necessary to accelerate clinical translation of novel immune-based cancer therapies. The critical hurdles identified by representatives of the collaborating organizations, now organized as the World Immunotherapy Council, are presented and discussed in this report. Some of the identified hurdles impede all investigators; others hinder investigators only in certain regions or institutions or are more relevant to specific types of immunotherapy or first-in-humans studies. Each of these hurdles can significantly delay clinical translation of promising advances in immunotherapy yet if overcome, have the potential to improve outcomes of patients with cancer.</p>

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Enhanced cancer immunotherapy using STAT3-depleted dendritic cells with high Th1-inducing ability and resistance to cancer cell-derived inhibitory factors

Iwata-Kajihara Tomoko, Sumimoto Hidetoshi, Kawamura Naoshi, Ueda Ryo, Takahashi Tomomi, Mizuguchi Hiroyuki, Miyagishi Makoto, Takeda Kiyoshi, Kawakami Yutaka

Journal of Immunology 187 ( 1 ) 27 - 36 2011.07

ISSN  0022-1767

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<p>STAT3 signaling constitutes an important negative feedback mechanism for the maintenance of immune homeostasis, a suppressive signal for the Th1 immune response in murine macrophages, and a cancer immune evasion signal in various immune cells. The strategy for STAT3 signal inhibition should be considered, because these features could impede effective cancer immunotherapy. We have evaluated the effects of STAT3 inactivation in dendritic cells (DCs) on immune responses in mice and humans. DCs derived from LysMcre/STAT3^flox/flox mice displayed higher cytokine production in response to TLR stimulation, activated T cells more efficiently, and were more resistant to the suppression of cytokine production by cancer-derived immunosuppressive factors compared with DCs from control littermates. Antitumor activities of STAT3-depleted and control DCs were compared by intratumoral administration of gp70 Ag peptide-pulsed DCs in the therapeutic MC38 tumor model. Intratumoral administration of STAT3-depleted DCs significantly inhibited MC38 tumor growth of both injected and nontreated remote tumors. The inhibition was accompanied by an increase in gp70-specific T cell response as well as in systemic Th1 immune response. STAT3-depleted human DCs with adenoviral STAT3 short hairpin RNAwere also capable of producing more cytokines with TLR stimulation and more resistant to cancer-derived factors, and they induced tumor Ag-specific T cells more efficiently than control DCs. The identified role of DC STAT3 signaling in both in vivo therapeutic tumor models in mice and in vitro-specific T cell induction in humans indicates that STAT3-inactivated DCs may be a promising approach for cancer immunotherapy.</p>

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Functional analysis of HOXD9 in human gliomas and glioma cancer stem cells

Tabuse Masanao, Ohta Shigeki, Ohashi Yohei, Fukaya Raita, Misawa Aya, Yoshida Kazunari, Kawase Takeshi, Saya Hideyuki, Thirant Cécile, Chneiweiss Hérve, Matsuzaki Yumi, Okano Hideyuki, Kawakami Yutaka, Toda Masahiro

Molecular Cancer 10 2011.05

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<p>Background: HOX genes encode a family of homeodomain-containing transcription factors involved in the determination of cell fate and identity during embryonic development. They also behave as oncogenes in some malignancies.Results: In this study, we found high expression of the HOXD9 gene transcript in glioma cell lines and human glioma tissues by quantitative real-time PCR. Using immunohistochemistry, we observed HOXD9 protein expression in human brain tumor tissues, including astrocytomas and glioblastomas. To investigate the role of HOXD9 in gliomas, we silenced its expression in the glioma cell line U87 using HOXD9-specific siRNA, and observed decreased cell proliferation, cell cycle arrest, and induction of apoptosis. It was suggested that HOXD9 contributes to both cell proliferation and/or cell survival. The HOXD9 gene was highly expressed in a side population (SP) of SK-MG-1 cells that was previously identified as an enriched-cell fraction of glioma cancer stem-like cells. HOXD9 siRNA treatment of SK-MG-1 SP cells resulted in reduced cell proliferation. Finally, we cultured human glioma cancer stem cells (GCSCs) from patient specimens found with high expression of HOXD9 in GCSCs compared with normal astrocyte cells and neural stem/progenitor cells (NSPCs).Conclusions: Our results suggest that HOXD9 may be a novel marker of GCSCs and cell proliferation and/or survival factor in gliomas and glioma cancer stem-like cells, and a potential therapeutic target.</p>

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Recommendations from the iSBTc-SITC/FDA/NCI workshop on immunotherapy biomarkers

Butterfield Lisa H., Palucka A. Karolina, Britten Cedrik M., Dhodapkar Madhav V., Håkansson Leif, Janetzki Sylvia, Kawakami Yutaka, Kleen Thomas Oliver, Lee Peter P., Maccalli Cristina, Maecker Holden T., Maino Vernon C., Maio Michele, Malyguine Anatoli, Masucci Giuseppe, Pawelec Graham, Potter Douglas M., Rivoltini Licia, Salazar Lupe G., Schendel Dolores J., Slingluff Craig L., Song Wenru, Stroncek David F., Tahara Hideaki, Thurin Magdalena, Trinchieri Giorgio, Van Der Burg Sjoerd H., Whiteside Theresa L., Wigginton Jon M., Marincola Francesco, Khleif Samir, Fox Bernard A., Disis Mary L.

Clinical Cancer Research 17 ( 10 ) 3064 - 3076 2011.05

ISSN  1078-0432

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<p>Purpose: To facilitate development of innovative immunotherapy approaches, especially for treatment concepts exploiting the potential benefits of personalized therapy, there is a need to develop and validate tools to identify patients who can benefit from immunotherapy. Despite substantial effort, we do not yet know which parameters of antitumor immunity to measure and which assays are optimal for those measurements. Experimental Design: The iSBTc-SITC (International Society for Biological Therapy of Cancer-Society for Immunotherapy of Cancer), FDA (Food and Drug Administration), and NCI (National Cancer Institute) partnered to address these issues for immunotherapy of cancer. Here, we review the major challenges, give examples of approaches and solutions, and present our recommendations. Results and Conclusions: Although specific immune parameters and assays are not yet validated, we recommend following standardized (accurate, precise, and reproducible) protocols and use of functional assays for the primary immunologic readouts of a trial; consideration of central laboratories for immune monitoring of large, multi-institutional trials; and standardized testing of several phenotypic and functional potential potency assays specific to any cellular product. When reporting results, the full QA (quality assessment)/QC (quality control) should be conducted and selected examples of truly representative raw data and assay performance characteristics should be included. Finally, to promote broader analysis of multiple aspects of immunity, and gather data on variability, we recommend that in addition to cells and serum, RNA and DNA samples be banked (under standardized conditions) for later testing. We also recommend that sufficient blood be drawn to allow for planned testing of the primary hypothesis being addressed in the trial, and that additional baseline and posttreatment blood is banked for testing novel hypotheses (or generating new hypotheses) that arise in the field.</p>

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AMP kinase-related kinase NUAK2 affects tumor growth, migration, and clinical outcome of human melanoma

Namiki Takeshi, Tanemura Atsushi, Valencia Julio C., Coelho Sergio G., Passeron Thierry, Kawaguchi Masakazu, Vieira Wilfred D., Ishikawa Masashi, Nishijima Wataru, Izumo Toshiyuki, Kaneko Yasuhiko, Katayama Ichiro, Yamaguchi Yuji, Yin Lanlan, Polley Eric C., Liu Hongfang, Kawakami Yutaka, Eishi Yoshinobu, Takahashi Eishi, Yokozeki Hiroo, Hearing Vincent J.

Proceedings of the National Academy of Sciences of the United States of America 108 ( 16 ) 6597 - 6602 2011.04

ISSN  0027-8424

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<p>The identification of genes that participate in melanomagenesis should suggest strategies for developing therapeutic modalities. We used a public array comparative genomic hybridization (CGH) database and real-time quantitative PCR (qPCR) analyses to identify the AMP kinase (AMPK)-related kinase NUAK2 as a candidate gene for melanomagenesis, and we analyzed its functions in melanoma cells. Our analyses had identified a locus at 1q32 where genomic gain is strongly associated with tumor thickness, and we used real-time qPCR analyses and regression analyses to identify NUAK2 as a candidate gene at that locus. Associations of relapse-free survival and overall survival of 92 primary melanoma patients with NUAK2 expression measured using immunohistochemistry were investigated using Kaplan-Meier curves, log rank tests, and Cox regression models. Knockdown of NUAK2 induces senescence and reduces S-phase, decreases migration, and down-regulates expression of mammalian target of rapamycin (mTOR). In vivo analysis demonstrated that knockdown of NUAK2 suppresses melanoma tumor growth in mice. Survival analysis showed that the risk of relapse is greater in acral melanoma patients with high levels of NUAK2 expression than in acral melanoma patients with low levels of NUAK2 expression (hazard ratio = 3.88; 95% confidence interval = 1.44-10.50; P = 0.0075). These data demonstrate that NUAK2 expression is significantly associated with the oncogenic features of melanoma cells and with the survival of acral melanoma patients. NUAK2 may provide a drug target to suppress melanoma progression. This study further supports the importance of NUAK2 in cancer development and tumor progression, while AMPK has antioncogenic properties.</p>

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The mechanisms of cancer immunoescape and development of overcoming strategies

Yaguchi Tomonori, Sumimoto Hidetoshi, Kudo-Saito Chie, Tsukamoto Nobuo, Ueda Ryo, Iwata-Kajihara Tomoko, Nishio Hiroshi, Kawamura Naoshi, Kawakami Yutaka

International Journal of Hematology 93 ( 3 ) 294 - 300 2011.03

ISSN  0925-5710

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<p>Cancer-induced immunosuppression is a major problem as it reduces the anti-tumor effects of immunotherapies. In cancer tissues, cancer cells, immune cells, and other stromal cells interact and create an immunosuppressive microenvironment through a variety of immunosuppressive factors. Some cancer subpopulations such as cancer cells undergoing epithelial-mesenchymal transition and cancer stem-like cells have immunosuppressive and immunoresistant properties. The production of immunosuppressive factors by cancer cells is mechanistically attributed to oncogenic signals frequently activated in cancer cells, including the STAT3, MAPK, NF-κB, and Wnt/β-catenin signals, which are upstream events leading to immunosuppressive cascades. Moreover, some of these signals are also activated in immunosuppressive immune cells stimulated by cancer-derived factors and contribute to their immunosuppressive activities. Therefore, targeting these signals both in cancer cells and immunosuppressive immune cells may result in the restoration of immuno-competence in cancer patients and improve current immunotherapy.</p>

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Adjuvant effects of formalin-inactivated HSV through activation of dendritic cells and inactivation of myeloid-derived suppressor cells in cancer immunotherapy

Ohkusu-Tsukada Kozo, Ohta Shigeki, Kawakami Yutaka, Toda Masahiro

International Journal of Cancer 128 ( 1 ) 119 - 131 2011.01

ISSN  0020-7136

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<p>Use of adequate adjuvant is necessary for induction of effective antitumor immune responses. To develop an effective adjuvant for cancer immunotherapy, we selected formalin-inactivated (f)-HSV as an adjuvant component, and analyzed the mechanisms underlying its adjuvant effects. First, we found that f-HSV can induce the tumor antigen-specific CTLs by enhancing antigen cross-presentation by dendritic cells (DCs), mainly through TLR2, but not TLR9. Next, f-HSV was also found to prevent the accumulation of myeloid-derived suppressor cells (MDSCs). We demonstrated that the expansion of MDSCs in the blood and spleen during tumor progression required B cells producing the inflammatory angiogenesis factors, vascular endothelial growth factor (VEGF)-A and neuropilin-1 (NRP-1), a co-receptor for VEGF receptor-2 (VEGFR-2). Interestingly, the transmembrane-type NRP-1 on B cells changed to soluble-type NRP-1 (sNRP-1) by f-HSV treatment. We further showed that the sNRP-1 and VEGF-A secreted from B cells by f-HSV treatment could abrogate the immunosuppressive ability of MDSCs. These results suggest that f-HSV can enhance antitumor immune responses as an adjuvant, not only through activation of DCs, but also inactivation of MDSCs via B cells.</p>

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Generation of human melanocytes from induced pluripotent stem cells

Ohta Shigeki, Imaizumi Yoichi, Okada Yohei, Akamatsu Wado, Kuwahara Reiko, Ohyama Manabu, Amagai Masayuki, Matsuzaki Yumi, Yamanaka Shinya, Okano Hideyuki, Kawakami Yutaka

PLoS One 6 ( 1 )  2011

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<p>Epidermal melanocytes play an important role in protecting the skin from UV rays, and their functional impairment results in pigment disorders. Additionally, melanomas are considered to arise from mutations that accumulate in melanocyte stem cells. The mechanisms underlying melanocyte differentiation and the defining characteristics of melanocyte stem cells in humans are, however, largely unknown. In the present study, we set out to generate melanocytes from human iPS cells in vitro, leading to a preliminary investigation of the mechanisms of human melanocyte differentiation. We generated iPS cell lines from human dermal fibroblasts using the Yamanaka factors (SOX2, OCT3/4, and KLF4, with or without c-MYC). These iPS cell lines were subsequently used to form embryoid bodies (EBs) and then differentiated into melanocytes via culture supplementation with Wnt3a, SCF, and ET-3. Seven weeks after inducing differentiation, pigmented cells expressing melanocyte markers such as MITF, tyrosinase, SILV, and TYRP1, were detected. Melanosomes were identified in these pigmented cells by electron microscopy, and global gene expression profiling of the pigmented cells showed a high similarity to that of human primary foreskin-derived melanocytes, suggesting the successful generation of melanocytes from iPS cells. This in vitro differentiation system should prove useful for understanding human melanocyte biology and revealing the mechanism of various pigment cell disorders, including melanoma.</p>

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Phase I clinical trial of the vaccination for the patients with metastatic melanoma using gp100-derived epitope peptide restricted to HLA-A*2402

Baba Toshiyuki, Sato-Matsushita Marimo, Kanamoto Akira, Itoh Akihiko, Oyaizu Naoki, Inoue Yusuke, Kawakami Yutaka, Tahara Hideaki

Journal of Translational Medicine 8 2010.09

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<p>Background: The tumor associated antigen (TAA) gp100 was one of the first identified and has been used in clinical trials to treat melanoma patients. However, the gp100 epitope peptide restricted to HLA-A*2402 has not been extensively examined clinically due to the ethnic variations. Since it is the most common HLA Class I allele in the Japanese population, we performed a phase I clinical trial of cancer vaccination using the HLA-A*2402 gp100 peptide to treat patients with metastatic melanoma.Methods: The phase I clinical protocol to test a HLA-A*2402 gp100 peptide-based cancer vaccine was designed to evaluate safety as the primary endpoint and was approved by The University of Tokyo Institutional Review Board. Information related to the immunologic and antitumor responses were also collected as secondary endpoints. Patients that were HLA-A*2402 positive with stage IV melanoma were enrolled according to the criteria set by the protocol and immunized with a vaccine consisting of epitope peptide (VYFFLPDHL, gp100-in4) emulsified with incomplete Freund's adjuvant (IFA) for the total of 4 times with two week intervals. Prior to each vaccination, peripheral blood mononuclear cells (PBMCs) were separated from the blood and stored at -80°C. The stored PBMCs were thawed and examined for the frequency of the peptide specific T lymphocytes by IFN-γ- ELISPOT and MHC-Dextramer assays.Results: No related adverse events greater than grade I were observed in the six patients enrolled in this study. No clinical responses were observed in the enrolled patients although vitiligo was observed after the vaccination in two patients. Promotion of peptide specific immune responses was observed in four patients with ELISPOT assay. Furthermore, a significant increase of CD8⁺ gp100-in4⁺ CTLs was observed in all patients using the MHC-Dextramer assay. Cytotoxic T lymphocytes (CTLs) clones specific to gp100-in4 were successfully established from the PBMC of some patients and these CTL clones were capable of lysing the melanoma cell line, 888 mel, which endogenously expresses HLA-restricted gp100-in4.Conclusion: Our results suggest this HLA-restricted gp100-in4 peptide vaccination protocol was well-tolerated and can induce antigen-specific T-cell responses in multiple patients. Although no objective anti-tumor effects were observed, the effectiveness of this approach can be enhanced with the appropriate modifications.</p>

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Cancer treatment by comprehensive regulation of anti-tumor immune network

Kawakami Yutaka

Nippon rinsho. Japanese journal of clinical medicine 68 ( 6 ) 1094 - 1099 2010.06

ISSN  0047-1852

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<p>Anti-tumor effects of current cancer vaccines are limited. However, detailed analyses of clinical trials revealed several important points to be improved towards immunological tumor rejection. These are, (1) identification of tumor antigens involved in tumor cell proliferation/survival and expressed in cancer stem cells, (2) development of in situ tumor destruction methods which release tumor antigens in an immunogenic manner to induce immune responses to multiple endogenous tumor antigens, (3) development of methods to augment antigen processing and presentation by dendritic cells, including effective adjuvants, (4) development of methods to expand CTL and helper T cells in vivo, (5) understanding of mechanisms underlying cancer induced immunosuppression and development of methods to overcome them. Comprehensive interventions on these points in the antitumor immune network will lead to effective cancer immunotherapy.</p>

Isolation of cancer stem-like cells from a side population of a human glioblastoma cell line, SK-MG-1

Fukaya Raita, Ohta Shigeki, Yamaguchi Masayuki, Fujii Hirofumi, Kawakami Yutaka, Kawase Takeshi, Toda Masahiro

Cancer Letters 291 ( 2 ) 150 - 157 2010.05

ISSN  0304-3835

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<p>Accumulating evidence suggests that in several types of brain tumors, including glioma, only a phenotypic subset of tumor cells called brain cancer stem cells (BCSCs) may be capable of initiating tumor growth. Recently, the isolation of side population (SP) cells using Hoechst dye has become a useful method for obtaining cancer stem cells in various tumors. In this study, we isolated cancer stem-like cells from human glioma cell lines using the SP technique. Flow cytometry analysis revealed that SK-MG-1, a human glioblastoma cell line, contained the largest number of SP cells among the five glioma cell lines that were analyzed. The SP cells had a self-renewal ability and were capable of forming spheres in a neurosphere culture medium containing EGF and FGF2. Spheres derived from the SP cells differentiated into three different lineage cells: neurons, astrocytes and oligodendrocytes. RT-PCR analysis revealed that the SP cells expressed a neural stem cell marker, Nestin. The SP cells generated tumors in the brains of NOD/SCID mice at 8 weeks after implantation, whereas the non-SP cells did not generate any tumors in the brain. These results indicate that SP cells isolated from SK-MG-1 possess the properties of cancer stem cells, including their self-renewal ability, multi-lineage differentiation, and tumorigenicity. Therefore, the SP cells from SK-MG-1 may be useful for analyzing BCSCs because of the ease with which they can be handled and their yield.</p>

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Targeted inhibition of IL-10-secreting CD25^- Treg via p38 MAPK suppression in cancer immunotherapy

Ohkusu-Tsukada Kozo, Toda Masahiro, Udono Heiichiro, Kawakami Yutaka, Takahashi Kimimasa

European Journal of Immunology 40 ( 4 ) 1011 - 1021 2010.04

ISSN  0014-2980

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<p>Cancer-induced immunotolerance mediated by inducible Treg (iTreg) is a major obstacle to cancer immunotherapy. In a basic study of immunotolerance, injection of an endogenous superantigen, i.e. the minor lymphocyte stimulatory (Mls)-1^a, into specific TCR Vβ8.1-Tg mice enabled generation of anergic CD25^- iTreg, the immunosuppressive function of which was maintained by IL-10 production via p38-MAPK activation. Interestingly, although p38-chemical inhibitor (p38-inhibitor) is capable of breaking CD25^- iTreg-induced immunotolerance, the p38-inhibitor had hardly any immunotolerance breaking effect when CD25⁺ Treg were present, suggesting that depletion of CD25⁺ Treg is necessary for p38-inhibitor to be effective. Peptide OVA_323-339 iv.-injection into its specific TCR-Tg (OT-II) mice also induced adaptive tolerance by iTreg. Peptide immunotherapy with p38-inhibitor after CD25⁺ Treg-depletion was performed in an OVA-expressing lymphoma E.G7-bearing tolerant model established by adoptive transfer of OT-II CD25^- iTreg, which resulted in suppression of tumor growth. Similarly, the antitumor immunity induced by peptide immunotherapy in colon carcinoma CT26-bearingmice, in which the number of IL-10-secreting iTreg is increased, was augmented by treatment with p38-inhibitor after CD25 ⁺ Treg-depletion and resulted in inhibition of tumor progression. These results suggest that simultaneous inhibition of two distinct Treg-functions may be important to the success of cancer immunotherapy.</p>

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Identification of HLA-A2- And A24-restricted T-cell epitopes derived from SOX6 expressed in glioma stem cells for immunotherapy

Ueda Ryo, Ohkusu-Tsukada Kozo, Fusaki Noemi, Soeda Akio, Kawase Takeshi, Kawakami Yutaka, Toda Masahiro

International Journal of Cancer 126 ( 4 ) 919 - 929 2010.02

ISSN  0020-7136

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<p>Malignant gliomas are the most aggressive human primary brain tumors and are currently incurable. Immunotherapies have the potential to target glioma and glioma stem cells (GSCs) that are resistant to conventional therapies. We previously identified SOX6 as a human glioma antigen and demonstrated that vaccination with SOX6 DNA induced cytotoxic T lymphocytes (CTLs) specific for glioma, thereby exerting therapeutic antitumor responses in glioma-bearing mice. In this study, we attempted to identify SOX6-derived peptides as specific targets for effective and safe T-cell-mediated immunotherapy targeting SOX6-positive glioma and GSCs. In vitro stimulation with human leukocyte antigen (HLA)-A*2402 (A24)-restricted peptides, RFENLGPQL (SOX6₅₀₄) and PYYEEQARL (SOX6₆₂₈) or the HLA-A*0201 (A2)-restricted peptide, ALFGDQDTV (SOX6₄₄₇) was capable of inducing SOX6 peptide-specific CTLs in peripheral blood mononuclear cells derived from healthy donors and glioma patients. These CTLs were able to lyse a majority of glioma cell lines and a GSC line derived from human glioblastoma in an HLA Class I-restricted and an antigen-dependent manner. Furthermore, peptide vaccines of SOX6₆₂₈, which was conserved in the murine SOX6 protein and expected to bind to major histocompatibility complex (MHC) H-2^d, induced CTLs specific for SOX6₆₂₈ in H-2^d mice. Normal autologous cells from mice, in which SOX6-specific immune responses were generated, were not destroyed. These results suggest that these SOX6 peptides are potnetially immunogenic in HLA-A24 or -A2 positive glioma patients and should be considered as a promising strategy for safe and effective T-cell-based immunotherapy of patients with gliomas.</p>

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Aberrant fatty acid-binding protein-7 gene expression in cutaneous malignant melanoma

Goto Yasufumi, Koyanagi Kazuo, Narita Norihiko, Kawakami Yutaka, Takata Minoru, Uchiyama Aya, Nguyen Linhda, Nguyen Tung, Ye Xing, Morton Donald L., Hoon Dave S B

Journal of Investigative Dermatology 130 ( 1 ) 221 - 229 2010.01

ISSN  0022-202X

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<p>Fatty acid-binding protein-7 (FABP7) has been shown to be expressed in cutaneous melanoma; however, its role in tumor progression is unclear. Expression of FABP7 was assessed during melanoma progression through assessment of various clinicopathology stages of primary tumor progression and metastasis. FABP7 mRNA was highly expressed in 60 of 87 (69%) primary melanomas, compared with significant (P0.0001) reduction in 13 of 68 (19%) metastatic melanomas. Analysis of 37 paired primary and metastatic melanomas by immunohistochemistry with anti-FABP7 Ab showed 73 and 27% positivity, respectively (P0.001). FABP7 detection of metastatic tissues was inversely correlated with relapse-free (P0.0001) and overall (P0.0001) survival. To examine FABP7 expression loss in advanced melanomas, loss of heterozygosity (LOH) was assessed using microsatellite markers encompassing the FABP7 gene. LOH was identified in 10 of 20 (50%) metastatic melanomas at 6q22.31, compared with 0 of 14 primary melanomas (P0.0017). FABP7 as a surrogate biomarker for circulating tumor cells (CTCs) in the blood was assessed by quantitative real-time (qRT)-PCR from melanoma patients blood (n134). Assessment of patients blood showed that FABP7() CTC decreased with disease progression. FABP7 may function as a tumor progression gene and can be used as a potential diagnostic biomarker of early-stage melanoma systemic spreading in blood.</p>

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The RNA silencing technology applied by lentiviral vectors in oncology.

Sumimoto Hidetoshi, Kawakami Yutaka

Methods in molecular biology (Clifton, N.J.) 614   187 - 199 2010

ISSN  1064-3745

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<p>Since the discovery of RNA interference (RNAi) in Caenorhabditis elegans in 1998, this mechanism has been found to be conserved in a wide variety of species, including insects, plants, and mammals. In mammals, small (or short) interfering RNA (siRNA) or short hairpin RNA (shRNA) can be expressed by using several expression vectors including lentiviral vectors. The lentiviral vector has several useful characteristics for RNAi experiments including broad host tropism and stable gene transduction to both dividing and nondividing cells, which permits stable depletion of target genes. This technology can be useful for several applications, including basic cancer research.</p>

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Simultaneous suppression of MITF and BRAF^V600E enhanced inhibition of melanoma cell proliferation

Kido Kenji, Sumimoto Hidetoshi, Asada Sakiyo, Okada Starlyn M., Yaguchi Tomonori, Kawamura Naoshi, Miyagishi Makoto, Saida Toshiaki, Kawakami Yutaka

Cancer Science 100 ( 10 ) 1863 - 1869 2009.10

ISSN  1347-9032

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<p>Microphthalmia-associated transcription factor (MITF) is a master gene regulating differentiation of melanocytes, and a lineage survival oncogene mediating pro-proliferative function in malignant melanoma. However, high expression of MITF also has an anti-proliferative effect. To clarify the therapeutic implication of MITF as a molecular target for human melanoma, we evaluated the role of MITF in cell proliferation in a panel of human melanoma cell lines which express different levels of MITF. We found that both MITF depletion and forced expression of MITF significantly inhibited proliferation, suggesting that endogenous MITF is regulated at an appropriate level for melanoma cell proliferation, and could be a molecular target for melanoma. However, half of the melanoma cell lines in this study were relatively resistant to MITF depletion, indicating other treatment strategies are required for therapy. Our microarray analysis indicated that regulation of several cell growth-associated molecules may be independent of MITF and dependent on BRAF^V600E. Thus to enhance the anti-proliferative effect of MITF down-regulation, we combined shRNA-mediated MITF depletion with BRAF^V600E inactivation, another known molecular target for melanoma. Indeed, simultaneous depletion of both MITF and BRAFV600E significantly inhibited melanoma growth even for the melanoma cell lines resistant to MITF depletion. These results suggest MITF may be an important molecular target for human melanoma and simultaneous inhibition of MITF and MAPK signaling may be an attractive strategy for melanoma treatment. (Cancer Sci 2009; 100: 1863-1869).</p>

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Impairment of plasmacytoid dendritic cells for IFN production by the ligand for immunoglobulin-like transcript 7 expressed on human cancer cells

Tsukamoto Nobuo, Okada Starlyn, Onami Yoko, Sasaki Yusuke, Umezawa Kazuo, Kawakami Yutaka

Clinical Cancer Research 15 ( 18 ) 5733 - 5743 2009.09

ISSN  1078-0432

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<p>Purpose: Plasmacytoid dendritic cells (pDC) are specialized cells to produce type I IFN. Infiltration of pDCs in cancer tissues that have impaired ability to produce IFN-α has been suggested to play immunosuppressive roles in tumor immunity. To identify potential mechanisms causing pDC impairment in the cancer microenvironment, expression of immunoglobulin-like transcript 7 ligands (ILT7L), which inhibits pDC production of type I IFNs on the surface of various human cancer and noncancer cells, was examined. Experimental Design: To detect unidentified ILT7L, reporter cells, which express green fluorescent protein on interaction with ILT7L, were constructed. ILT7L expression on various human cancer cell lines as well as various noncancerous stromal cells and immune cells was examined. Cytokines and signals involved in the ILT7L expression were also investigated. Results: ILT7L was detected on all of the various types of human cancer cell lines tested. IFN-α, IFN-β, IFN-γ, tumor necrosis factor-α, interleukin-1β, transforming growth factor-β, lipopolysaccharide, and imiquimod induced ILT7L expression on cancer and noncancer cells. High ILT7L-expressing cancer cells inhibited production of IFN-α and tumor necrosis factor-α by pDC stimulated with CpG. ILT7L does not appear to be a member of classic or nonclassic HLAs. Additionally, NF-κB and mammalian target of rapamycin are involved in regulating ILT7L expression. Conclusions: ILT7L expression on cancer cells may be one of the mechanisms for impairment of pDCs in the cancer microenvironment. ILT7/ILT7L signaling may normally enable a negative immune response feedback following viral infection. Intervention of the ILT7L/ILT7 system may be useful for enhancing antitumor immunity as well as antiviral immunity.</p>

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RNA helicase encoded by melanoma differentiation-associated gene 5 is a major autoantigen in patients with clinically amyopathic dermatomyositis

Sato Shinji, Hoshino Kana, Satoh Takashi, Fujita Tomonobu, Kawakami Yutaka, Fujita Takashi, Kuwana Masataka

Arthritis and Rheumatism 60 ( 7 ) 2193 - 2200 2009.07

ISSN  0004-3591

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<p>Objective. To identify the autoantigen recognized by the autoantibody that is associated with clinically amyopathic dermatomyositis (C-ADM) and rapidly progressive interstitial lung disease (ILD). Methods. An anti-CADM-140 antibody-positive prototype serum sample was used to screen a HeLa cell-derived complementary DNA (cDNA) library. Selected cDNA clones were further evaluated by immunoprecipitation of their in vitro-transcribed and in vitro-translated products using anti-CADM-140 antibody-positive and anti-CADM-140 antibody-negative sera. The lysates of COS-7 cells transfected with the putative antigen were similarly tested. An enzyme-linked immunosorbent assay (ELISA) to detect the anti-CADM-140 antibody was established using a recombinant CADM-140 antigen, and its specificity and sensitivity for C-ADM and rapidly progressive ILD were assessed in 294 patients with various connective tissue Results. By cDNA library screening and immunoprecipitation of in vitro-transcribed and in vitro-translated products, we obtained a cDNA clone encoding melanoma differentiation-associated gene 5 (MDA-5). The anti-CADM-140 antibodies in patients' sera specifically reacted with MDA-5 protein expressed in cells transfected with full-length MDA-5 cDNA, confirming the identity of MDA-5 as the CADM-140 autoantigen. The ELISA, using recombinant MDA-5 protein as the antigen, showed an analytical sensitivity of 85% and analytical specificity of 100%, in comparison with the "gold standard" immunoprecipitation assay, and was useful for identifying patients with C-ADM and/or rapidly progressive ILD. Conclusion. Given that RNA helicase encoded by MDA-5 is a critical molecule involved in the innate immune defense against viruses, viral infection may play an important role in the pathogenesis of C-ADM and rapidly progressive ILD. Moreover, our ELISA using recombinant MDA-5 protein makes detection of the anti-CADM-140 antibody routinely available.</p>

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Emerging concepts in biomarker discovery; The US-Japan workshop on immunological molecular markers in oncology

Tahara Hideaki, Sato Marimo, Thurin Magdalena, Wang Ena, Butterfield Lisa H., Disis Mary L., Fox Bernard A., Lee Peter P., Khleif Samir N., Wigginton Jon M., Ambs Stefan, Akutsu Yasunori, Chaussabel Damien, Doki Yuichiro, Eremin Oleg, Fridman Wolf Herevé, Hirohashi Yoshihiko, Imai Kohzoh, Jacobson James, Jinushi Masahisa, Kanamoto Akira, Kashani-Sabet Mohammed, Kato Kazunori, Kawakami Yutaka, Kirkwood John M., Kleen Thomas O., Lehmann Paul V., Liotta Lance, Lotze Michael T., Maio Michele, Malyguine Anatoli, Masucci Giuseppe, Matsubara Hisahiro, Mayrand-Chung Shawmarie, Nakamura Kiminori, Nishikawa Hiroyoshi, Karolina A. Karolina, Petricoin Emanuel F., Pos Zoltan, Ribas Antoni, Rivoltini Licia, Sato Noriyuki, Shiku Hiroshi, Slingluff Craig L., Streicher Howard, Stroncek David F., Takeuchi Hiroya, Toyota Minoru, Wada Hisashi, Wu Xifeng, Wulfkuhle Julia, Yaguchi Tomonori, Zeskind Benjamin, Zhao Yingdong, Zocca Mai Britt, Marincola Francesco M.

Journal of Translational Medicine 7 2009.06

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<p>Supported by the Office of International Affairs, National Cancer Institute (NCI), the "US-Japan Workshop on Immunological Biomarkers in Oncology" was held in March 2009. The workshop was related to a task force launched by the International Society for the Biological Therapy of Cancer (iSBTc) and the United States Food and Drug Administration (FDA) to identify strategies for biomarker discovery and validation in the field of biotherapy. The effort will culminate on October 28th 2009 in the "iSBTc-FDA-NCI Workshop on Prognostic and Predictive Immunologic Biomarkers in Cancer", which will be held in Washington DC in association with the Annual Meeting. The purposes of the US-Japan workshop were a) to discuss novel approaches to enhance the discovery of predictive and/or prognostic markers in cancer immunotherapy; b) to define the state of the science in biomarker discovery and validation. The participation of Japanese and US scientists provided the opportunity to identify shared or discordant themes across the distinct immune genetic background and the diverse prevalence of disease between the two Nations. Converging concepts were identified: enhanced knowledge of interferon-related pathways was found to be central to the understanding of immune-mediated tissue-specific destruction (TSD) of which tumor rejection is a representative facet. Although the expression of interferon-stimulated genes (ISGs) likely mediates the inflammatory process leading to tumor rejection, it is insufficient by itself and the associated mechanisms need to be identified. It is likely that adaptive immune responses play a broader role in tumor rejection than those strictly related to their antigen-specificity; likely, their primary role is to trigger an acute and tissue-specific inflammatory response at the tumor site that leads to rejection upon recruitment of additional innate and adaptive immune mechanisms. Other candidate systemic and/or tissue-specific biomarkers were recognized that might be added to the list of known entities applicable in immunotherapy trials. The need for a systematic approach to biomarker discovery that takes advantage of powerful high-throughput technologies was recognized; it was clear from the current state of the science that immunotherapy is still in a discovery phase and only a few of the current biomarkers warrant extensive validation. It was, finally, clear that, while current technologies have almost limitless potential, inadequate study design, limited standardization and cross-validation among laboratories and suboptimal comparability of data remain major road blocks. The institution of an interactive consortium for high throughput molecular monitoring of clinical trials with voluntary participation might provide cost-effective solutions.</p>

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Activation of dendritic-like cells and neural stem/progenitor cells in injured spinal cord by GM-CSF

Hayashi Kaori, Ohta Shigeki, Kawakami Yutaka, Toda Masahiro

Neuroscience Research 64 ( 1 ) 96 - 103 2009.05

ISSN  0168-0102

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<p>Previously, we demonstrated that implanted dendritic cells (DCs) in the injured spinal cord of adult mice exert a neurotrophic effect, resulting in the activation of endogenous neural stem/progenitor cells (NSPCs) and neurogenesis. Granulocyte-macrophage colony stimulating factor (GM-CSF), which is an essential cytokine for the generation of DCs from haematopoietic progenitor cells, has been shown to be beneficial for the treatment of spinal cord injury (SCI). In the present study, to evaluate the mechanisms underlying this therapeutic efficacy of GM-CSF, we investigated the effects of GM-CSF on the DC-like cells and NSPCs in the injured spinal cord. When GM-CSF was injected into the injured spinal cords of mice, the numbers of DC-like cells and activated microglia/macrophages around the lesion site increased, accompanied by an increase in BDNF expression. A significant increase in endogenous NSPCs was observed around the lesion site in the GM-CSF-treated mice compared with that in the controls. A neurosphere forming assay revealed that GM-CSF also induced the proliferation of NSPCs in vitro. Moreover, injection of GM-CSF into the lesion immediately after the SCI resulted in early recovery of the locomotor function of the injured mice. In conclusion, GM-CSF activated DC-like cells and NSPCs in the injured spinal cord, which was probably involved in its beneficial effects in cases of spinal cord injury.</p>

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Single nucleotide polymorphism of interleukin-1β associated with Helicobacter pylori infection in immune thrombocytopenic purpura

Satoh T., Pandey J. P., Okazaki Y., Asahi A., Kawakami Y., Ikeda Y., Kuwana M.

Tissue Antigens 73 ( 4 ) 353 - 357 2009.04

ISSN  0001-2815

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<p>To examine the role of genetic factors in development of immune thrombocytopenic purpura (ITP) in association with Helicobacter pylori infection, gene polymorphisms within the loci for human leukocyte antigen class II, interleukin (IL)-1β (-511), tumor necrosis factor-β (+252), immunoglobulin (Ig)G1 heavy chain (+643), and Igκ light chain (+573) were determined in 164 adults with ITP and 75 healthy controls. Of these gene polymorphisms, the IL-1β (-511) T allele was less frequently detected in H. pylori-infected than in H. pylori-uninfected (58% vs 81%, P = 0.01, odds ratio = 0.31) ITP patients diagnosed before age 50. These findings suggest that a single nucleotide polymorphism within the IL-1β (-511) may affect susceptibility to early-onset ITP associated with H. pylori infection.</p>

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Cancer Metastasis Is Accelerated through Immunosuppression during Snail-Induced EMT of Cancer Cells

Kudo-Saito Chie, Shirako Hiromi, Takeuchi Tadashi, Kawakami Yutaka

Cancer Cell 15 ( 3 ) 195 - 206 2009.03

ISSN  1535-6108

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<p>Epithelial-mesenchymal transition (EMT) is a key step toward cancer metastasis, and Snail is a major transcription factor governing EMT. Here, we demonstrate that Snail-induced EMT accelerates cancer metastasis through not only enhanced invasion but also induction of immunosuppression. Murine and human melanoma cells with typical EMT features after snail transduction induced regulatory T cells and impaired dendritic cells in vitro and in vivo partly through TSP1 production. Although Snail⁺ melanoma did not respond to immunotherapy, intratumoral injection with snail-specific siRNA or anti-TSP1 monoclonal antibody significantly inhibited tumor growth and metastasis following increase of tumor-specific tumor-infiltrating lymphocytes and systemic immune responses. These results suggest that inhibition of Snail-induced EMT could simultaneously suppress both tumor metastasis and immunosuppression in cancer patients.</p>

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Epithelial mesenchymal transition in human ocular chronic graft-versus-host disease

Ogawa Yoko, Shimmura Shigeto, Kawakita Tetsuya, Yoshida Satoru, Kawakami Yutaka, Tsubota Kazuo

American Journal of Pathology 175 ( 6 ) 2372 - 2381 2009

ISSN  0002-9440

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<p>Chronic graft-versus-host disease (cGVHD) of the ocular surface and lacrimal gland is a vision-threatening condition that occurs after allogeneic bone marrow transplantation. In this study, we used immunohistochemistry and electron microscopy to investigate whether epithelial mesenchymal transition (EMT) contributed to the pathogenesis of ocular cGVHD. We detected down-regulation of E-cadherin and translocation of β-catenin from the intercellular junction to the cytoplasm and nucleus of cGVHD conjunctival basal epithelia and lacrimal gland myoepithelia. Notable findings included expression of Snail, an inducer of EMT, in the nucleus of ocular cGVHD epithelia. The fibrosis markers heat shock protein 47, α-smooth muscle actin, and fibroblast specific protein-1 were overexpressed in ocular cGVHD epithelia. In addition, p63, a marker of conjunctival basal epithelia, was observed in the nuclei of subconjunctival cells beneath disrupted basal lamina. Disrupted basal lamina and the presence of altered collagen bundles were observed in the cytoplasm and beneath cGVHD epithelia. In contrast, these observations were rarely observed in the normal conjunctiva and in Sjögren's syndrome lacrimal gland epithelia. These findings together indicate that ocular cGVHD epithelia gain the mesenchymal phenotype and the capacity to migrate into the subepithelial stroma. Our findings suggest that EMT may be partially responsible for the conjunctival and lacrimal gland fibrosis found in patients with cGVHD.</p>

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Involvement of hyaluronan and its receptor CD44 with choroidal neovascularization

Mochimaru Hiroshi, Takahashi Eri, Tsukamoto Nobuo, Miyazaki Junichiro, Yaguchi Tomonori, Koto Takashi, Kurihara Toshihide, Noda Kousuke, Ozawa Yoko, Ishimoto Takatsugu, Kawakami Yutaka, Tanihara Hidenobu, Saya Hideyuki, Ishida Susumu, Tsubota Kazuo

Investigative Ophthalmology and Visual Science 50 ( 9 ) 4410 - 4415 2009

ISSN  0146-0404

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<p>PURPOSE. CD44 is a cell-surface adhesion molecule and receptor for hyaluronan (HA), one of the major extracellular matrix components. The purpose of the present study was to clarify a role of HA and CD44 in the development of choroidal neovascularization (CNV). METHODS. Laser photocoagulation was used to induce CNV in C57BL/6 mice or CD44-deficient mice. The mRNA expression of CD44 and HA synthase (HAS)-2 in the retinal pigment epithelium (RPE)-choroid complex was evaluated by DNA microarray and real-time RT-PCR analyses 3 days after laser treatment. HA synthesis and CD44 expression were examined by immunohistochemistry 1 week after photocoagulation. Mice with laser-induced CNV were systemically administered the HA synthesis inhibitor 4-methylumbelliferone (MU) or an anti- CD44-neutralizing antibody. The response of CNV was analyzed by volumetric measurements 1 week after photocoagulation. Macrophage infiltration into CNV lesions was evaluated by real-time RT-PCR for F4/80 3 days after laser-induced injury. RESULTS. The induction of CNV led to a significant increase in expression of CD44 and HAS2 mRNA. HA and CD44 were immunopositive in the CNV lesions. Compared with vehicle treatment, the systemic application of MU significantly attenuated CNV volume in a dose-dependent fashion, together with macrophage infiltration into the lesions. Consistently, antibody- based blockade of CD44 resulted in a significant reduction of CNV volume, compared with the isotype control. In contrast, genetic ablation of CD44 significantly augmented CNV formation together with HA accumulation and macrophage infiltration, compared with wild-type mice. CONCLUSIONS. These results indicate a significant role of HA and its receptor CD44 in the development of CNV. (Invest Ophthalmol Vis Sci.</p>

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Identification of a WT1 protein-derived peptide, WT1₁₈₇, as a HLA-A*0206-restricted, WT1-specific CTL epitope

Li Zheyu, Oka Yoshihiro, Tsuboi Akihiro, Fujiki Fumihiro, Harada Yukie, Nakajima Hiroko, Masuda Tomoki, Fukuda Yoko, Kawakatsu Mai, Morimoto Soyoko, Katagiri Takamasa, Tatsumi Naoya, Hosen Naoki, Shirakata Toshiaki, Nishida Sumiyuki, Kawakami Yutaka, Udaka Keiko, Kawase Ichiro, Oji Yusuke, Sugiyama Haruo

Microbiology and Immunology 52 ( 11 ) 551 - 558 2008.11

ISSN  0385-5600

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<p>The Wilms' tumor gene WT1 is overexpressed in various kinds of hematopoietic malignancies as well as solid cancers, and this protein has been demonstrated to be an attractive target antigen for cancer immunotherapy. WT1-specific CTL epitopeswith a restriction of HLA-A*2402 or HLA-A*0201 have been already identified. In the present study it has been demonstrated that a 9-mer WT1-derived WT1₁₈₇ peptide, which had already been shown to elicit a WT1-specific CTL response with a restriction of HLA-A*0201, can also elicit a CTL response with a restriction of HLA-A*0206. In all three different HLA-A*0206⁺ healthy donors examined, WT1187 peptide-specific CTL could be generated from peripheral blood mononuclear cells, and the CTL showed cytotoxic activity that depended on dual expression of WT1 and HLA-A*0206 molecules. The present study describes the first identification of a HLA-A*0206-restricted, WT1-specific CTL epitope. The present results should help to broaden the application of WT1 peptide-based immunotherapy fromonly HLA-A*0201- positive to HLA-A*0206-positive cancer patients as well.</p>

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Helicobacter pylori eradication shifts monocyte Fcγ receptor balance toward inhibitory FcγRIIB in immune thrombocytopenic purpura patients

Asahi Atsuko, Nishimoto Tetsuya, Okazaki Yuka, Suzuki Hidekazu, Masaoka Tatsuhiro, Kawakami Yutaka, Ikeda Yasuo, Kuwana Masataka

Journal of Clinical Investigation 118 ( 8 ) 2939 - 2949 2008.08

ISSN  0021-9738

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<p>Immune thrombocytopenia purpura (ITP) is a bleeding disorder in which platelet-specific autoantibodies cause a loss of platelets. In a subset of patients with ITP and infected with Helicobacter pylori, the number of platelets recovers after eradication of H. pylori. To examine the role of H. pylori infection in the pathogenesis of ITP, the response of 34 ITP patients to treatment with a standard H. pylori eradication regimen, irrespective of whether they were infected with H. pylori, was evaluated. Eradication of H. pylori was achieved in all H. pylori-positive patients, and a significant increase in platelets was observed in 61% of these patients. By contrast, none of the H. pylori-negative patients showed increased platelets. At baseline, monocytes from the H. pylori-positive patients exhibited an enhanced phagocytic capacity and low levels of the inhibitory Fcγ receptor IIB (FcγRIIB). One week after starting the H. pylori eradication regimen, this activated monocyte phenotype was suppressed and improvements in autoimmune and platelet kinetic parameters followed. Modulation of monocyte FcγR balance was also found in association with H. pylori infection in individuals who did not have ITP and in mice. Our findings strongly suggest that the recovery in platelet numbers observed in ITP patients after H. pylori eradication is mediated through a change in FcγR balance toward the inhibitory FcγRIIB.</p>

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Novel system evaluating in vivo pathogenicity of desmoglein 3-reactive T cell clones using murine pemphigus vulgaris

Takahashi Hayato, Amagai Masayuki, Nishikawa Takeji, Fujii Yoshiko, Kawakami Yutaka, Kuwana Masataka

Journal of Immunology 181 ( 2 ) 1526 - 1535 2008.07

ISSN  0022-1767

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<p>Autoreactive T cells are thought to be involved in the pathogenesis of autoimmune diseases, but evidence for their direct pathogenicity is almost lacking. Herein we established a unique system for evaluating the in vivo pathogenicity of desmoglein 3 (Dsg3)-reactive T cells at a clonal level in a mouse model for pemphigus vulgaris (PV), an autoimmune blistering disease induced by anti-Dsg3 autoantibodies. Dsg3-reactive CD4⁺ T cell lines generated in vitro were adoptively transferred into Rag-2^-/- mice with primed B cells derived from Dsg3-immunized Dsg3^-/- mice. Seven of 20 T cell lines induced IgG anti-Dsg3 Ab production and acantholytic blister, a typical disease phenotype, in recipient mice. Comparison of the characteristics between pathogenic and nonpathogenic Dsg3-reactive T cell lines led to the identification of IL-4 and IL-10 as potential factors associated with pathogenicity. Further in vitro analysis showed that IL-4, but not IL-10, promoted IgG anti-Dsg3 Ab production by primed B cells. Additionally, adenoviral expression of soluble IL-4Rα in vivo suppressed IgG anti-Dsg3 Ab production and the PV phenotype, indicating a pathogenic role of IL-4. This strategy is useful for evaluating the effector function of autoreactive T cells involved in the pathogenesis of various autoimmune diseases.</p>

Preferential activation of circulating CD8⁺ and γδ T cells in patients with active Behçet's disease and HLA-B51

Yasuoka Hidekata, Yamaguchi Yukie, Mizuki Nobuhisa, Nishida Tomomi, Kawakami Yutaka, Kuwana Masataka

Clinical and Experimental Rheumatology 26 ( 4 SUPPL. 50 )  2008.07

ISSN  0392-856X

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<p>Objective. To evaluate the activation status of circulating CD4⁺, CD8⁺, and γδ T cells in patients with active and inactive Behçet's disease (BD). Methods. We studied 11 subjects with active BD, 28 with inactive BD, and 13 healthy controls. The expression of CD4, CD8, pan-γd, Vδ1, and Vδ2 along with the early activation marker CD69 was analyzed by 3-color flow cytometry. Results. Proportions of activated CD8⁺ and γδ T cells were significantly greater in patients with active BD than in those with inactive BD or healthy control subjects, but the proportion of activated CD4⁺ T cells did not differ among these 3 groups. In addition, significantly greater proportions of the Vδ1⁺ and Vδ2⁺ γδ T-cell subsets were activated in patients with active BD than in those with inactive BD or healthy controls; in active BD, the balance of activation between these subsets favored the Vδ1⁺ T cells. No significant differences in these proportions were found between subjects with inactive BD and healthy controls. These findings were observed exclusively in patients with HLA-B51. A comparison of samples from 5 patients taken during active BD and after resolution of BD-related symptoms showed the proportions of activated CD8⁺ and γδ T cells dropped when the patients' BD became inactive. Conclusion. CD8⁺ and γδ T cells, rather than CD4⁺ T cells, were activated in vivo in patients with active BD and HLA-B51, but not in those with inactive BD, suggesting that these potentially cytotoxic T cells play a critical role in BD flares.</p>

Functional recovery after spinal cord injury in mice through activation of microglia and dendritic cells after IL-12 administration

Yaguchi Masae, Ohta Shigeki, Toyama Yoshiaki, Kawakami Yutaka, Toda Masahiro

Journal of Neuroscience Research 86 ( 9 ) 1972 - 1980 2008.07

ISSN  0360-4012

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<p>We have previously reported that the transplantation of dendritic cells (DCs) brings about functional recovery after spinal cord injury in mice through the activation of endogenous microglia/macrophages and neural stem/progenitor cells. In this study, the effect of interleukin-12 (IL-12), which is secreted from DCs, was evaluated for the treatment of spinal cord injury in mice. Administration of IL-12 into the injured site significantly increased the number of activated microglia/macrophages and DCs as well as the expression of brain-derived neurotrophic factor surrounding the lesion site. Immunohistochemical analyses showed that de novo neurogenesis and remyelination were induced by IL-12 treatment. Furthermore, an open field test using Basso-Beattie-Brenham scoring revealed a significant improvement of locomotor function in mice treated with IL-12. These results suggest that IL-12 administration into the injured spinal cord results in a functional recovery through the activation of microglia/macrophages and DCs.</p>

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Induction of protective and therapeutic antitumor immunity by a DNA vaccine with a glioma antigen, SOX6

Ueda Ryo, Kinoshita Eiko, Ito Rena, Kawase Takeshi, Kawakami Yutaka, Toda Masahiro

International Journal of Cancer 122 ( 10 ) 2274 - 2279 2008.05

ISSN  0020-7136

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<p>We previously reported identifying SOX6 as a glioma antigen by serological screening using a testis cDNA library. Its preferential expression and frequent IgG responses in glioma patients indicate that SOX6 may be a useful target for immunotherapy. To examine whether cytotoxic T-lymphocyte (CTL) responses specific for SOX6 to destroy glioma can be generated in vivo, we treated glioma-bearing mice by vaccination with a plasmid DNA encoding murine full-length SOX6 protein. Following SOX6-DNA vaccination, CTLs specific for SOX6-expressing glioma cells were induced, while normal autologous-cells that had restrictedly expressed SOX6 during embryogenesis were not destroyed. Furthermore, DNA vaccination with SOX6 exerted protective and therapeutic antitumor responses in the glioma-bearing mice. This antitumor activity was abrogated by the depletion of CD4 positive T cells and/or CD8 positive T cells. These results suggest that the SOX6 protein has multiple CTL and helper epitopes to induce antitumor activity and the effectiveness of SOX6-DNA vaccine for the prevention and treatment of glioma.</p>

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Isolation and characterization of dendritic cells from common marmosets for preclinical cell therapy studies

Ohta Shigeki, Ueda Yoko, Yaguchi Masae, Matsuzaki Yumi, Nakamura Masaya, Toyama Yoshiaki, Tanioka Yoshikuni, Tamaoki Norikazu, Nomura Tatsuji, Okano Hideyuki, Kawakami Yutaka, Toda Masahiro

Immunology 123 ( 4 ) 566 - 574 2008.04

ISSN  0019-2805

　View Summary

<p>Dendritic cells (DCs) have important functions as modulators of immune responses, and their ability to activate T cells is of great value in cancer immunotherapy. The isolation of DCs from the peripheral blood of rhesus and African green monkeys has been reported, but the immune system in the common marmoset remains poorly characterized, although it offers many potential advantages for preclinical studies. In the present study, we devised methods, based on techniques developed for mouse and human DC preparation, for isolating DCs from three major tissue sources in the common marmoset: bone marrow (BM), spleen and peripheral blood. Each set of separated cells was analysed using the cell surface DC-associated markers CD11c, CD80, CD83, CD86 and human leucocyte antigen (HLA)-DR, all of which are antibodies against human antigens, and the cells were further characterized both functionally and morphologically as antigen-presenting cells. BM proved to be an excellent cell source for the isolation of DCs intended for preclinical studies on cell therapy, for which large quantities of cells are required. In the BM-derived CD11c⁺ cell population, cells exhibiting the characteristic features of DCs were enriched, with the typical DC morphology and the abilities to undergo endocytosis, to secrete interleukin (IL)-12, and to stimulate Xenogenic T cells. Moreover, BM-derived DCs produced the neurotrophic factor NT-3, which is also found in murine splenic DCs. These results suggest that BM-derived DCs from the common marmoset may be useful for biological analysis and for preclinical studies on cell therapy for central nervous system diseases and cancer.</p>

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Suppression of alkali burn-induced corneal neovascularization by dendritic cell vaccination targeting VEGF receptor 2

Mochimaru Hiroshi, Usui Tomohiko, Yaguchi Tomonori, Nagahama Yasuharu, Hasegawa Go, Usui Yoshihiko, Shimmura Shigeto, Tsubota Kazuo, Amano Shiro, Kawakami Yutaka, Ishida Susumu

Investigative Ophthalmology and Visual Science 49 ( 5 ) 2172 - 2177 2008.04

ISSN  0146-0404

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<p>Purpose. To investigate whether the induction of cytotoxic T lymphocytes (CTLs) targeting VEGF receptor 2 inhibits corneal neovascularization caused by alkali injury. Methods. H-2Db-restricted peptide corresponding to amino acids 400 to 408 of VEGF receptor 2 (VEGFR2_400-408) was used as an epitope peptide. Dendritic cells (DCs) were harvested from bone marrow progenitors of C57BL/6 mice. Six- week-old C57BL/6 mice received subcutaneous injections of VEGFR2_400-408- or gp70-pulsed mature DCs three times at 6-day intervals. After the third immunization, corneal neovas- cularization was induced by alkali injury. Two weeks after the injury, the corneal vascularized area was evaluated by lectin angiography. To confirm the peptide-specific CTL activities in C57BL/6 mice, CD8⁺ T cells from immunized mice were subjected to ELISA for interferon (IFN)-γ and tumor necrosis factor (TNF)-α production and ⁵¹Cr-release cytotoxicity assay. To determine the in vivo effector T cells, the immunized mice were intraperitoneally injected with an anti-CD4 or -CD8 depletion antibody. Results. Corneal neovascularization was significantly attenuated in mice immunized with VEGFR2_400-408 compared with those not immunized or immunized with gp70. VEGFR2_400-408 or gp70, but not β-gal_96-103 application led to dose-dependent induction of IFN-γ and TNF-α in the CD8⁺ T cells cocultured with stimulator cells. Cytotoxicity assays showed the specific lysis of major histocompatibility complex-matched cells expressing VEGFR2, but not β-gal_96-103. In vivo depletion of CD8⁺, but not CD4⁺, T cells significantly reversed the suppressive effect of VEGFR2_400-408 immunization on corneal neovascularization to the level observed in nonimmunized or gp70-immunized animals. Conclusions. These results indicate the possibility of DC vaccination targeting VEGFR2 as a novel therapeutic strategy for corneal chemical injury.</p>

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Dendritic cell based personalized immunotherapy based on cancer antigen research

Kawakami Yutaka, Fujita Tomonobu, Kudo Chie, Sakurai Toshiharu, Udagawa Masaru, Yaguchi Tomonori, Hasegawa Go, Hayashi Emiko, Ueda Yoko, Iwata Tomoko, Wang Qinfu, Okada Starlyn, Tsukamoto Nobuo, Matsuzaki Yuriko, Sumimoto Hidetoshi

Frontiers in Bioscience 13 ( 5 ) 1952 - 1958 2008

ISSN  1093-9946

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<p>Human tumor antigens were identified using various immunological and genetic methods, and immune responses to the identified antigens were evaluated in cancer patients. Autologous tumor specific unique antigens derived from genetic alterations in cancer cells were isolated from patients with favorable prognosis after immunotherapy, indicating that they are attractive targets for immunotherapy. Immunogenicity of shared antigens was found to differ among patients due to antigen expression in cancer cells and patients' immunoreactivity. These observations suggest that personalization may be applied for cancer immunotherapy. We therefore developed intratumoral DC administration protocols that are able to induce immune responses to both unique and shared tumor antigens expressed in each individual cancer. By combining cryoablative tumor pretreatment and TLR stimulated DC, the anti-tumor effect of the intratumoral DC administration was significantly augmented in a murine tumor model. This improved protocol enhanced systemic induction of anti-tumor CD8 ⁺ CTL, and was able to regress relatively large remote untreated tumors. In clinical trials, systemic immune induction was observed by intraturmoral DC administration following cryoablative tumor treatment, although anti-tumor effects are relatively weak, indicating that additional interventions are required for more effective immunotherapy.</p>

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Autoreactive T-cell responses to myeloperoxidase in patients with antineutrophil cytoplasmic antibody-associated vasculitis and in healthy individuals

Seta Noriyuki, Tajima Michiko, Kobayashi Shigeto, Kawakami Yutaka, Hashimoto Hiroshi, Kuwana Masataka

Modern Rheumatology 18 ( 6 ) 593 - 600 2008

ISSN  1439-7595

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<p>The aim of this study was to evaluate the characteristics of autoreactive T cells to myeloperoxidase (MPO) in patients with MPO-antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. Peripheral blood T cells from 15 patients with MPO-ANCA-associated vasculitis and 14 healthy individuals were cultured with three recombinant proteins that together comprised the entire MPO sequence (L, all 112 amino acids (AA) of the light chain; HI, AA 1-227 of the heavy chain; HII, AA 212-467 of the heavy chain), and the antigen-specific T-cell proliferative response was measured by ³H-thymidine incorporation. T-cell responses to MPO-L and HI were both detected in four patients and three healthy donors, and responses to MPO-HII were detected in four patients and seven healthy donors. These findings indicate that at least three independent T-cell epitopes exist on the MPO molecule. Interestingly, the patients whose T cells showed these MPO-induced responses were mainly in remission. Peripheral blood T cells reactive with MPO were primarily of the HLA-DR-restricted CD4⁺ phenotype. In summary, we successfully used recombinant MPO fragments to detect autoreactive CD4⁺ T cells to multiple MPO epitopes in blood samples from patients with MPO-ANCA-associated vasculitis and healthy individuals.</p>

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A pilot study of human interferon β gene therapy for patients with advanced melanoma by in vivo transduction using cationic liposomes

Matsumoto Kazuhiko, Kubo Hitomi, Murata Hiroshi, Uhara Hisashi, Takata Minoru, Shibata Shinichi, Yasue Satoshi, Sakakibara Akihiro, Tomita Yasushi, Kageshita Toshiro, Kawakami Yutaka, Mizuno Masaaki, Yoshida Jun, Saida Toshiaki

Japanese Journal of Clinical Oncology 38 ( 12 ) 849 - 856 2008

ISSN  0368-2811

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<p>Background: Cationic liposomes containing the human interferon β (HuIFNβ) gene (IAB-1) was used for the clinical trial for glioma patients. HuIFNβ gene therapy showed much higher anti-tumor activity compared with the administration of HuIFNβ protein for melanoma. These results suggest that HuIFNβ gene therapy is an attractive strategy for the treatment of melanoma. Methods: Stage IV or III melanoma patients with cutaneous or subcutaneous metastatic lesions were enrolled in this pilot study. IAB-1 was dissolved by sterile PBS at a concentration of 30 μg DNA/ml and was injected into cutaneous or subcutaneous metastatic nodules three times a week for 2 weeks and the effect on the injected and non-injected metastatic lesions was evaluated. Results: Clinical responses were as follows (five patients): mixed response (MR) and no change in each one patient, and progressive disease in three patients. In the MR patient, the IAB-1 injected lesion disappeared clinically and histopathologically and one-half of IAB-1 non-injected skin metastases were transiently inflamed and mostly regressed. In the responded non-injected lesions of this patient, histopathologically, infiltration of CD4 positive T cells was observed around the melanoma cells in the dermis, which expressed the HLA-Class II antigen. Adverse events due to this gene therapy were not recognized in any of the patients. Conclusions: The efficacy of this gene therapy was generally insufficient; however, some immunological responses were recognized in one patient. No adverse events were observed. HuIFNβ gene therapy could be an attractive strategy for treatment of a variety of malignancies, including melanoma, though some modifications should be required.</p>

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Identification of a neuron-specific human gene, KIAA1110, that is a guanine nucleotide exchange factor for ARF1

Hattori Yujiro, Ohta Shigeki, Hamada Kenji, Yamada-Okabe Hisafumi, Kanemura Yonehiro, Matsuzaki Yumi, Okano Hideyuki, Kawakami Yutaka, Toda Masahiro

Biochemical and Biophysical Research Communications 364 ( 4 ) 737 - 742 2007.12

ISSN  0006-291X

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<p>To identify neuron-specific genes, we performed gene expression profiling, cDNA microarray and in silico ESTs (expressed sequence tags) analyses. We identified a human neuron-specific gene, KIAA1110 (homologue of rat synArfGEF (Po)), that is a member of the guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF). RT-PCR analysis showed that the KIAA1110 gene was expressed specifically in the brain among adult human tissues, whereas no apparent expression was observed in immature neural tissues/cells, such as fetal brain, glioma tissues/cells, and neural stem/precursor cells (NSPCs). The KIAA1110 protein was shown to be expressed in mature neurons but not in undifferentiated NSPCs. Immunohistochemical analysis also showed that KIAA1110 was expressed in neurons of the human adult cerebral cortex. Furthermore, the pull-down assay revealed that KIAA1110 has a GEF activity toward ARF1 that regulates transport along the secretion pathway. These results suggest that KIAA1110 is expressed specifically in mature neurons and may play an important role in the secretion pathway as a GEF for ARF1.</p>

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Lentiviral vector-mediated RNAi and its use for cancer research

Sumimoto Hidetoshi, Kawakami Yutaka

Future Oncology 3 ( 6 ) 655 - 664 2007.12

ISSN  1479-6694

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<p>RNAi is a useful tool for functional analysis of genes and developing a potential therapeutic strategy for various diseases including cancers. RNAican be applied in various forms. HIV vectors are useful for the stable transduction of genes to both replicating and quiescent cells with a broad host tropism, and have been developed for basic and clinical research of gene therapy. HIV vectors can deliver shRNAs for post-transcriptional silencing of specific genes with high efficiency, and have been used to evaluate various genes far their potential involvement in cancer development and malignant features, and may be useful for future cancer gene therapy. Here we describe the development of shRNA-expressing HIV vectors and their use in cancer research, as well as perspectives for their future use in cancer gene therapy.</p>

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Dendritic cells transduced with autoantigen FCRLA induce cytotoxic lymphocytes and vaccinate against murine B-cell lymphoma

Inozume Takashi, Mitsui Hiroshi, Okamoto Takashi, Matsuzaki Yuriko, Kawakami Yutaka, Shibagaki Naotaka, Shimada Shinji

Journal of Investigative Dermatology 127 ( 12 ) 2818 - 2822 2007.12

ISSN  0022-202X

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<p>We previously reported early evidence of the human Fc receptor-like A (hFCRLA), an antigen (Ag) that was specifically expressed in melanocytes, melanoma cells, and some B-cell states, and was recognized by IgG antibodies from melanoma patients. Recently, it has been demonstrated that hFCRLA is expressed in most human B-cell lymphoma tissues. In this report, we investigated the potential of FCRLA as a tumor-associated Ag of B-cell lymphoma for immunotherapy. We confirmed that murine FCRLA (mFCRLA) was expressed and distributed in murine tissues similar to hFCRLA. Recombinant mFCRLA fusion protein was constructed with a polyarginine (R9)-protein-transduction domain (PTD) (rR9-HA-mFCRL), and was transduced into bone marrow-derived dendritic cells (DC) ex vivo. Mice immunized with rR9-HA-mFCRL-treated DC primed cytotoxic T-lymphocyte (CTL) that killed the B-cell lymphoma cell line (A20), which express mFCRLA abundantly. In a tumor challenging study, A20 tumor growth inoculated in skin was significantly suppressed in mice vaccinated with rR9-HA-mFCRL-treated DC, compared with control mice. These results indicated that FCRLA is a potential target Ag in immunotherapy for B-cell lymphoma. In addition, our experimental system using R9-PTD-containing full-length proteins might be a useful method to analyze the immunogenicity of novel candidates of tumor-associated Ags in vivo.</p>

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Identification of a novel cancer-testis antigen CRT2 frequently expressed in various cancers using representational differential analysis

Hayashi Emiko, Matsuzaki Yuriko, Hasegawa Go, Yaguchi Tomonori, Kurihara Sachiko, Fujita Tomonobu, Kageshita Toshiro, Sano Makoto, Kawakami Yutaka

Clinical Cancer Research 13 ( 21 ) 6267 - 6274 2007.11

ISSN  1078-0432

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<p>Purpose: Cancer-testis antigens are promising targets for cancer immunotherapy. Identification of additional cancer-testis antigens with frequent expression in various cancers was attempted using representational differential analysis (RDA) and immunogenicity evaluation. Experimental Design: cDNAs preferentially expressed in testis were enriched using RDA by subtraction between testis and normal tissues. Thirty clones showing cancer-testis - like expression based on EST database analysis were evaluated by reverse transcription-PCR. A potential antigen, CRT2, was identified and its expression was analyzed with a newly generated anti-CRT2 antibody. The immunogenicity of CRT2 was examined based on reactivity with serum immunoglobulin G (IgG) from cancer patients, using Western blot and ELISA analysis, and on in vitro induction of tumor-reactive CTLs from HLA-A24 transgenic mice and human peripheral blood lymphocytes. Results: CRT2 was expressed in elongated spermatids of testis among normal tissues and in various cancer cell lines and tissues. The recombinant CRT2 protein was recognized by serum IgG from patients with various cancers in Western blot and ELISA analyses. ACRT2-derived peptide was identified as an HLA-A24-restricted T-cell epitope that induced tumor-reactive CTLs. Conclusion: CRT2 was identified as a new cancer-testis antigen expressed in elongated spermatids of testis and in cancer tissues (particularly melanoma) that is recognized by serum IgG from cancer patients. An HLA-A24-restricted T-cell epitope capable of inducing tumor-reactive CTLs was identified, suggesting that CRT2 may be useful for cancer diagnosis and immunotherapy.</p>

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Suppression of choroidal neovascularization by dendritic cell vaccination targeting VEGFR2

Mochimaru Hiroshi, Nagai Norihiro, Hasegawa Go, Kudo-Saito Chie, Yaguchi Tomonori, Usui Yoshihiko, Kurihara Toshihide, Koto Takashi, Satojuka Shingo, Shinoda Hajime, Ozawa Yoko, Tsubota Kazuo, Kawakami Yutaka, Ishida Susumu

Investigative Ophthalmology and Visual Science 48 ( 10 ) 4795 - 4801 2007.10

ISSN  0146-0404

　View Summary

<p>PURPOSE. To investigate whether the induction of cellular immunity against vascular endothelial growth factor receptor (VEGFR) 2 inhibits the development of choroidal neovascularization (CNV). METHODS. H-2Db-restricted peptide corresponding to amino acids 400 to 408 of VEGFR2 was used as an epitope peptide. Dendritic cells (DCs) were harvested from bone marrow progenitors of C57BL/6 mice. Six-week-old C57BL/6 mice received subcutaneous injections of the epitope peptide-pulsed mature DCs three times at 6-day intervals. After the third immunization, laser photocoagulation was performed to induce CNV. One week after photocoagulation, mice were killed to harvest the choroid and splenocytes. CNV volume was evaluated by volumetric measurements. To confirm the specific immunogenicity of the epitope peptides in C57BL/6 mice, CD8 T cells isolated from harvested splenocytes were restimulated to measure interferon (IFN)-γ and tumor necrosis factor (TNF)-α production through enzyme-linked immunospot assay and ELISA. To determine the T-cell subset responsible for the immunotherapy, mice were intraperitoneally injected with an anti-CD4 or anti-CD8 depletion antibody. RESULTS. CNV volume was significantly lower in mice immunized with the VEGFR2 epitope peptide than in those not immunized or immunized with a control peptide gp70. Cytokine assays showed the peptide-specific production of IFN-γ and TNF-α from the CD8 T cells in a dose-dependent manner. In vivo depletion of CD8, but not CD4, T cells significantly reversed the suppressive effect of the VEGFR2 peptide-pulsed DC vaccination on CNV to the level observed in nonimmunized or gp70-immunized animals. CONCLUSIONS. These results indicate that the VEGFR2 peptide-specific induction of cellular immunity inhibits CNV through the cytotoxicity of CD8 T cells. Results of the present study suggested the possibility of DC vaccination targeting VEGFR2 as a novel therapeutic strategy for CNV.</p>

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The possibility and limitations of cell therapy. 2) The current status and overview of tumor-immune cell therapy--T cell-dendritic cell therapy

Kawakami Yutaka

Nihon Naika Gakkai zasshi. The Journal of the Japanese Society of Internal Medicine 96 ( 9 ) 1923 - 1928 2007.09

ISSN  0021-5384

Clinical significance of serum p53 antibodies in patients with ulcerative colitis and its carcinogenesis

Yoshizawa Shigeo, Matsuoka Katsuyoshi, Inoue Nagamu, Takaishi Hiromasa, Ogata Haruhiko, Iwao Yasushi, Mukai Makio, Fujita Tomonobu, Kawakami Yutaka, Hibi Toshifumi

Inflammatory Bowel Diseases 13 ( 7 ) 865 - 873 2007.07

ISSN  1078-0998

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<p>Background: For early detection of ulcerative colitis (UC)-associated colorectal cancer (CRC), surveillance colonoscopy is recommended in UC patients at high risk. However, poor acceptability deteriorates its effectiveness and a suitable marker for selecting patients at high risk is needed. Here we evaluated clinical usefulness of the measurement of anti-p53 antibodies (Abs) by enzyme-linked immunosorbent assay (ELISA) using sera samples from UC patients. Methods: Sera from 286 patients with UC, 82 patients with sporadic CRC, and 63 healthy controls (HC) were obtained. Serum anti-p53 antibodies were detected with ELISA. Immunohistochemical detection was also performed in patients who developed dysplasia or CRC. Results: Serum p53 Ab was positive in 15.0% of UC, while it was positive only in 1.6% of HCs. In sporadic CRCs, 52.4% of 82 patients were positive. In UC patients with disease duration equal to or longer than 8 years, positivity of serum p53 Ab was significantly higher than those in patients with shorter duration. Eight of 13 (61.5%) UC patients with CRC or dysplasia were positive for serum p53 Abs, which was significantly higher than that in patients without neoplasia. All UC patients with CRC were positive for p53 staining, while 2 were negative for serum p53 Ab. Finally, levels of serum p53 Ab had fallen in 4 patients with CRC we could monitor after surgery. Conclusions: This study revealed that p53 Ab developed in the progression of UC-associated CRC but not in all patients with neoplasia, suggesting that serological detection of p53 Abs by ELISA is not suitable in primarily selecting patients at high risk; however, it is helpful in salvaging patients who drop from a surveillance program.</p>

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Preferential expression and frequent IgG responses of a tumor antigen, SOX5, in glioma patients

Ueda Ryo, Yoshida Kazunari, Kawase Takeshi, Kawakami Yutaka, Toda Masahiro

International Journal of Cancer 120 ( 8 ) 1704 - 1711 2007.04

ISSN  0020-7136

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<p>We previously reported to identify SOX5 as a glioma antigen by serological screening using a testis cDNA library. The present study was designed to analyze SOXS expression, its immunoreactivity, and the correlation between SOX5 IgG responses and clinical features in glioma patients to evaluate the possibility of its use as a diagnostic marker. Quantitative RT-PCR and Western blot analysis revealed that SOX5 was expressed in glioma tissues, but not in normal adult tissues, except in the testis. An immunohistochemical analysis showed that SOX5 was expressed in glioma cells, but only a few SOX5-positive cells were detected in non-neoplastic tissues from the cerebral cortex. IgG antibodies against SOX5 were detected in sera from 8 of the 27 glioma patients (27.6%), 0 of the 14 patients with other brain diseases (0%), 1 of the 54 other cancer patients (1.9%) and 1 of the 37 healthy individuals (2.7%). Patients with glioblastotna (GBM) who showed IgG responses against SOX5 exhibited significantly better survival periods than GBM patients without SOX5 antibodies. In summary, SOX5 is aberrantly expressed in glioma and can be recognized as a glioma antigen using IgGs from the sera of glioma patients. Furthermore, there is a statistically significant correlation between the presence of SOX5 IgGs and survival in GBM patients, suggesting that the glioma antigen SOX5 may be useful not only as a diagnostic marker, but also as a prognostic marker in glioma patients.</p>

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Role of heat shock protein 47, a collagen-binding chaperone, in lacrimal gland pathology in patients with cGVHD

Ogawa Yoko, Razzaque Mohammed S., Kameyama Kaori, Hasegawa Go, Shimmura Shigeto, Kawai Masataka, Okamoto Shinichiro, Ikeda Yasuo, Tsubota Kazuo, Kawakami Yutaka, Kuwana Masataka

Investigative Ophthalmology and Visual Science 48 ( 3 ) 1079 - 1086 2007.03

ISSN  0146-0404

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<p>PURPOSE. Uncontrolled fibrosis due to excessive accumulation of extracellular matrix proteins in the lacrimal glands of patients with chronic graft-versus-host disease (cGVHD) is well documented. Heat-shock protein 47 (HSP47) is involved in the molecular maturation of collagen and has been shown to have a fibrogenic role in various fibrotic diseases. In this study, the role of HSP47 in the pathogenesis of lacrimal gland of patients with cGVHD was investigated. METHODS. The expression of HSP47, Ki67 (a proliferation marker), types I and III collagen, and α-smooth muscle actin (α-SMA) was examined in tissue sections and in primary cultures of fibroblasts obtained from the lacrimal glands of patients with cGVHD (n = 8) and Sjögren's syndrome (SS; n = 7). RESULTS. Tissue sections of the lacrimal glands of patients with cGVHD showed markedly increased expression of HSP47 in fibroblasts around the medium-sized ducts than did those from patients with SS. The elevated expression of HSP47 in patients with cGVHD was mostly detected in Ki67-positive fibroblasts and was associated with increased accumulation of types I and III collagen in and around the fibrotic areas. Primary fibroblast cultures generated from cGVHD lacrimal gland showed higher HSP47 mRNA expression than did fibroblasts isolated from SS biopsy tissue, as determined by RT-PCR (P &lt; 0.05). In contrast, α-SMA was higher in the SS than cGVHD fibroblasts at both mRNA and protein levels, and more lacrimal gland fibroblasts in the SS were positive for α-SMA than cGVHD (P &lt; 0.01). CONCLUSIONS. In cGVHD, increased expression of HSP47 may promote excessive collagen assembly in and around the periductal areas where fibroblasts are mostly in an active state. The less α-SMA in the cGVHD lacrimal gland fibroblasts suggests a relative lack of myofibroblastic transformation. It is likely that fibroblasts incapable of myofibroblastic transformation are the main source of HSP47 and collagen production, and the resultant effect is the periductal fibrotic changes seen in lacrimal glands of patients with cGVHD.</p>

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T helper type 2-biased natural killer cell phenotype in patients with pemphigus vulgaris

Takahashi Hayato, Amagai Masayuki, Tanikawa Akiko, Suzuki Shigeaki, Ikeda Yasuo, Nishikawa Takeji, Kawakami Yutaka, Kuwana Masataka

Journal of Investigative Dermatology 127 ( 2 ) 324 - 330 2007.02

ISSN  0022-202X

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<p>Pemphigus vulgaris (PV) is an autoantibody-mediated bullous disease, but the role of natural killer (NK) cells in its pathogenic process has never been examined in detail. Circulating CD56⁺CD3^- NK cells as well as CD69⁺-activated NK cells were increased in PV patients compared with healthy controls and patients with other autoantibody-mediated autoimmune diseases, including immune thrombocytopenic purpura and myasthenia gravis. Gene expression analysis of highly purified NK cells demonstrated an increased expression of IL-10 and decreased expression of IL-12Rβ2, perforin, and granzyme B ex vivo in PV patients versus healthy controls. The NK cells from PV patients also showed impaired signal transducer and activator of transduction4 phosphorylation upon in vitro IL-12 stimulation. Moreover, NK cells from PV patients exhibited reduced IL-10 production in response to in vitro stimulation with IL-2/IL-12. Finally, IL-5 expression in NK cells was exclusively detected ex vivo in PV patients with active disease, and was lost in subsequent analyses performed during disease remission. Together these findings suggest that NK cells contribute to a T helper type 2-biased immune response in PV patients through impaired IL-12 signaling and an upregulation of IL-10 and IL-5.</p>

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Evaluation of platelet kinetics in patients with liver cirrhosis

Kajihara Mikio, Okazaki Yuka, Kato Shinzo, Ishii Hiromasa, Kawakami Yutaka, Ikeda Yasuo, Kuwana Masataka

Journal of Gastroenterology and Hepatology (Australia) 22 ( 1 ) 112 - 118 2007

ISSN  0815-9319

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<p>Background: Thrombocytopenia is a common manifestation of liver cirrhosis (LC), but its underlying mechanism is not fully understood. The purpose of the present paper was to evaluate the platelet kinetics in LC patients by examining several non-invasive convenient markers. Methods: Fifty-seven LC patients, 32 patients with idiopathic thrombocytopenic purpura (ITP), 12 with aplastic anemia (AA), and 29 healthy individuals were studied. Plasma thrombopoietin was measured by enzyme-linked immunosorbent assay. Absolute reticulated platelet (RP) count and plasma glycocalicin were used as indices for thrombopoiesis, and the indices for platelet turnover were the RP proportion and the plasma glycocalicin normalized to the individual platelet count (GCI). Results: There was no difference in thrombopoietin levels between LC patients and healthy controls. The RP proportion and GCI were significantly higher and the absolute RP count and glycocalicin significantly lower in LC patients than in healthy controls. These markers in ITP and LC patients were comparable, but significantly different from those in AA patients. The bone marrow megakaryocyte density in LC and ITP patients was similar, and significantly higher than in AA patients. Conclusions: Cirrhotic thrombocytopenia is a multifactorial condition involving accelerated platelet turnover and moderately impaired thrombopoiesis. Thrombopoietin deficiency is unlikely to be the primary contributor to cirrhotic thrombocytopenia.</p>

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Enhancement of immunologic tumor regression by intratumoral administration of dendritic cells in combination with cryoablative tumor pretreatment and Bacillus Calmette-Guerin cell wall skeleton stimulation

Udagawa Masaru, Kudo-Saito Chie, Hasegawa Go, Yano Kazuhito, Yamamoto Aiko, Yaguchi Masae, Toda Masahiro, Azuma Ichiro, Iwai Takehisa, Kawakami Yutaka

Clinical Cancer Research 12 ( 24 ) 7465 - 7475 2006.12

ISSN  1078-0432

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<p>Purpose: We developed an effective immunotherapy, which could induce antitumor immune responses against shared and unique tumor antigens expressed in autologous tumors. Experimental Design: Intratumoral administration of dendritic cells is one of the individualized immunotherapies; however, the antitumor activity is relatively weak. In this study, we attempted to enhance the antitumor efficacy of the i.t. dendritic cell administration by combining dendritic cells stimulated with Bacillus Calmette-Guerin cell wall skeleton (BCG-CWS) additionally with cryoablative pretreatment of tumors and analyzed the therapeutic mechanisms. Results: These two modifications (cryoablation of tumors and BCG-CWS stimulation of dendritic cells) significantly increases the antitumor effect on both the treated tumor and the untreated tumor, which was distant at the opposite side, in a bilateral s.c. murine CT26 colon cancer model. Further analysis of the augmented antitumor effects revealed that the cryoablative pretreatment enhances the uptake of tumor antigens by the introduced dendritic cells, resulting in the induction of tumor-specific CD8⁺ T cells responsible for the in vivo tumor regression of both treated and remote untreated tumors. This novel combination i.t. dendritic cell immunotherapy was effective against well-established large tumors. The antitumor efficacy was further enhanced by depletion of CD4⁺CD25 ⁺FoxP3⁺ regulatory T cells. Conclusions: This novel dendritic cell immunotherapy with i.t. administration of BCG-CWS-treated dendritic cells following tumor cryoablation could be used for the therapy of cancer patients with multiple metastases.</p>

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Endothelial differentiation potential of human monocyte-derived multipotential cells

Kuwana Masataka, Okazaki Yuka, Kodama Hiroaki, Satoh Takashi, Kawakami Yutaka, Ikeda Yasuo

Stem Cells 24 ( 12 ) 2733 - 2743 2006.12

ISSN  1066-5099

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<p>We previously reported a unique CD14⁺CD45⁺CD34 ⁺ type I collagen⁺ cell fraction derived from human circulating CD14⁺ monocytes, named monocyte-derived multipotential cells (MOMCs). This primitive cell population contains progenitors capable of differentiating along the mesenchymal and neuronal lineages. Here, we investigated whether MOMCs can also differentiate along the endothelial lineage. MOMCs treated with angiogenic growth factors for 7 days changed morphologically and adopted a caudate appearance with rod-shaped microtubulated structures resembling Weibel-Palade bodies. Almost every cell expressed CD31, CD144, vascular endothelial growth factor (VEGF) type 1 and 2 receptors, Tie-2, von Willebrand factor (vWF), endothelial nitric-oxide synthase, and CD146, but CD14/CD45 expression was markedly downregulated. Under these culture conditions, the MOMCs continued to proliferate for up to 7 days. Functional characteristics, including vWF release upon histamine stimulation and upregulated expression of VEGF and VEGF type 1 receptor in response to hypoxia, were indistinguishable between the MOMC-derived endothelial-like cells and cultured mature endothelial cells. The MOMCs responded to angiogenic stimuli and promoted the formation of mature endothelial cell tubules in Matrigel cultures. Finally, in xenogenic transplantation studies using a severe combined immunodeficient mouse model, syngeneic colon carcinoma cells were injected subcutaneously with or without human MOMCs. Cotransplantation of the MOMCs promoted the formation of blood vessels, and more than 40% of the tumor vessel sections incorporated human endothelial cells derived from MOMCs. These findings indicate that human MOMCs can proliferate and differentiate along the endothelial lineage in a specific permissive environment and thus represent an autologous transplantable cell source for therapeutic neovasculogenesis.</p>

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Effects of a Helicobacter pylori eradication regimen on anti-platelet autoantibody response in infected and uninfected patients with idiopathic thrombocytopenic purpura

Asahi Atsuko, Kuwana Masataka, Suzuki Hidekazu, Hibi Toshifumi, Kawakami Yutaka, Ikeda Yasuo

Haematologica 91 ( 10 ) 1436 - 1437 2006.10

ISSN  0390-6078

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<p>Thirty-seven patients with idiopathic thrombocytopenic purpura (TTP) were treated with a standard Helicobacter pylori eradication regimen irrespective of H. pylori infection. Our results indicate that platelet recovery results from the disappearance of H. pylori itself, and is mediated, in part, through suppression of anti-platelet autoantibody production.</p>

Prolonged thrombocytopenia after allogeneic hematopoietic stem cell transplantation

Yamazaki R., Kuwana M., Mori T., Okazaki Y., Kawakami Y., Ikeda Y., Okamoto S.

Bone Marrow Transplantation 38 ( 5 ) 377 - 384 2006.09

ISSN  0268-3369

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<p>To evaluate the mechanisms underlying prolonged thrombocytopenia after allogeneic hematopoietic stem cell transplantation (SCT), an index for plasma glycocalicin normalized for the individual platelet count (GCI), plasma thrombopoietin (TPO), and circulating B cells producing anti-GPIIb-IIIa antibodies were measured in 50 SCT recipients with or without prolonged thrombocytopenia, 42 patients with idiopathic thrombocytopenic purpura, nine patients with aplastic anemia, and 22 healthy individuals. All three indices were significantly higher in the SCT recipients with thrombocytopenia than in those without (P&lt;0.01 for all comparisons), and were significantly correlated with the platelet count in SCT recipients. Stepwise multiple regression analysis of the samples from the SCT recipients revealed that GCI and TPO independently pointed to specific mechanisms of thrombocytopenia. The GCI and TPO status in SCT recipients with thrombocytopenia had a pattern similar to that seen in aplastic anemia, suggesting a major role for impaired thrombopoiesis. An antiplatelet antibody response was frequently detected in SCT recipients, but the development of thrombocytopenia is likely to depend on additional factors, such as reticuloendothelial function. In summary, post transplant prolonged thrombocytopenia is associated with complex mechanisms, including impaired thrombopoiesis and increased platelet turnover.</p>

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The BRAF-MAPK signaling pathway is essential for cancer-immune evasion in human melanoma cells

Sumimoto Hidetoshi, Imabayashi Fumie, Iwata Tomoko, Kawakami Yutaka

Journal of Experimental Medicine 203 ( 7 ) 1651 - 1656 2006.07

ISSN  0022-1007

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<p>The mitogen-activated protein kinase (MAPK) pathway is frequently activated in human cancers, leading to malignant phenotypes such as autonomous cellular proliferation. Here, we demonstrate a novel role of the activated MAPK pathway in immune evasion by melanoma cells with the mutation of BRAF, which encodes a MAPKKs, (BRAF^V600E). MEK inhibitor U0126 or RNA interference (RNAi) for BRAF V600E decreased production of the immunosuppressive soluble factors interleukin (IL)-10, VEGF, or IL-6 from melanoma cells to levels comparable to those after signal transducer and activator of transcription (STAT)3 inactivation. The suppressive activity of the culture supernatants from the melanoma cells on the production of inflammatory cytokines IL-12 and tumor necrosis factor α by dendritic cells upon lipopolysaccharide stimulation was markedly reduced after transduction with BRAF^V600E RNAi, comparable to the effects observed with STAT3 RNAi transduction. No additive or synergistic effects were observed by the simultaneous transduction of RNAi for both BRAF^V600E and STAT3. Furthermore, specific DNA binding and transcriptional activity of STAT3 were not affected by down-regulation of the MAPK signaling with the BRAF RNAi. These results indicate that the MAPK signal, along with the STAT3 signal, is essential for immune evasion by human melanomas that have constitutively active MAPK signaling and is a potential molecular target for overcoming melanoma cell evasion of the immune system. JEM</p>

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Frequent immune response to a melanocyte specific protein KU-MEL-1 in patients with Vogt-Koyanagi-Harada disease

Otani S., Sakurai T., Yamamoto K., Fujita T., Matsuzaki Y., Goto Y., Ando Y., Suzuki S., Usui M., Takeuchi M., Kawakami Yutaka

British Journal of Ophthalmology 90 ( 6 ) 773 - 777 2006.06

ISSN  0007-1161

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<p>Aim: To isolate autoantigens possibly involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. Methods: Autoantigens recognised by immunoglobulin G antibodies (IgG Ab) in sera from VKH patients were isolated by screening the lambda phage cDNA libraries made from melanocytes and a highly pigmented melanoma cell line with the patients' sera. Presence of IgG specific for the autoantigens in sera from patients with various panuveitis and healthy individuals was evaluated. Relation between the specific IgG and various clinicopathological features was examined. Results: KU-MEL-1 was found to be one of the 81 isolated positive clones representing 35 distinct genes, which is a previously isolated melanoma antigen preferentially expressed in melanocytes. The IgG Ab specific for KU-MEL-1 was detected in sera from patients with VKH in significantly higher amounts than in sera from patients with Behçet's disease, sarcoidosis, and from healthy individuals. Positive serum KU-MEL-1 Ab was significantly associated with HLA-DRB1*0405 and male VKH patients. Conclusion: KU-MEL-1 was identified as a new autoantigen for VKH. The highly frequent induction of IgG Ab for KU-MEL-1 in HLA-DRB1*0405 positive VKH patients may suggest the possible involvement of KU-MEL-1 specific CD4 ⁺ T cells in the pathogenesis of VKH, suggesting the possible use in the development of diagnostic and therapeutic treatments for VKH patients.</p>

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Increase in circulating endothelial precursors by atorvastatin in patients with systemic sclerosis

Kuwana Masataka, Kaburaki Junichi, Okazaki Yuka, Yasuoka Hidekata, Kawakami Yutaka, Ikeda Yasuo

Arthritis and Rheumatism 54 ( 6 ) 1946 - 1951 2006.06

ISSN  0004-3591

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<p>Objective. To evaluate whether atorvastatin can increase bone marrow-derived circulating endothelial precursors (CEPs) and improve the vascular symptoms in patients with systemic sclerosis (SSc; scleroderma). Methods. The study was designed as an open-label, prospective study involving 14 patients with SSc who received 10 mg/day of atorvastatin for 12 weeks and were followed up for the subsequent 4 weeks. CEPs were quantified at weeks 0 (pretreatment), 4, 8, 12 (during treatment), and 16 (posttreatment) by cell sorting followed by 3-color flow cytometry. Raynaud's phenomenon variables, global measures, and psychological scales as well as circulating angiogenic factors and endothelial activation/injury markers were serially assessed. The potential of CEPs to differentiate into mature endothelial cells was examined in cultures with angiogenic stimuli. Results. None of the patients experienced an adverse event, but 1 dropped out because of an excessive decrease in serum total cholesterol. Atorvastatin treatment resulted in a 1.7- to 8.0-fold increase in CEPs from baseline levels (P &lt; 0.0001), but the numbers returned to within baseline levels at posttreatment. However, 8 patients (62%) experienced a gradual decrease in the number of CEPs, even while taking atorvastatin. Variables indicating the extent of Raynaud's phenomenon improved significantly, and up-regulated levels of angiogenic factors and vascular endothelial activation/injury markers decreased significantly during atorvastatin treatment. These variables returned to within baseline levels after discontinuation of the drug. In contrast, atorvastatin failed to improve the in vitro maturation potential of CEPs. Conclusion. The results of this pilot study suggest that atorvastatin treatment can increase CEPs and may be effective in improving Raynaud's phenomenon, even in SSc patients who have CEP dysfunction intrinsically.</p>

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A new melanoma antigen fatty acid-binding protein 7, involved in proliferation and invasion, is a potential target for immunotherapy and molecular target therapy

Goto Yasufumi, Matsuzaki Yuriko, Kurihara Sachiko, Shimizu Ayako, Okada Tsutomu, Yamamoto Kazuhiko, Murata Hiroshi, Takata Minoru, Aburatani Hiroyuki, Hoon Dave S B, Saida Toshiaki, Kawakami Yutaka

Cancer Research 66 ( 8 ) 4443 - 4449 2006.04

ISSN  0008-5472

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<p>The identification of molecules that are preferentially expressed in melanoma cells and involved in their malignant phenotypes is important for understanding melanoma biology and the development of new diagnostic and therapeutic methods. By comparing the expression profile of a melanoma cell line with those of various normal tissues using GeneChip and by confirming the actual expression of the selected genes by reverse transcription-PCR and Northern and Western blot analyses, fatty acid-binding protein 7 (FABP7), which is frequently expressed in melanomas, was identified. Immunohistochemical examination revealed that FABP7 was expressed in 11 of 15 melanoma tissues. By down-regulating the FABP7 expression with FABP7-specific small interfering RNAs, in vitro cell proliferation and Matrigel invasion were suppressed in two of six melanoma cell lines. Overexpression of FABP7 in a FABP7-negative embryonic kidney cell line 293T by transfecting with the FABP7 cDNA resulted in enhanced cell proliferation and Matrigel invasion, indicating that FABP7 plays a role in the malignant phenotype of some melanoma cell lines. IgG antibodies specific for the phage or bacterial recombinant FABP7 protein were detected in 14 of 25 (56%) or in 8 of 31 (26%) sera from melanoma patients, respectively, but not in sera from healthy individuals, indicating that FABP7 is an immunogenic antigen in melanoma patients. These results showed that FABP7 is frequently expressed in melanoma, may be involved in cell proliferation and invasion, and may be a potential target for development of diagnostic and therapeutic methods.</p>

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Neurogenic potential of progenitors derived from human circulating CD14⁺ monocytes

Kodama Hiroaki, Inoue Takafumi, Watanabe Ryuichi, Yasutomi Daisuke, Kawakami Yutaka, Ogawa Satoshi, Mikoshiba Katsuhiko, Ikeda Yasuo, Kuwana Masataka

Immunology and Cell Biology 84 ( 2 ) 209 - 217 2006.04

ISSN  0818-9641

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<p>We previously reported a primitive cell fraction derived from human circulating CD14⁺ monocytes, named monocyte-derived multipotential cells (MOMC), that can differentiate along mesenchymal lineages, including bone, cartilage, fat, skeletal muscle and cardiac muscle. In this study, we investigated whether MOMC can differentiate into the neuronal lineage. MOMC were fluorescently labelled and cocultivated with a primary culture of rat neurons for up to 4 weeks. The protein and gene expressions of neuron-specific markers in the human MOMC were evaluated over time using immunohistochemistry, in situ hybridization and reverse transcription followed by PCR. Shortly after cocultivation with rat neurons, nearly all the MOMC expressed early neuroectodermal markers, Mash1, Neurogenin2 and NeuroD, together with nestin, an intermediate filament expressed in neurogenesis. After 14 days of coculture, a subpopulation of MOMC displayed a multipolar morphology with elongated neurites and expressed mature neuron-specific markers, including neurofilament, microtubule-associated protein type 2, β3-tubulin, NeuN and Hu. Transdifferentiation of monocytes into the neuroectodermal lineage was shown by the simultaneous expression of proneural markers and CD45/CD14 early in the differentiation process. The cocultivated MOMC retained their proliferative capacity for at least 16 days. Finally, the neuronal differentiation of MOMC was observed when they were cultured with neurons without cell-to-cell contact. The capacity of MOMC to differentiate into both mesodermal and neuroectodermal lineages suggests that circulating CD14⁺ monocytes are more multipotential than previously thought.</p>

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Identification of a glioma antigen, GARC-1, using cytotoxic T lymphocytes induced by HSV cancer vaccine

Iizuka Yukihiko, Kojima Hidefumi, Kobata Tetsuji, Kawase Takeshi, Kawakami Yutaka, Toda Masahiro

International Journal of Cancer 118 ( 4 ) 942 - 949 2006.02

ISSN  0020-7136

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<p>Despite several ongoing clinical trials of immunotherapies against glioma, few glioma-specifk antigens recognized by cytotoxic T lymphocytes (CTLs) have been identified. We recently demonstrated that intratumoral inoculation with herpes simplex virus (HSV) as a cancer vaccine activates tumor-specific CTLs. To identify glioma antigens recognized by CTLs, we used the HSV cancer vaccine to vaccinate mice harboring a syngeneic mouse glioma cell line, GL261. From the splenocytes of the immunized mice, we generated an H-2D^b-restricted CTL line, GCL-1, that was specific for GL261. Then, a cDNA expression library generated from GL261 was screened with GCL-1, and a new gene encoding glioma antigen, GARC-1, was isolated. Sequence analysis revealed that the GARC-1 gene isolated from GL261 had a point mutation causing an amino acid change (Asp to Asn at position 81). T-cell epitope analysis revealed that the mutated peptide GARC-1_77-85 (AALLNKLYA) but not the wild-type peptide (AALLDKLYA), was recognized by GCL-1. These results suggest that HSV cancer vaccination may be a useful method for inducing tumor-specific CTLs and identifying tumor antigens. Furthermore, this GL261/GARC-1 murine glioma model may be useful for the development of immunotherapy for brain tumors.</p>

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Autoantibody to CD40 ligand in systemic lupus erythematosus

Nakamura M., Tanaka Y., Satoh T., Kawai M., Hirakata M., Kaburaki J., Kawakami Y., Ikeda Y., Kuwana M.

Rheumatology 45 ( 2 ) 150 - 156 2006.02

ISSN  1462-0324

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<p>Objectives. To examine the prevalence, clinical associations and pathogenic roles of autoantibodies to CD40 ligand (CD40L) in patients with systemic lupus erythematosus (SLE). Methods. Plasma anti-CD40L antibodies from 125 patients with SLE, 24 with primary antiphospholipid syndrome (APS) and 90 with idiopathic thrombocytopenic purpura (ITP) and from 62 healthy individuals were measured with an enzyme-linked immunosorbent assay (ELISA). HeLa cells transfected with human CD40L cDNA (HeLa/CD40L) were used to confirm the presence of anti-CD40L autoantibodies. The effect of anti-CD40L antibodies on the CD40L-CD40 interaction was evaluated by observing CD40L-induced IκB activation in CD40-expressing fibroblasts. Results. Anti-CD40L autoantibody was detected in seven (6%) SLE, three (13%) primary APS and 11 (12%) ITP patients, but in no healthy controls. Antibody binding in an ELISA was competitively inhibited by membrane components of HeLa/CD40L. Anti-CD40L antibody-positive IgG specifically bound the surface of living HeLa/CD40L, as shown by flow cytometry. The frequency of thrombocytopenia was significantly higher in SLE patients with the anti-CD40L antibody than in those without (100 vs 14% P&lt;0.00001), whereas there was no association between the anti-CD40L antibody and thrombosis. Binding of the anti-CD40L antibodies in patients' plasma to CD40L was competitively inhibited by a series of mouse anti-CD40L monoclonal antibodies. Anti-CD40L antibody-positive IgG failed to inhibit CD40L-induced IκB activation. Conclusions. Anti -CD40L autoantibody is associated with thrombocytopenia but not thromboembolism. Our findings are potentially useful in understanding the complex roles of CD40L in the pathophysiology of thrombosis and haemostasis as well as the thromboembolic complications that occur during treatment with anti-CD40L humanized antibody.</p>

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Effective inhibition of cell growth and invasion of melanoma by combined suppression of BRAF (V599E) and Skp2 with lentiviral RNAi

Sumimoto Hidetoshi, Hirata Kenro, Yamagata Shizuko, Miyoshi Hiroyuki, Miyagishi Makoto, Taira Kazunari, Kawakami Yutaka

International Journal of Cancer 118 ( 2 ) 472 - 476 2006.01

ISSN  0020-7136

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<p>p27^Kip1 that regulates the G1/S transition of cell cycle and inhibits Rho A signaling is frequently lost in several cancers leading to the deregulation of cell growth and cell motility. Mitogen-activated protein kinases (MAPK) regulate the export of p27^Kip1 from nucleus to cytoplasm, followed by the degradation with proteases. Skp-2, a subunit of an SCF ubiquitin-protein ligase complex responsible for the ubiquitination of p27 ^Kip1, is upregulated frequently in several cancers, leading to the decrease of p27^Kip1. We applied human immunodeficiency virus (HIV) lentivirus-mediated RNA interference (RNAi) to melanoma cells with the BRAF mutation (V599E) and overexpressed Skp-2 and found that the simultaneous suppression of these activated oncogenes resulted in the effective inhibition of in vitro cell growth and invasive ability of melanoma cells accompanied by the additional increase of p27^Kip1. Our results suggest that gene therapy against melanoma with the enhanced MAPK and ubiquitin-proteasomal pathways could be a specific and effective therapeutic strategy for cancers.</p>

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A novel cancer testis antigen that is frequently expressed in pancreatic, lung, and endometrial cancers

Okada Takaho, Akada Masanori, Fujita Tomonobu, Iwata Takashi, Goto Yasufumi, Kido Kenji, Okada Tsutomu, Matsuzaki Yuriko, Kobayashi Kouichi, Matsuno Seiki, Sunamura Makoto, Kawakami Yutaka

Clinical Cancer Research 12 ( 1 ) 191 - 197 2006.01

ISSN  1078-0432

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<p>Purpose: To isolate cancer testis antigens that are expressed in pancreatic cancers and may be useful in clinical applications. Experimental Design: To efficiently isolate cancer testis antigens, a testis cDNA library was immunoscreened (SEREX) with serum from a patient with pancreatic ductal adenocarcinoma. The expression of isolated antigens in various cancer cell lines and tissues was evaluated by reverse transcription-PCR and Northern blot analyses. The immunogenicity of the antigen in cancer patients was evaluated by detection of the IgG antibody in sera from patients with various cancers. Results: Of the three clones isolated through screening of a total of 2 × 10⁶ cDNA library clones, one clone (KU-CT-1) was found to be expressed in various cancers but only in testis among normal tissues, indicating that it was a novel cancer testis antigen. The KU-CT-1 gene is located on chromosome 10p12 and produces two splice variants, which encode proteins of 397 and 872 amino acids, respectively. KU-CT-1 was expressed in pancreatic cancer tissues (3 of 9, 33%), lung cancer tissues (9 of 24, 38%), and endometrial cancer tissues (7 of 11, 64%). Specific serum IgG antibodies were detected in 3 of 20 pancreatic cancer patients, 2 of 12 endometrial cancer patients, 1 of 18 colon cancer patients, and 1 of 10 prostate cancer patients but not detected in 30 healthy individuals. Conclusions: KU-CT-1 is a new cancer testis antigen that is expressed in pancreatic, lung, and endometrial cancers and may be useful for diagnosis and immunotherapy for patients with various cancers.</p>

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Possible involvement of allogeneic antigens recognised by donor-derived CD4 ⁺ cytotoxic T cells in selective GVL effects after stem cell transplantation of patients with haematological malignancy

Matsushita Maiko, Yamazaki Rie, Ikeda Hideyuki, Mori Takehiko, Sumimoto Hidetoshi, Fujita Tomonobu, Okamoto Shinichiro, Ikeda Yasuo, Kawakami Yutaka

British Journal of Haematology 132 ( 1 ) 56 - 65 2006.01

ISSN  0007-1048

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<p>Cytotoxic T lymphocyte (CTL) lines specific for allogeneic antigens were generated by in vitro stimulation of donor-derived peripheral blood mononuclear cells obtained from patients who received human leucocyte antigen (HLA)-matched allogeneic haematopoietic stem cell transplantation (HSCT). One of the allogeneic antigen-specific CD4 ⁺ CTL lines, CTL-A, generated from a patient with T cell acute lymphoblastic leukaemia, recognised HLA-DPB1*0501-positive Epstein-Barr virus-immortalised human B cell line (EBV-B cells), phytohaemagglutinin blasts and leukaemia cells, but not interferon-γ (IFN-γ) treated HLA-DPB1*0501-positive fibroblasts, indicating that this CD4 ⁺ T-cell line recognised a minor histocompatibility antigen (mHa) that is preferentially expressed in haematopoietic cells in an HLA-DPB1*0501-restricted manner. The other CD4 ⁺ CTL line, CTL-B, generated from a patient with chronic myeloid leukaemia, recognised mismatched HLA-DQB1*0303 on EBV-B cells and phytohaemagglutinin (PHA) blasts. Interestingly, this CTL line did not recognise IFN-γ-treated recipient's skin fibroblasts, as HLA-DQ was merely upregulated even after IFN-γ stimulation in non-haematopoietic cells including fibroblasts, endothelial cells and hepatocytes. These results suggest that these CD4 positive CTLs, specific for mismatch HLA-DQ and mHa that are preferentially expressed on haematopoietic cells, may play an important role in induction of selective graft-versus-leukaemia effect without development of graft-versus-host disease after allogeneic HSCT.</p>

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Novel autoantibodies to a voltage-gated potassium channel K_V1.4 in a severe form of myasthenia gravis

Suzuki Shigeaki, Satoh Takashi, Yasuoka Hidekata, Hamaguchi Yasuhito, Tanaka Kortaro, Kawakami Yutaka, Suzuki Norihiro, Kuwana Masataka

Journal of Neuroimmunology 170 ( 1-2 ) 141 - 149 2005.12

ISSN  0165-5728

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<p>Sera from patients with myasthenia gravis (MG) were screened for autoantibodies to skeletal muscle-specific antigens by immunoprecipitation assay, using rhabdomyosarcoma and leukemia cell lines. Eleven of 61 MG sera immunoprecipitated a rhabdomyosarcoma-specific 70-kDa protein, which was identified as the voltage-gated K⁺ channel 1.4 (K_V1.4). This antibody specificity was not detected in 30 patients with polymyositis/dermatomyositis, 9 with thymoma alone, or 30 healthy controls. Clinical features associated with anti-K_V1.4 antibody included bulbar involvement, myasthenic crisis, thymoma, myocarditis, and QT prolongation on electrocardiogram. These findings suggest that anti-K_V1.4 antibody is a novel autoantibody associated with a severe MG subset and thymoma.</p>

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Cardiomyogenic potential of mesenchymal progenitors derived from human circulating CD14⁺ monocytes

Kodama Hiroaki, Inoue Takafumi, Watanabe Ryuichi, Yasuoka Hidekata, Kawakami Yutaka, Ogawa Satoshi, Ikeda Yasuo, Mikoshiba Katsuhiko, Kuwana Masataka

Stem Cells and Development 14 ( 6 ) 676 - 686 2005.12

ISSN  1547-3287

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<p>Previously, we reported a unique CD14⁺CD45⁺CD34 ⁺type I collagen⁺ cell fraction derived from human circulating CD14⁺ monocytes, named monocyte-derived mesenchymal progenitors (MOMPs). These primitive cells differentiate along mesenchymal lineages, including bone, cartilage, fat, and skeletal muscle. Here, we demonstrate that CD14⁺ monocytes generate MOMPs that differentiate into cardiomyocytes. MOMPs labeled with a fluorescent marker and co-cultivated with rat cardiomyocytes for 4 weeks expressed the cardiomyocyte-specific transcription factors Nkx2.5, GATA-4, eHAND, and MEF2 and the hematopoietic/monocytic markers CD45 and CD14 within 10 days and retained their proliferative capacity for up to 16 days. A subpopulation of MOMPs subsequently expressed the cardiomyocyte-specific markers α-sarcomeric actinin, troponin I, and atrial natriuretic peptide on day 21. Furthermore, fluorescence-labeled, spontaneously beating cells that formed gap junctions with adjacent rat cardiomyocytes appeared in these cultures and these cells exhibited electrophysiological properties typical of ventricular myocytes. The co-cultivation of human MOMPs with rat GFP-tagged cardiomyocytes resulted in the generation of human cardiomyocytes lacking green fluorescent protein (GFP) staining, suggesting that our observations could not solely be explained by cell fusion. Our results demonstrate for the first time that human circulating CD14⁺ monocytes include progenitors capable of proliferating and differentiating along the cardiomyogenic lineage via their differentiation into MOMPs.</p>

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Donor fibroblast chimerism in the pathogenic fibrotic lesion of human chronic graft-versus-host disease

Ogawa Yoko, Kodama Hiroaki, Kameyama Kaori, Yamazaki Kazuto, Yasuoka Hidekata, Okamoto Shinichiro, Inoko Hidetoshi, Kawakami Yutaka, Kuwana Masataka

Investigative Ophthalmology and Visual Science 46 ( 12 ) 4519 - 4527 2005.12

ISSN  0146-0404

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<p>PURPOSE. Tissue atrophy and excessive fibrosis are prominent histologic features of chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation, but the underlying mechanism remains unknown. The current study was undertaken to investigate whether the increase in fibroblasts at the site of pathogenic fibrosis originated from transplanted donor cells in patients with chronic GVHD. METHODS. Lacrimal gland biopsy specimens were obtained from nine patients with chronic GVHD. The male-specific sequences detected by fluorescein in situ hybridization (FISH) and in situ hybridization (ISH) were used as markers for the donor cells in seven female patients who had received a transplant from male donors. Primary fibroblast cultures were generated from lacrimal gland biopsy specimens and examined for mismatched genetic markers between recipients and donors. RESULTS. In lacrimal gland specimens obtained from seven female patients who received a sex-mismatched transplant, 13.4% to 26.7% of CD34⁺ fibroblasts that accumulated in the fibrotic lesion were donor derived, as determined by FISH for the Y-chromosome. The male-specific mRNA was also detected in the lacrimal gland fibroblasts by ISH. Primary lacrimal gland fibroblast cultures were generated from four patients with chronic GVHD and further examined for mismatched genetic markers between recipients and donors. As a result, the presence of donor origin of the fibroblasts was demonstrated by detecting the Y-chromosome sequence and donor-specific microsatellite genetic markers. CONCLUSIONS. These findings together indicate the chimeric status of accumulated CD34⁺ fibroblasts in the lacrimal gland of patients with chronic GVHD. Fibroblasts originating from circulating donor-derived precursors may participate in the excessive fibrosis in these patients.</p>

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Carbonic anhydrase II is a tumor vessel endothelium-associated antigen targeted by dendritic cell therapy

Yoshiura Kenta, Nakaoka Takashi, Nishishita Toshihide, Sato Katsuaki, Yamamoto Akifumi, Shimada Shinji, Saida Toshiaki, Kawakami Yutaka, Takahashi Tsuneo A., Fukuda Hiroyuki, Imajoh-Ohmi Shinobu, Oyaizu Naoki, Yamashita Naohide

Clinical Cancer Research 11 ( 22 ) 8201 - 8207 2005.11

ISSN  1078-0432

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<p>Tumor-associated antigens are promising candidates as target molecules for immunotherapy and a wide variety of tumor-associated antigens have been discovered through the presence of serum antibodies in cancer patients. We previously conducted dendritic cell therapy on 10 malignant melanoma patients and shrinkage or disappearance of metastatic tumors with massive necrosis occurred in two patients. In this study, we found a 29-kDa protein against which antibody was elicited by dendritic cell therapy in one of the two patients. Matrix-assisted laser desorption ionization-time of flight/mass spectrometry analysis of the protein isolated by two-dimensional electrophoresis combined with Western blots revealed that the 29-kDa protein was carbonic anhydrase II (CA-II). Immunohistochemistry of the tumors and normal tissues showed that CA-II was expressed in the tumor vessel but not in normal vessel endothelium. CA-II expression in tumor endothelium was observed as well in other cancers including esophageal, renal, and lung cancers. In an in vitro angiogenesis model, CA-II expression of normal human vein endothelial cells was significantly up-regulated when cells were cultured in the acidic and hypoxic conditions indicative of a tumor environment. These findings suggest that CA-II is a tumor vessel endothelium-associated antigen in melanoma and other cancers, and elicitation of serum anti-CA-II antibody by dendritic cell therapy may be associated with good clinical outcome including tumor reduction.</p>

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Immune responses to DNA mismatch repair enzymes hMSH2 and hPMS1 in patients with pancreatic cancer, dermatomyositis and polymyositis

Okada Takaho, Noji Shinobu, Goto Yasufumi, Iwata Takashi, Fujita Tomonobu, Okada Tsutomu, Matsuzaki Yuriko, Kuwana Masataka, Hirakata Michito, Horii Akira, Matsuno Seiki, Sunamura Makoto, Kawakami Yutaka

International Journal of Cancer 116 ( 6 ) 925 - 933 2005.10

ISSN  0020-7136

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<p>To identify tumor antigens useful for diagnosis and immunotherapy of patients with pancreatic ductal adenocarcinoma, we applied a SEREX approach with a cDNA library made from 5 pancreatic cancer cell lines and sera obtained from 8 patients with pancreatic cancer, and isolated total 32 genes, including 14 previously characterized genes and 18 genes with unknown functions. Among these isolated antigens, serum IgG antibodies for 2 isolated DNA mismatch repair enzymes, Homo sapiens mutS homolog 2 (HMSH2) and Homo sapiens postmeiotic segregation increased 1 (hPMS1), were detected in patients with pancreatic ductal adenocarcinoma and dermatomyositis (DM), and polymyositis (PM), but not in sera from healthy individuals. Immunohistochemical study demonstrated that hMSH2 and hPMS1 were over-expressed in pancreatic ductal adenocarcinoma compared to normal pancreatic ducts. These results suggested that hMSH2 and hPMS1 may be useful as CD4+ helper T cell antigens for immunotherapy of pancreatic cancer patients and that serum IgG antibodies may be useful for diagnosis of patients with pancreatic ductal adenocarcinoma and DM/PM.</p>

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Immunological detection of altered signaling molecules involved in melanoma development

Kawakami Yutaka, Sumimoto Hidetoshi, Fujita Tomonobu, Matsuzaki Yuriko

Cancer and Metastasis Reviews 24 ( 2 ) 357 - 366 2005.06

ISSN  0167-7659

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<p>To understand immune responses to human cancer and develop more effective immunotherapy, human tumor antigens has been isolated using various immunological methods with tumor reactive T cells or antibodies obtained from patients with melanoma. During the process of tumor antigen isolation, various molecules with genetic alterations or over-expression in tumor cells, which may be involved in proliferation, differentiation, or survival of various cancer cells, were identified. In melanoma, abnormal molecules with mutations including β -catenin, CDK4, and BRAF, and molecules with increased expression including Survivin, were immunologically detected. Therefore, immunological isolation of human tumor antigens contributes to the identification of important molecules including altered signaling molecules involved in melanoma formation.</p>

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Frequent immune responses to a cancer/testis antigen, CAGE, in patients with microsatellite instability - Positive endometrial cancer

Iwata Takashi, Fujita Tomonobu, Hirao Nobumaru, Matsuzaki Yuriko, Okada Tsutomu, Mochimaru Hiroshi, Susumu Nobuyuki, Matsumoto Eri, Sugano Kokichi, Yamashita Naohide, Nozawa Shiro, Kawakami Yutaka

Clinical Cancer Research 11 ( 10 ) 3949 - 3957 2005.05

ISSN  1078-0432

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<p>Purpose: Identification of cancer/testis antigens useful for diagnosis or immunotherapy of cancers was attempted by cDNA expression cloning with patients' sera (SEREX). Experimental Design: cDNA expression libraries made from testis or endometrial cancer cell lines were screened using sera from patients with endometrial cancer or melanoma patients immunized with dendritic cells pulsed with autologous tumor lysates. Tissue-specific expression by RT-PCR and immunogenicity by Western blotting of the bacterial recombinant antigen with sera from cancer patients were evaluated. Results: A cancer/testis antigen, CAGE, was isolated by two independently performed SEREX. CAGE was expressed in various cancer cell lines including endometrial cancer, colon cancer, and melanoma in 7 of 10 endometrial cancer tissues and in 1 of 3 atypical endometrial hyperplasia, but not in normal tissues including the endometrium and testis. The protein expression on cancer cells was confirmed by Western blot analysis with the recombinant CAGE protein, anti-CAGE IgG antibody was detected in sera from 5 of 45 endometrial cancer, 2 of 24 melanoma, and 2 of 33 colon cancer patients, but not in sera from healthy individuals. By ELISA analysis, anti-CAGE antibody was detected in 12 of 45 endometrial cancer, 2 of 20 melanoma, and 4 of 33 colon cancer patients. Intriguingly, anti-CAGE antibody was highly positive in 7 of the 13 (53.8%) microsatellite instability (MSI)-H patients with endometrial cancer, but negative in 20 non-MSI-H patients (P = 0.001). Conclusion: CAGE may be useful for immunotherapy and diagnosis of various cancers particularly MSI-positive endometrial cancer.</p>

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Novel melanoma antigen, FCRL/FREB, identified by cDNA profile comparison using DNA chip Are immunogenic in multiple melanoma patients

Inozume Takashi, Matsuzaki Yuriko, Kurihara Sachiko, Fujita Tomonobu, Yamamoto Akifumi, Aburatani Hiroyuki, Shimada Shinji, Kawakami Yutaka

International Journal of Cancer 114 ( 2 ) 283 - 290 2005.03

ISSN  0020-7136

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<p>We applied a strategy that utilized a combination of systematic gene expression analysis with various tissues and immunological detection with sera from melanoma patients to identify melanoma antigens expressed preferentially in melanoma and melanocytes. We selected 101 genes by comparing cDNA profiles obtained by GeneChip analysis of a highly pigmented melanoma cell line, SKmel23, primary cultured melanocytes, HUVECs cultured endothelial cells, keratinocytes, liver and stomach. After the additional selection with criterion of high registered frequency of each cDNA in melanocyte-related cDNA libraries in the NCBI database, 15 genes including 12 known melanocyte specific genes were identified. One of the remaining 3 genes, FCRL/FREB, encoding a member of the Fc receptor family that was previously reported to express in germinal center B cells, was found to express preferentially in melanocytes and melanoma tissues by RT-PCR and Northern blot analysis. The FCRL/FREB protein was detected in the cytoplasm of melanoma cells by staining with the murine polyclonal antibody and by transfection with GFP-fused FCRL/FREB cDNA. The bacterial recombinant protein was recognized by serum IgG antibody obtained from some patients with melanoma. These results suggest that FCRL/FREB may function in melanocytes and melanoma and may be useful for development of diagnostic methods for various pigment disorders and immunotherapy of melanoma.</p>

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Binding of β₂-glycoprotein I to anionic phospholipids facilitates processing and presentation of a cryptic epitope that activates pathogenic autoreactive T cells

Kuwana Masataka, Matsuura Eiji, Kobayashi Kazuko, Okazaki Yuka, Kaburaki Junichi, Ikeda Yasuo, Kawakami Yutaka

Blood 105 ( 4 ) 1552 - 1557 2005.02

ISSN  0006-4971

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<p>Antiphospholipid syndrome (APS) is an autoimmune prothrombotic disorder in association with autoantibodies to phospholipid (PL)-binding plasms proteins, such as β₂-glycoprotein I (β₂GPI). We have recently found that CD4⁺ T cells autoreactive to β ₂GPI in patients with APS preferentially recognize a cryptic peptide encompassing amino acid residues 276-290 (p276-290), which contains the major PL-binding site, in the context of DR53. However, it is not clear how previously cryptic p276-290 becomes visible to the immune system and elicits a pathogenics autoimmune response to β₂GPI. Here we show that presentation of a disease-relevant cryptic T-cell determinant in β₂GPI is induced as a direct consequence of antigen processing from β₂GPI bound to anionic PL. Dendritic cells or macrophages pulsed with PL-bound β₂GPI induced a response of p276-290-specific CD4⁺ T-cell lines generated from the patients in an HLA-DR-restricted and antigen-processing-dependent manner but those with β₂GPI or PL alone did not. In addition, the p276-290-reactive T-cell response was primed by stimulating peripheral blood T cells from DR53-carrying healthy individuals with dendritic cells bearing PL-bound β₂GPI in vitro. Our finding is the first demonstration of an in vitro mechanism eliciting pathogenic autoreactive T-cell responses to β₂GPI and should be useful in clarifying the pathogenesis of APS.</p>

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Genomic alterations in primary cutaneous melanomas detected by metaphase comparative genomic hybridization with laser capture or manual microdissection

Namiki Takeshi, Yanagawa Shigeru, Izumo Toshiyuki, Ishikawa Masashi, Tachibana Masayoshi, Kawakami Yutaka, Yokozeki Hiroo, Nishioka Kiyoshi, Kaneko Yasuhiko

Cancer Genetics and Cytogenetics 157 ( 1 ) 1 - 11 2005.02

ISSN  0165-4608

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<p>To clarify the correlation of genomic alterations with clinical and histological features, we performed metaphase comparative genomic hybridization analysis on 20 primary cutaneous melanomas, which were obtained by laser capture or manual microdissection, and 16 melanoma cell lines. There were no differences in the average number of aberrations between acral melanomas (AM) and non-AM, although gains of 5q and 11q13 were more frequent (P = 0.05) and 10q loss was less frequent (P = 0.01) in AM than in non-AM. Although tumor thickness is considered a measurable estimate of clinical expression, there were no differences in the average number of aberrations among 4 groups, classified by thickness of the tumor. While the majority of aberrations were equally distributed among the 4 groups, 6p gains were found only in the thickest tumors. Patients with 6p or 1q gains had a lower overall survival rate than those without them (P = 0.0002 or P = 0.013). While gains of 1q, 2q, 3p, 3q, 7q, 20p, and 20q were more frequent in the cell lines than in the primary tumors (P &lt; 0.01), losses of 6q, 9p, 10p, and 10q were equally found in both cell lines and primary tumors. The present study showed that chromosomal aberrations had already occurred in the thinner tumors, and that 6p and 1q gains may be a prognostic factor.</p>

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Lack of circulating autoantibodies to bone morphogenetic protein receptor-II or activin receptor-like kinase 1 in mixed connective tissue disease patients with pulmonary arterial hypertension

Satoh T., Kimura K., Okano Y., Hirakata M., Kawakami Y., Kuwana M.

Rheumatology 44 ( 2 ) 192 - 196 2005.02

ISSN  1462-0324

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<p>Objectives. To examine whether autoantibodies against bone morphogenetic protein receptor-II (BMPR-II) or activin receptor-like kinase 1 (ALK-1) are associated with pulmonary arterial hypertension (PAH) in patients with mixed connective tissue disease (MCTD). Methods. We studied sera from 37 MCTD patients with or without PAH, six patients with idiopathic PAH, and 30 healthy controls. Circulating anti-BMPR-II and anti-ALK-1 antibodies were detected using immunoprecipitation of recombinant antigens generated by in vitro transcription/translation and indirect immunofluorescence of cultured cells that were induced to express these antigens by gene transfer. Anti-BMPR-II antibodies were further examined by immunoprecipitation and immunoblotting using a recombinant fragment of the extracellular domain of BMPR-II. Results. Serum anti-BMPR-II and anti-ALK-1 autoantibodies were not detected in MCTD patients irrespective of the presence or absence of PAH, or in patients with idiopathic PAH. Conclusions. Our finding does not support the hypothesis that autoantibody-mediated dysregulation of signals through BMPR-II or ALK-1 contributes to the development of PAH in patients with connective tissue diseases.</p>

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Systematic identification of human melanoma antigens using Serial Analysis of Gene Expression (SAGE)

Matsuzaki Yuriko, Hashimoto Shin Ichi, Fujita Tomonobu, Suzuki Takuji, Sakurai Toshiharu, Matsushima Kouji, Kawakami Yutaka

Journal of Immunotherapy 28 ( 1 ) 10 - 19 2005.01

ISSN  1524-9557

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<p>To identify new melanoma antigens using systematic gene expression analysis combined with rapid screening of patient sera for immunogenicity, a serial analysis of gene expression (SAGE) method was applied to profile transcripts in a highly pigmented melanoma cell line SKmel23. 25,997 SAGE tags consisting of 10,382 unique transcripts were sequenced. This melanoma SAGE library was compared with a testis SAGE library and the colon SAGE database, and to the cDNA database obtained by random sequencing of a melanocyte cDNA library. Among the 15 tags finally selected with criteria of preferential expression on melanoma and melanocytes at relatively high frequency, two tags were further analyzed for their structure and immunogenicity. One was identified as PAX3, and its isoform, PAX3d, was found to be dominantly expressed in melanoma and melanocytes. The other was derived from a novel gene and its full-length cDNA clone was isolated. Preferential expression of these genes in melanoma and melanocytes was confirmed by RT-PCR and Northern blot analysis. The recombinant bacterial PAX3d protein was recognized by serum IgG from some patients with melanoma and Vogt-Koyanagi-Harada (VKH) disease but not from healthy individuals, indicating that PAX3d is a new melanocyte-specific antigen immunogenic in patients with melanoma or VKH disease. The authors report two melanocyte/melanoma-specific molecules, which may be useful for development of diagnosis or treatment of these pigment disorders. In addition, a system using SAGE and immunoscreening with patients' sera is shown to be an efficient method for the systematic identification of tumor antigens.</p>

Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference

Sumimoto H., Yamagata S., Shimizu A., Miyoshi H., Mizuguchi H., Hayakawa T., Miyagishi M., Taira K., Kawakami Y.

Gene Therapy 12 ( 1 ) 95 - 100 2005.01

ISSN  0969-7128

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<p>Increase of Skp-2, which is involved in the degradation of cell cycle regulators including p27^Kip1, p21 and c-myc, is one of the important mechanisms for dysregulation of cell cycles in various cancers. We applied RNA interference (RNAi) for Skp-2 by using HIV-lentiviral or adenoviral vectors for a human small-cell lung carcinoma cell line with increased Skp-2 to evaluate RNAi strategy for cancer gene therapy. HIV-lentivirus-mediated RNAi for Skp-2 resulted in efficient inhibition of the in vitro cell growth of cancer cells with increased Skp-2 through the increase of p27^Kip1 and p21, but no significant effect on the growth of cells without high Skp-2 expression. Furthermore, intratumoral administration of adenovirus siRNA vector for Skp-2 efficiently inhibited growth of established subcutaneous tumor on NOD/SCID mice. These results indicate that the Skp-2 RNAi may be a useful strategy for gene therapy of cancers with high Skp-2 expression.</p>

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Identification of human tumor antigens recognized by T cells

Kawakami Yutaka

Nippon rinsho. Japanese journal of clinical medicine 63 Suppl 4   562 - 567 2005

ISSN  0047-1852

Involvement of overexpressed wild-type BRAF in the growth of malignant melanoma cell lines

Tanami Hideaki, Imoto Issei, Hirasawa Akira, Yuki Yasuhiro, Sonoda Itaru, Inoue Jim, Yasui Kohichiro, Misawa-Furihata Akiko, Kawakami Yutaka, Inazawa Johji

Oncogene 23 ( 54 ) 8796 - 8804 2004.11

ISSN  0950-9232

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<p>Comparative genomic hybridization (CGH) using 40 cell lines derived from malignant melanomas (MMs) revealed frequent amplification at 7q33-q34 containing BRAF gene, which often is mutated in MM. We found this gene to be amplified to a remarkable degree in the MM cell lines that exhibited high-level gains at 7q33-q34 in CGH. Among 40 cell lines, the eight lines that revealed neither BRAF nor NRAS mutations showed even higher levels of BRAF mRNA expression than the 32 mutated lines, although DNA amplification at 7q33-q34 was not detected in every lines overexpressing BRAF. MM cells that carried wild-type BRAF and NRAS showed constitutive over-expression of B-Raf protein and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), even after serum starvation. Not only downregulation of the endogenously overexpressed wild-type B-Raf by antisense oligonucleotide but also a treatment with an inhibitor of mitogen-activated protein kinase kinase (MAPKK, MEK) reduced phosphorylated ERK1/2 and cell growth, whereas the exogenously expressed wild-type B-Raf promoted cell growth in MM cells. Our results provide the evidence that overexpression of wild-type B-Raf, in part but not always as a result of gene amplification, is one of the mechanisms underlying constitutive activation of the MAPK pathway that stimulates growth of MM cells.</p>

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Autoreactive CD8+ cytotoxic T lymphocytes to major histocompatibility complex class I chain-related gene A in patients with Behçet's disease

Yasuoka Hidekata, Okazaki Yuka, Kawakami Yutaka, Hirakata Michito, Inoko Hidetoshi, Ikeda Yasuo, Kuwana Masataka

Arthritis and Rheumatism 50 ( 11 ) 3658 - 3662 2004.11

ISSN  0004-3591

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<p>Objective. To detect and characterize the autoreactive CD8+ T cells to major histocompatibility complex class 1 chain-related gene A (MICA), a stress-inducible antigen preferentially expressed on the epithelium and endothelium, in patients with Behçet's disease (BD). Methods. A candidate for the antigenic MICA peptide was selected based on its predicted binding affinity for HLA-B51 and proteasomal cleavage sites. Peripheral blood T cells from 14 patients with BD and 15 healthy controls were repeatedly stimulated with the MICA peptide, and the specific T cell response was measured by peptide-induced interferon-γ. Cytotoxic T lymphocyte activity was examined by chromium-51 release from an BLA-151-transfected B cell line in the presence of the MICA peptide. Results. A 9-mer peptide AAAAAIFVI (termed MICA transmembrane [MICA-TM]) was selected as a candidate for the antigenic peptide presented by HLA-B51. A specific T cell response to MICA-TM was detected in 4 patients with BD (29%) but in none of the 15 healthy donors. All 4 responders had HLA-B51 and active disease, and the specific T cell response was lost after the BD-related symptoms disappeared. The MICA-induced T cell response was specifically inhibited by anti-HLA class I antibody or by CD8+ cell depletion. MICA-reactive T cells recognized an HLA-B51-transfected B cell line pulsed with MICA-TM or a B cell line transfected with both HLA-B51 and MICA in the absence of exogenous peptides. Finally, MICA-stimulated T cell lines lysed the HLA-B51-expressing B cell line in the presence of MICA-TM. Conclusion. HLA-B51-restricted cytotoxic T lymphocytes autoreactive to MICA may be involved in the pathogenesis of BD.</p>

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Recent progress in identification methods for human tumor antigens recognized by T cells

Hayashi Emiko, Matsuzaki Yuriko, Kawakami Yutaka

Biotherapy 18 ( 6 ) 497 - 502 2004.11

ISSN  0914-2223

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<p>Recent identification of human tumor antigens recognized by T cells made it possible to analyze anti-tumor immune responses quantitatively and qualitatively in human and to develop immunotherapy in a more scientific way. The identification methods have been being improved during the past 10 years. When tumor reactive T-cells are available, DNA expression cloning or direct peptide sequencing from HPLC fractions extracted from MHC class I/peptide complex can be performed. However, it is difficult to establish tumor reactive T cells in many tumors. In those cases, we can apply the reverse immunology approach, in which tumor antigen candidates are first identified by various genetic or immunological methods, including cDNA subtraction between cancers and various tissues with cDNA profiles obtained through systematic gene analysis such as DNA chip/microarrays, SAGE, or public gene databases, or cDNA expression cloning with patients' sera (SEREX). Then antigenicity of the identified candidates is confirmed by demonstration of T cell induction against candidates through in vitro T cell induction or immunization of HLA transgenic mice. We have made various modifications on these techniques. Identification of human tumor antigens in various cancers will lead to development of more effective immunotherapy in the future.</p>

Identification of an epigenetically silenced gene, RFX1, in human glioma cells using restriction landmark genomic scanning

Ohashi Yohei, Ueda Masakazu, Kawase Takeshi, Kawakami Yutaka, Toda Masahiro

Oncogene 23 ( 47 ) 7772 - 7779 2004.10

ISSN  0950-9232

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<p>To identify the CpG islands differentially methylated in human glioma, we performed restriction landmark genomic scanning with a CpG methylation-sensitive enzyme. We found 12 spots, the intensity of which was entirely lost or decreased in both the human glioma tissues examined as compared with that in matched normal lymphocytes, indicating aberrant methylation of these CpG islands in gliomas. The expression of RFX1, one of the genes associated with the methylated CpG islands, was frequently decreased in human glioma cell lines and tissues. We also demonstrated that the isolated CpG island located in the seventh intron of the RFX1 gene had enhancer activity and was hypermethylated in all of the glioma tissues and cell lines analysed, but not in normal brains or lymphocytes. Treatment of glioma cells with a demethylating agent, 5-azacytidine, resulted in the expression of RFX1, indicating that the silencing of the RFX1 gene may be attributable to its methylation. RFX1 has been implicated in transcriptional downregulation of the proto-oncogene c-myc. By expression of the RFX1 gene, the cellular proliferative activity of glioma cells was suppressed. Taken together, these results suggest that the RFX1 gene may be epigenetically silenced in human gliomas and involved in glioma tumorigenesis.</p>

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Identification of human tumor antigens and its implications for diagnosis and treatment of cancer

Kawakami Yutaka, Fujita Tomonobu, Matsuzaki Yuriko, Sakurai Toshiharu, Tsukamoto Makoto, Toda Masahiro, Sumimoto Hidetoshi

Cancer Science 95 ( 10 ) 784 - 791 2004.10

ISSN  1347-9032

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<p>Human tumor antigens recognized by T cells have been identified by means of various molecular biological and immunological methods, including cDNA expression cloning with patients' T cells and antibodies, cDNA subtraction using RDA and PCR differential display, systematic gene analysis such as DNA sequencing, CGH, DNA chip/microarray and SAGE, in vitro T cell induction and immunization of HLA transgenic mice. The identification of human tumor antigens has led to a better understanding of the nature of tumor antigens, anti-tumor immune responses in patients before and after immunotherapy, and tumor escape mechanisms. The information obtained from these researches has enabled us to develop and improve immunotherapy by attempting to overcome the identified problems, including intrinsically low immunogenicity of tumor antigens and several escape mechanisms, such as regulatory T cell induction. The existence of immunogenic unique antigens derived from genetic alterations in tumor cells, and the varied immunogenicity of shared tumor antigens among patients due to differing expression in tumor cells and immunoreactivity of patients, indicates that individualized immunotherapy should ideally be performed. The identified antigens will also be useful for development of diagnostic methods and molecular targeting therapy for cancer.</p>

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Defective vasculogenesis in systemic sclerosis

Kuwana Masataka, Okazaki Yuka, Yasuoka Hidekata, Kawakami Yutaka, Ikeda Yasuo

Lancet 364 ( 9434 ) 603 - 610 2004.08

ISSN  0140-6736

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<p>Background Typical vascular features of systemic sclerosis include low capillary density and vascular obliteration. The formation and repair of blood vessels in adults involve vasculogenesis, which is mediated through the recruitment of bone-marrow-derived circulating endothelial precursors (CEP). We investigated whether vasculogenesis is impaired in patients with systemic sclerosis. Methods Peripheral blood was obtained from 11 patients with systemic sclerosis, 11 with rheumatoid arthritis, and 11 healthy controls. Factors potentially affecting the CEP number were matched among the three groups. CEP (identified as circulating cells positive for CD34, CD133, and the type 2 receptor for vascular endothelial growth factor) were quantified by cell sorting and three-colour flow cytometry. The circulating concentrations of angiogenic factors were measured by ELISA. The potential of CEP to differentiate into endothelial cells was assessed by the upregulation of von Willebrand factor after in-vitro maturation treatment. Findings The absolute number of CEP was much lower in patients with systemic sclerosis than in patients with rheumatoid arthritis or healthy controls (median 274 [IQR 178-395] vs 1154 [653-1524] and 1074 [713-1186] per 20 mL peripheral blood, respectively; p&lt;0·0001 by Kruskal-Wallis test. Paradoxically, circulating concentrations of most angiogenic factors were significantly higher in patients with systemic sclerosis than in healthy controls. The proportion of CEP that differentiated into endothelial cells was significantly lower in patients with systemic sclerosis than in healthy controls (p&lt;0·0001, Mann-Whitney test). Interpretation Insufficient vascular repair machinery due to defective vasculogenesis might contribute to vasculopathy in systemic sclerosis. Relevance to practice As well as providing an important insight into the pathogenesis of this disorder, these findings suggest that dysregulated vasculogenesis might be important in other disorders with abnormalities in vascular formation, including those with excessive formation of new vessels such as cancer and those with deficient vessel formation such as atherosclerosis. Circulating endothelial precursors could be a novel target for therapeutic strategies for ischaemic complications in patients with systemic sclerosis.</p>

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Inhibition of growth and invasive ability of melanoma by inactivation of mutated BRAF with lentivirus-mediated RNA interference

Sumimoto Hidetoshi, Miyagishi Makoto, Miyoshi Hiroyuki, Yamagata Shizuko, Shimizu Ayako, Taira Kazunari, Kawakami Yutaka

Oncogene 23 ( 36 ) 6031 - 6039 2004.08

ISSN  0950-9232

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<p>Oncogenic mutations of molecules involved in the mitogen-activated protein kinase (MAPK) pathways provide signals mediating both tumor growth and invasion in various cancers including melanomas. BRAF somatic mutations, found in 66% of melanomas, have NIH3T3 transforming ability with the elevated kinase activity in vitro. We attempted to mediate RNA interference (RNAi) with HIV lentiviral vectors specific for either wild type or the most frequently mutated form of BMAF (V599E) in 10 melanoma cell lines, and found that RNAi inhibited the growth of most melanoma cell lines in vitro as well as in vivo, which was accompanied by decrease of both BRAF protein and ERK phosphorylation. Interestingly, the mutated BRAF (V599E)-specific siRNA inhibited the growth and MAPK activity of only melanoma cell lines with this mutation. Furthermore, BRAF RNAi inhibited matrigel invasion of melanoma cells accompanied with a decrease of matrix metalloproteinase activity and β₁ integrin expression. These results clarify that the mutated BRAF (V599E) is essentially involved in malignant phenotype of melanoma cells through the MAPK activation and is an attractive molecular target for melanoma treatment. The lentivirus-mediated RNAi specific for oncogenic mutations may be a powerful technique for gene therapy of cancer.</p>

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Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells

Miyagishi Makoto, Sumimoto Hidetoshi, Miyoshi Hiroyuki, Kawakami Yutaka, Taira Kazunari

Journal of Gene Medicine 6 ( 7 ) 715 - 723 2004.07

ISSN  1099-498X

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<p>Background. RNA interference (RNAi) is a phenomenon in which expression of an individual gene can be specifically silenced by introducing a double-stranded RNA, one complementary to the gene, into cells. This phenomenon can be observed in mammalian cells when small interfering RNAs (siRNAs) are used, and is receiving attention as the most powerful tool for reverse genetics in the post genome era. Several groups have developed vector-based siRNA-expression systems that can induce RNAi in living cells. Methods. We describe here a comparative analysis of various siRNA-expression systems, in which we examined the effects of stem length, loop sequence and insertion of mutation(s) and/or bulges in the stem sequence on silencing effects and on the stability of the vectors. Results. As a result of the comparative analysis, we determined the following optimized siRNA-expression system: U6 promoter-driven hairpin-type dsRNA with 21-nt stem length, three to four mutations in the sense strand only, and the optimized 9-nt loop sequence, derived from microRNA. Moreover, we demonstrate that the siRNA-expression system with a tetracycline-regulated U6 promoter(s) could have the potential to control RNAi in cells, and that the HTV vector-mediated transfer of an siRNA-expression cassette into cells resulted in efficient silencing of a target gene at a multiplicity of infection as low as five. Conclusion. The mutated hairpin siRNAs and their genetically stable coding vectors could be very useful for gene knockdown experiments, and could further benefit gene therapy using RNAi.</p>

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Implantation of Dendritic Cells in Injured Adult Spinal Cord Results in Activation of Endogenous Neural Stem/Progenitor Cells Leading to De Novo Neurogenesis and Functional Recovery

Mikami Yuji, Okano Hideyuki, Sakaguchi Masanori, Nakamura Masaya, Shimazaki Takuya, Okano Hirotaka James, Kawakami Yutaka, Toyama Yoshiaki, Toda Masahiro

Journal of Neuroscience Research 76 ( 4 ) 453 - 465 2004.05

ISSN  0360-4012

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<p>We report a treatment for spinal cord injury involving implantation of dendritic cells (DCs), which act as antigen-presenting cells in the immune system. The novel mechanisms underlying this treatment produce functional recovery. Among the immune cells tested, DCs showed the strongest activity inducing proliferation and survival of neural stem/progenitor cells (NSPCs) in vitro. Furthermore, in DC-implanted adult mice, endogenous NSPCs in the injured spinal cord were activated for mitotic de novo neurogenesis. These DCs produced neurotrophin-3 and activated endogenous microglia in the injured spinal cord. Behavioral analysis revealed the locomotor functions of DC-implanted mice to have recovered significantly as compared to those of control mice. Our results suggest that DC-implantation exerts trophic effects, including activation of endogenous NSPCs, leading to repair of the injured adult spinal cord.</p>

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Identification of human cancer antigens by SEREX

Okada Tsutomu, Kawakami Yutaka

Biotherapy 18 ( 3 ) 205 - 211 2004.05

ISSN  0914-2223

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<p>SEREX (serological identification of tumor antigens by recombinant cDNA expression cloning) is a powerful cDNA expression cloning technique for isolation of tumor antigens recognized by T cells using serum antibodies from cancer patients from whom it is difficult to raise tumor-reactive T cells. To efficiently identify tumor antigens, various modifications have been attempted for SEREX because of frequent isolation of antigens not associated with immune responses to tumor cells. These include use of a cDNA library made from testis, use of sera from patients who are immunized with autologous tumor-derived products who demonstrate tumor regression after treatment, and use of human IgG containing mice sera from immunodeficient mice implanted with human tumor tissues.</p>

Development of Immunotherapy for Pancreatic Cancer

Kawakami Yutaka, Okada Takaho, Akada Masanori

Pancreas 28 ( 3 ) 320 - 325 2004.04

ISSN  0885-3177

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<p>Human tumor antigens recognized by T cells have been recently identified in various cancers, including pancreatic cancer. With the identified antigens, new immunotherapies can be developed using more efficient immunologic intervention (due to sufficient amounts of antigens in a more immunogenic form), as well as more quantitative and qualitative immunomonitoring. Various immunotherapies for patients with various cancers, including pancreatic cancer, are currently under evaluation in clinical trials. These include adoptive transfer of tumor reactive T cells and LAK cells; nonmyeloablative stem cell transplantation; active immunization with the identified tumor antigens, various tumor-derived products, dendritic cells pulsed with tumor antigens, and gene-modified tumor cells. Although these efforts in the realm of pancreatic cancer are still limited, various groups in Japan continue to be actively involved in this field of research.</p>

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Single nucleotide polymorphisms of the inflammatory cytokine genes in adults with chronic immune thrombocytopenic purpura

Satoh Takashi, Pandey Janardan P., Okazaki Yuka, Yasuoka Hidekata, Kawakami Yutaka, Ikeda Yasuo, Kuwana Masataka

British Journal of Haematology 124 ( 6 ) 796 - 801 2004.03

ISSN  0007-1048

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<p>Single nucleotide polymorphisms (SNPs) of inflammatory cytokine genes were examined in 84 adult Japanese patients with chronic immune thrombocytopenic purpura (ITP) and 56 race-matched healthy controls. The SNPs examined were within the genes encoding tumour necrosis factor (TNF)-α (-238 G/A and -308 G/A), TNF-β (+252 G/A), and interleukin (IL)-1β (-511 C/T and +3953 T/C). Of these SNPs, the frequency of the TNF-β (+252) G/G phenotype was significantly higher in ITP patients than in healthy controls (21% vs. 7%, P = 0.04, odds ratio = 3.6, 95% confidence interval 1.1-11.1), while no significant association was detected for the other SNPs. The distribution of the TNF-β (+252) phenotype was not associated with human leucocyte antigen class II alleles or the therapeutic response in ITP patients. The frequency of circulating anti-glycoprotein IIb/IIIa antibody-producing B cells was significantly higher in ITP patients with the TNF-β (+252) G/G phenotype than in those with the G/A or A/A phenotype (11.9 ± 4.9 vs. 6.8 ± 4.9 and 3.7 ± 2.8 per 10⁵ peripheral blood mononuclear cells; P = 0.02 and P &lt; 0.001, respectively). These findings suggest that the SNP located at TNF-β (+252) contributes to susceptibility to chronic ITP by controlling the autoreactive B-cell responses to platelet membrane glycoproteins.</p>

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Augmentation of Antitumor Immune Responses by Multiple Intratumoral Inoculations of Replication-Conditional HSV and Interleukin-12

Iizuka Yukihiko, Suzuki Ayuko, Kawakami Yutaka, Toda Masahiro

Journal of Immunotherapy 27 ( 2 ) 92 - 98 2004.03

ISSN  1524-9557

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<p>Intratumoral inoculation with a herpes simplex virus (HSV) mutant, G207, as an in situ cancer vaccine has been shown to inhibit tumor growth by inducing tumor-specific immune responses. Here, as a step toward the clinical application of this therapeutic approach, we evaluated different protocols for enhancing the antitumor effect. First, in a bilaterally established tumor model with CT26 colon carcinoma, we demonstrated that multiple intratumoral inoculations with G207 induced a greater antitumor effect on both the inoculated and distant tumors than did 1 or 2 inoculations. Second, to boost this antitumor effect, we developed 2 strategies: multiple in situ cancer vaccines with G207 in combination with systemic administration of recombinant interleukin-12 (rIL-12) (G207/systemic rIL-12) or local administration of rIL-12 (G207/local rIL-12). The antitumor effects in both the inoculated and distant tumors by the combined treatments were significantly greater than by either G207 or rIL- 12 treatment alone. G207/systemic rIL-12 and G207/local rIL-12 mediated the complete regression of both the inoculated and distant tumors in 67% and 79% of the animals, respectively. These results indicate that multiple intratumoral inoculations of G207 and systemic or local rIL- 12 administration work synergistically to facilitate tumor regression and that this combination of treatments may have potential for treating cancer metastasis.</p>

Identification of bladder cancer antigens recognized by IgG antibodies of a patient with metastatic bladder cancer

Ito Keiichi, Fujita Tomonobu, Akada Masanori, Kiniwa Yukiko, Tsukamoto Makoto, Yamamoto Aiko, Matsuzaki Yuriko, Matsushita Maiko, Asano Takako, Nakashima Jun, Tachibana Masaaki, Hayakawa Masamichi, Ikeda Hideyuki, Murai Masaru, Kawakami Yutaka

International Journal of Cancer 108 ( 5 ) 712 - 724 2004.02

ISSN  0020-7136

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<p>To identify tumor antigens useful for the diagnosis and treatment of patients with bladder cancer, a lambda phage cDNA library constructed from a high-grade bladder cancer cell line was screened with autologous serum from a patient with metastatic bladder cancer. Forty-eight distinct antigens were isolated. By evaluating the immunogenicity and the tissue-specific expression, KU-BL-1 and KU-BL-2 were identified as immunogenic antigens with restricted tissue expression. KU-BL-1 was found to be a putative human lipoic acid synthetase with a metal-binding site, CXXXCXXC, that was expressed in bladder cancer cell lines and most bladder cancer tissues, as well as normal bladder mucosa and testis tissues. Immunoglobulin (Ig)G antibody to KU-BL-1 was detected in 2 of 28 patients with bladder cancer, but not in 30 healthy individuals. KU-BL-2 was found to be a putative human kelch-like protein that was homologous to Drosophila kelch, with a BTB/POZ domain and kelch repeats. KU-BL-2 was expressed in bladder cancer cell lines, most bladder cancer tissues, testis and heart, but not in normal bladder mucosa. IgG antibody to KU-BL-2 was detected in 8 of 28 patients with bladder cancer, but not in 16 healthy individuals. Tumor reactive T cells were induced from peripheral blood mononuclear cells (PBMC) by stimulation with one of the HLA-A24 binding KU-BL-2 peptides. Therefore, KU-BL-1 and KU-BL-2, which showed preferential expression in bladder cancer with restricted expression in normal tissues, as well as immunogenicity in multiple patients with bladder cancer, may be useful for the development of diagnostic and therapeutic methods for patients with bladder cancer.</p>

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Identification of a human glioma antigen, SOX6, recognized by patients' sera

Ueda Ryo, Iizuka Yukihiko, Yoshida Kazunari, Kawase Takeshi, Kawakami Yutaka, Toda Masahiro

Oncogene 23 ( 7 ) 1420 - 1427 2004.02

ISSN  0950-9232

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<p>To identify tumor antigens for glioma, a human testis cDNA library was screened by serological identification of antigens by recombinant expression cloning with sera from glioma patients. In this screening, the most frequently isolated antigen was SOX6, an Sry-related high-mobility group (HMG) box-containing gene. SOX6 is a transcriptional factor that is specifically expressed in the developing central nervous system and in the early stages of chondrogenesis in mouse embryos. IgG antibodies against SOX6 were detected in sera from 12 of 36 glioma patients (33.3%), 0 of 14 patients with other brain disease (0%), and one of 54 other cancer patients (1.9%). In sera from 37 healthy individuals, no IgG responses against SOX6 were detected, except in an elderly female. Furthermore, Western blot and ELISA analyses with sera from glioma patients revealed that the DNA-binding domain, the HMG box of SOX6, might be a dominant epitope of IgGs against SOX6. RT-PCR and Northern blot analysis revealed that the SOX6 gene was more highly expressed in glioma tissues than in normal adult tissues, except testis. Western blot analysis with an anti-SOX6 antibody demonstrated that the SOX6 protein was expressed in glioma tissues, but not in normal adult brain tissue. Immunohistochemical analysis with the anti-SOX6 antibody showed that all the glioma tissues analysed expressed SOX6 in tumor cells, but only a few SOX6-positive cells were detected in non-neoplastic tissues from the cerebral cortex. In summary, these results indicate that the developmentally regulated transcription factor SOX6 is aberrantly expressed in glioma and specifically recognized by IgGs from glioma patients' sera.</p>

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Quantitative analysis of PRAME for detection of minimal residual disease in leukemia.

Matsushita Maiko, Yamazaki Rie, Kawakami Yutaka

Methods in molecular medicine 97   267 - 275 2004

ISSN  1543-1894

Immunohistochemical analysis of SOX6 expression in human brain tumors

Ueda Ryo, Yoshida Kazunari, Kawakami Yutaka, Kawase Takeshi, Toda Masahiro

Brain Tumor Pathology 21 ( 3 ) 117 - 120 2004

ISSN  1433-7398

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<p>We previously demonstrated that the developmentally regulated gene, SOX6, is strongly expressed in glioma cells and in the fetal brain, but only faintly in the normal adult brain. Recent studies have indicated that brain tumor cells may share antigens, signaling systems, and behavior with neural stem/progenitor cells. To test the validity of this proposition, we analyzed the expression of SOX6 in various human central nervous system (CNS) tumors. Immunohistochemical analysis revealed that astrocytic and oligodendroglial tumors expressed SOX6; neuronal-glial cell tumors (central neurocytoma) and embryonal tumors (medulloblastoma), which arise from multipotential stem cell precursors, also showed a high intensity of SOX6 staining. In contrast, ependymal tumors (ependymoma and subependymoma), meningioma, and schwannoma, which are all well differentiated tumors, showed either no staining or only faint staining for SOX6. These results suggest that SOX6 may be expressed in bipotential or multipotential cells capable of neuronal and glial differentiation, but not in fully differentiated cells. SOX6 may be a useful marker for the diagnosis of tumors arising from immature bipotential cells that may differentiate into neuronal and glial cells.</p>

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Expression of a transcriptional factor, SOX6, in human gliomas

Ueda Ryo, Yoshida Kazunari, Kawakami Yutaka, Kawase Takeshi, Toda Masahiro

Brain Tumor Pathology 21 ( 1 ) 35 - 38 2004

ISSN  1433-7398

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<p>By screening a human testis cDNA library with glioma patients' sera, we isolated a transcriptional factor, SOX6. Here, we analyzed SOX6 expression in gliomas having a range of malignancy grades using immunostaining. Murine Sox6 is a transcriptional factor that is specifically expressed in the developing central nervous system and in the early stages of chondrogenesis in mouse embryos. The reverse transcription-polymerase chain reaction (RT-PCR) revealed that the SOX6 gene was more highly expressed in glioma tissues and fetal brain than in normal adult brain and other cancer cells, except melanoma cells. Immunohistochemical analysis with the anti-SOX6 antibody showed that all the glioma tissues analyzed (14 glioblastomas, 14 anaplastic astrocytomas, 3 anaplastic oligoastrocytomas, 5 diffuse astrocytomas, 1 oligodendroglioma, and 1 pilocytic astrocytoma) expressed SOX6 in tumor cells, but only a few SOX6-positive cells were detected in nonneoplastic tissues from the cerebral cortex. These results indicate that the developmentally regulated transcription factor SOX6 may be a potential diagnostic marker for gliomas.</p>

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Development of immunotherapy for cancer

Kawakami Yutaka

Nihon Rinshō Men'eki Gakkai kaishi = Japanese journal of clinical immunology 27 ( 2 ) 87 - 98 2004

ISSN  0911-4300

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<p>Identification of human melanoma antigens by various molecular biological and immunological techniques and evaluation of tumor reactive T cells in patients with the identified tumor antigen and HLA tetramer technology, not only provided us more profound understanding of anti-tumor immune responses in human, but also led to reveal basic problems in each step towards immunological tumor rejection, including systemic suppressive mechanisms such as regulatory T cell induction and local inhibitory environment in tumors. Based on these results obtained from the basic and clinical researches, various improvements have been applied for immunotherapy, including active immunization with modified antigenic peptides and recombinant virus, T cell adoptive transfer with lymphodepletive pretreatment, and administration of anti-CTLA-4 Ab, although further improvement is necessary. The translational research performed on melanoma, would facilitate development of immunotherapy for other cancers.</p>

A Novel Protein Highly Expressed in Testis Is Overexpressed in Systemic Sclerosis Fibroblasts and Targeted by Autoantibodies

Yasuoka Hidekata, Ihn Hironobu, Medsger Thomas A., Hirakata Michito, Kawakami Yutaka, Ikeda Yasuo, Kuwana Masataka

Journal of Immunology 171 ( 12 ) 6883 - 6890 2003.12

ISSN  0022-1767

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<p>Nearly all autoantibody specificities in sera from patients with systemic sclerosis (SSc) target proteins distributed ubiquitously, and Abs against proteins whose expression is restricted to the affected sites have not been identified. In this study we describe SSc-specific autoantibody to a novel testicular Ag, termed protein highly expressed in testis (PHET), which is ectopically over-expressed in SSc dermal fibroblasts. A partial cDNA encoding PHET was isolated by immunoscreening of a HepG2 cDNA library with an SSc serum. PHET appeared to be a member of the UniGene cluster Hs.129872, but had a unique exon composition and a characteristic mRNA expression profile restricted to the testis. Serum Abs to a recombinant PHET fragment were detected in nine (8.4%) of 107 SSc patients, but in none of 50 systemic lupus erythematosus patients or 77 healthy controls. In SSc patients, the presence of anti-PHET Abs was associated with diffuse cutaneous SSc and lung involvement (p = 0.02 and 0.01, respectively). PCR-based quantitative analysis of PHET mRNA expression in cultured dermal fibroblasts showed increased expression of PHET mRNA in SSc fibroblasts compared with control fibroblasts. PHET-reactive Abs purified from SSc sera stained the cytoplasm of SSc dermal fibroblasts, and the staining intensity tended to be more prominent on SSc compared with control fibroblasts. These findings suggest that the autoantibody response to PHET can be induced by ectopic overexpression of PHET in dermal fibroblasts in SSc patients.</p>

HLA Class II Alleles in Systemic Sclerosis Patients with Anti-RNA Polymerase I/III Antibody

Kuwana Masataka, Pandey Janardan P., Silver Richard M., Kawakami Yutaka, Kaburaki Junichi

Journal of Rheumatology 30 ( 11 ) 2392 - 2397 2003.11

ISSN  0315-162X

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<p>Objective. To examine HLA class II gene associations with anti-RNA polymerase (RNAP) I/III antibody responses in patients with systemic sclerosis (SSc). Methods. HLA-DRB1, DRB3, DRB4, and DQB1 alleles were determined using polymerase chain reaction-based methods in 257 SSc patients (129 Japanese and 128 Caucasians) and 271 race-matched regional controls (138 Japanese and 133 Caucasians). Anti-RNAP I/III antibodies were identified by immunoprecipitation assay, and reactivities to individual RNAP subunits were determined by immunoblots using affinity-purified RNAP I, II, and III. Results. Serum anti-RNAP I/III antibody was detected in 10 (8%) Japanese and 24 (19%) Caucasian patients with SSc. The presence of anti-RNAP I/III antibodies was associated with DRB1*0405, DRB4*01, and DQB1*0401 in Japanese, and with DRB3*02 in Caucasians, but these associations were weak and inconsistent between these 2 ethnic groups. When anti-RNAP I/III-positive SSc patients were divided into 2 groups based on the presence or absence of reactivities to individual RNAP subunit proteins, significant associations of anti-IIa/IIo reactivity with DRB3*02, anti-Ia reactivity with DRB1*04, anti-43-kDa subunit reactivity with DRB4*01, and anti-34-kDa subunit reactivity with DRB1*15 were detected. These HLA associations with subunit reactivities were generally shared by Japanese and Caucasian patients with SSc. Conclusion. Our results suggest that in patients with SSc, anti-RNAP I/III antibodies are composed of subsets defined by combinations of reactivities to individual RNAP subunits having specific HLA class II correlations.</p>

HLA class II alleles in systemic sclerosis patients with anti-RNA polymerase I/III antibody: associations with subunit reactivities.

Kuwana M, Pandey JP, Silver RM, Kawakami Y, and Kaburaki J.

Journal of Rheumatology. 30 ( 11 ) 2392-2397 2003.11

Research paper (scientific journal), Joint Work

Human circulating CD14⁺ monocytes as a source of progenitors that exhibit mesenchymal cell differentiation

Kuwana Masataka, Okazaki Yuka, Kodama Hiroaki, Izumi Keisuke, Yasuoka Hidekata, Ogawa Yoko, Kawakami Yutaka, Ikeda Yasuo

Journal of Leukocyte Biology 74 ( 5 ) 833 - 845 2003.11

ISSN  0741-5400

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<p>Circulating CD14⁺ monocytes are precursors of phagocytes, such as macrophages and dendritic cells. Here we report primitive cells with a fibroblast-like morphology derived from human peripheral blood CD14⁺ monocytes that can differentiate into several distinct mesenchymal cell lineages. We named this cell population monocyte-derived mesenchymal progenitor (MOMP). MOMPs were obtained in vitro from human peripheral blood mononuclear cells cultured on fibronectin in the presence of fetal bovine serum alone as a source of growth factors. MOMPs had a unique molecular phenotype-CD14 ⁺CD45⁺CD34⁺type I collagen⁺-and showed mixed morphologic and molecular features of monocytes and endothelial and mesenchymal cells. MOMPs were found to be derived from a subset of circulating CD14⁺ monocytes, and their differentiation required that they bind fibronectin and be exposed to one or more soluble factors derived from peripheral blood CD14^- cells. MOMPs could be expanded in culture without losing their original phenotype for up to five passages. The induction of MOMPs to differentiate along multiple limb-bud mesodermal lineages resulted in the expression of genes and proteins specific for osteoblasts, skeletal myoblasts, chondrocytes, and adipocytes. Our findings represent the first evidence that human circulating CD14⁺ monocytes are a source of progenitors that exhibit mesenchymal cell differentiation.</p>

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Results of a phase I clinical study using autologous tumour lysate-pulsed monocyte-derived mature dendritic cell vaccinations for stage IV malignant melanoma patients combined with low dose interleukin-2

Nagayama Hitomi, Sato Katsuaki, Morishita Mariko, Uchimaru Kaoru, Oyaizu Naoki, Inazawa Takeshi, Yamasaki Tomoko, Enomoto Makoto, Nakaoka Takashi, Nakamura Tetsuya, Maekawa Taira, Yamamoto Akifumi, Shimada Shinji, Saida Toshiaki, Kawakami Yutaka, Asano Shigetaka, Tani Kenzaburo, Takahashi Tsuneo A., Yamashita Naohide

Melanoma Research 13 ( 5 ) 521 - 530 2003.10

ISSN  0960-8931

　View Summary

<p>We conducted a pilot study to assess the feasibility and efficacy of immunotherapy for stage IV malignant melanoma patients resistant to conventional therapies involving vaccination with mature dendritic cells (mDCs) combined with administration of low dose interleukin-2, Autologous monocytes were harvested from a single apheresis and cultured for 7 days with granulocyte-macrophage colony-stimulating factor and interleukin-4, yielding immature dendritic cells (iDCs), which were then cryopreserved until use. For 4 days prior to vaccination, iDCs were exposed to autologous tumour lysate combined with tumour necrosis factor-α to induce terminal differentiation into mDCs. Patients were then vaccinated weekly with 10⁷ mDCs for 10 weeks and given 350-700 kIU of interleukin-2 three times per week. Of the 10 patients in the study, one showed stable disease, seven showed progressive disease, and two showed mixed responses, including partial tumour regression, and were therefore given 20 additional injections. Only minimal adverse events were noted, including localized skin reactions and mild fever (NIH-CTC grade 0-1). Median survival from the first vaccination was 240 days (range 31-735 days). In vitro, melanoma patient-derived dendritic cells (DCs) showed reduced cell surface expression of CD1a antigen on iDCs and reduced CD86 and HLA-DR expression on mDCs. In addition, antigen uptake, chemotaxis and antigen presentation were all attenuated in DCs from the patients. In summary, although improvement of clinical efficacy will require further research, autologous tumour lysate-pulsed monocytederived mDCs could be safely harvested, cryopreserved and administrated to patients without obvious complications.</p>

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Tumor-specific immunological recognition of frameshift-mutated peptides in colon cancer with microsatellite instability

Ishikawa Toshiaki, Fujita Tomonobu, Suzuki Yuriko, Okabe Satoshi, Yuasa Yasuhito, Iwai Takehisa, Kawakami Yutaka

Cancer Research 63 ( 17 ) 5564 - 5572 2003.09

ISSN  0008-5472

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<p>Colorectal cancers with microsatellite instability (MSI+ CRCs) caused by dysfunction of DNA mismatch repair have unique clinicopathological characteristics including good prognosis with T-cell infiltration in tumor. To identify tumor antigens that induce immune response against MSI+ CRC, SEREX (serological analysis of recombinant cDNA expression cloning) was applied. By screening a λ phage cDNA library constructed from three MSI+ CRC cell lines with serum from a patient with MSI+ CRC with abundant T-cell infiltrates in tumor, 64 antigens were isolated. Immunogenicity of each antigen was evaluated by screening sera from patients with various cancers and from healthy individuals, and specific IgG antibodies (Abs) for 49 antigens were detected only in MSI+ CRC patients. A frameshift mutation in the repetitive G sequences (microsatellite) in the coding region of CDX2, one of the identified antigens, was found in the tumor tissue of the patient who had anti-CDX2 serum Ab. The Ab recognized both the COOH-terminal tumor-specific peptides created by the frameshift mutation and the NH₂-terminal normal peptides of CDX2 when Western blot analysis was performed using various bacterial recombinant CDX2 proteins including the normal and altered peptides, which indicated that immune response could be raised against tumor-specific peptides generated through MSI. The anti-CDX2 Ab was detected only in the patient with the CDX2 frameshift mutation in tumor and disappeared 7 years after the curative resection, suggesting that this immune response may also be useful as a tumor marker. No altered subcellular localization and transcription ability was demonstrated in the mutated CDX2, although decreased expression was suggested in immunohistochemical analysis. Therefore, tumor-specific peptides generated by MSI may be involved in antitumor immune responses and may be useful for the development of diagnostic and therapeutic methods for patients with MSI+ CRC.</p>

A role of autoantibody-mediated platelet destruction in thrombocytopenia in patients with cirrhosis

Kajihara Mikio, Kato Shinzo, Okazaki Yuka, Kawakami Yutaka, Ishii Hiromasa, Ikeda Yasuo, Kuwana Masataka

Hepatology 37 ( 6 ) 1267 - 1276 2003.06

ISSN  0270-9139

　View Summary

<p>Thrombocytopenia is a common manifestation in patients with liver cirrhosis (LC), but its underlying mechanism remains controversial. This study examined the role of anti-platelet autoimmunity in cirrhotic thrombocytopenia by determining the autoantibody response to GPIIb-IIIa, a major platelet surface autoantigen recognized by anti-platelet antibodies in patients with idiopathic thrombocytopenic purpura (ITP). Circulating B cells producing anti-GPIIb-IIIa antibodies as well as platelet-associated and plasma anti-GPIIb-IIIa antibodies were examined in 72 patients with LC, 62 patients with ITP, and 52 healthy controls. In vitro anti-GPIIb-IIIa antibody production was induced in cultures of peripheral blood mononuclear cells (PBMCs) by stimulation with GPIIb-IIIa. The frequency of anti-GPIIb-IIIa antibody-producing B cells in patients with LC was significantly greater than in healthy controls (10.9 ± 6.2 vs. 0.4 ± 0.3/10⁵ PBMCs; P &lt; .0001) and was even higher than the frequency in patients with ITP (8.2 ± 5.2; P = .007). Anti-GPIIb-IIIa antibodies in the patients with LC and ITP were mainly present on the surfaces of circulating platelets rather than in the plasma in an unbound form. Furthermore, PBMCs from patients with LC and ITP produced anti-GPIIb-IIIa antibodies on antigenic stimulation with GPIIb-IIIa in vitro, and the specific antibodies produced had the capacity to bind normal platelet surfaces. In conclusion, the similar profile of the anti-GPIIb-IIIa autoantibody response in patients with LC and ITP suggests that autoantibody-mediated platelet destruction may contribute at least in part to cirrhotic thrombocytopenia.</p>

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Identification of human tumor antigens recognized by T-cells and their use for immunotherapy

Kawakami Yutaka

International Journal of Hematology 77 ( 5 ) 427 - 434 2003.06

ISSN  0925-5710

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<p>Identification of human tumor antigens led to development of new immunotherapies for patients with cancer. Identification of T-cell epitopes allowed us to monitor antitumor T-cell responses quantitatively and qualitatively during immunotherapy as well as to develop more efficient immunotherapies with sufficient amounts of antigens in more immunogenic forms. Various immunotherapies, passive immunotherapies, including adoptive transfer of tumor reactive T-cells or allogeneic antigen-specific donor T-cells, and active immunization with identified tumor antigens or dendritic cells pulsed with tumor antigens are being evaluated in clinical trials. Although tumor regression has been observed in some patients, further improvement is required.</p>

Genetic reversion in an acute myelogenous leukemia cell line from a Fanconi anemia patient with biallelic mutations in BRCA2

Ikeda Hideyuki, Matsushita Maiko, Waisfisz Quinten, Kinoshita Akitoshi, Oostra Anneke B., Nieuwint Aggie W M, De Winter Johan P., Hoatlin Maureen E., Kawai Yohko, Sasaki Masao S., D'Andrea Alan D., Kawakami Yutaka, Joenje Hans

Cancer Research 63 ( 10 ) 2688 - 2694 2003.05

ISSN  0008-5472

　View Summary

<p>A 2-year old boy was diagnosed with Fanconi anemia (FA) and acute myeloid leukemia (AML). A cell line (termed FA-AML1) was established from blast cells obtained after a second relapse after a successful bone marrow transplant. Histochemical and surface marker analysis confirmed that the cells were derived from the myeloid lineage. Cytogenetic analysis revealed multiple chromosomal aberrations, including a ring 7. Stable proliferation of the cultured cells was absolutely dependent on the presence of granulocyte macrophage colony-stimulating factor or interleukin 3. This is the first AML cell line successfully established from a FA patient. Remarkably, FA-AML1 cells appeared to lack the characteristic cellular FA phenotype, i.e., a hypersensitivity to growth inhibition and chromosomal breakage by the cross-linking agent mitomycin C. Genomic DNA from the patient showed biallelic mutations [8415G&gt;T (K2729N)and 8732C&gt;A (S2835STOP)] in the breast cancer susceptibility gene FANCD1/BRCA2 [N. Howlett et al., Science (Wash. DC), 297: 606-609, 2002]. In the AML cells, however, the 8732C&gt;A nonsense mutation was changed into a missense mutation by a secondary alteration, 8731T&gt;G, resulting in 2835E, which restored the open-reading frame of the gene and could explain the reverted phenotype of these cells. Loss of the FA phenotype by genetic correction of a FA gene mutation during AML progression may be a common late event in the pathogenesis of AML in FA patients, which may be treatment related. This finding suggests a novel mechanistic principle of tumor progression based on the genetic correction of an early caretaker gene defect.</p>

Periductal area as the primary site for T-cell activation in lacrimal gland chronic graft-versus-host disease

Ogawa Yoko, Kuwana Masataka, Yamazaki Kazuto, Mashima Yukihiko, Yamada Masakazu, Mori Takehiko, Okamoto Shinichiro, Oguchi Yoshihisa, Kawakami Yutaka

Investigative Ophthalmology and Visual Science 44 ( 5 ) 1888 - 1896 2003.05

ISSN  0146-0404

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<p>PURPOSE. To examine immune processes in the lacrimal gland of patients with chronic graft-versus-host disease (cGVHD) by evaluating the expression of surface molecules associated with T-cell activation. METHODS. Antibodies to CD4, CD8, CD34, CD40, CD54, CD80, CD86, CD154, and HLA-DR were used for immunohistochemical analysis of lacrimal gland biopsy specimens obtained from nine patients with cGVHD and five with Sjögren's syndrome (SS). The regions of interest were further assessed by transmission electron microscopy. RESULTS. CD4⁺ and CD8⁺ T cells were mainly detected in the periductal areas of the glands of patients with cGVHD, but were distributed throughout the acinar areas in patients with SS. In the periductal areas of patients with cGVHD, a subpopulation of CD4⁺ and CD8⁺ T cells expressed the activation marker CD154. In addition, CD4⁺ and CD8⁺ T cells were colocalized with mononuclear infiltrates and stromal fibroblasts expressing the full component of surface molecules necessary for antigen presentation, including HLA-DR, CD54, CD40, CD80, and CD86. Electron microscopy revealed activated fibroblasts that embraced lymphocytes and macrophages with their processes. Also, there were more CD8⁺ T cells in the glandular epithelia of patients with cGVHD than in those with SS. Intraepithelial T cells were attached to epithelial cells by several primitive contacts and colocalized with dead cells. CONCLUSIONS. The results strongly suggest that CD4⁺ and CD8⁺ T cells in the lacrimal glands of patients with cGVHD are primarily activated in the periductal area through antigenic stimulation by potent antigen-presenting cells and stromal fibroblasts, and exert various effector functions, including cytotoxic effects on glandular epithelial cells.</p>

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Autoreactive T cells to the P3A⁺ isoform of AChR α subunit in myasthenia gravis

Suzuki Shigeaki, Tanaka Kortaro, Yasuoka Hidekata, Fukuuchi Yasuo, Kawakami Yutaka, Kuwana Masataka

Journal of Neuroimmunology 137 ( 1-2 ) 177 - 186 2003.04

ISSN  0165-5728

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<p>In vitro T cell proliferative response to an alternative splicing variant of acetylcholine receptor α subunit (AChRα) with the P3A exon-encoded region was examined in peripheral blood samples from 28 myasthenia gravis (MG) patients and 14 healthy donors using recombinant fragments and synthetic peptides. T cells responsive to the P3A region-specific sequences were detected in five MG patients, all of whom were late-onset disease with thymoma, but in none of healthy donors. These autoreactive T cells may be involved in the pathogenic process in a subset of MG patients.</p>

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Preferentially expressed antigen of melanoma (PRAME) in the development of diagnostic and therapeutic methods for hematological malignancies

Matsushita Maiko, Yamazaki Rie, Ikeda Hideyuki, Kawakami Yutaka

Leukemia and Lymphoma 44 ( 3 ) 439 - 444 2003.03

ISSN  1042-8194

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<p>PRAME (Preferentially expressed antigen of melanoma), highly expressed in various solid tumor cells and normal testis, was first isolated as a human melanoma antigen recognized by cytotoxic T cells (CTL). This gene was also expressed in some of the hematological malignancies, including acute myelogenous leukemia (AML) and multiple myeloma. We and others have extensively evaluated the PRAME expression in various hematological malignancies and demonstrated high expression of the PRAME gene in subsets of AML, chronic myelogenous leukemia, acute lymphocytic leukemia, lymphoma and multiple myeloma. In addition, we have demonstrated that PRAME was a useful marker for detection of minimal residual disease (MRD) in patients with leukemia, particularly those leukemias in which tumor specific markers are currently unavailable. Since PRAME was first identified as a tumor antigen recognized by T cells, the possibility that PRAME is a leukemia antigen recognized by T cells was evaluated, and it was found that PRAME-positive leukemia cell lines and fresh leukemia cells were susceptible to lysis by the PRAME-specific CTL. Five CTL epitopes associated with either HLA-A*0201 or HLA-A*2402 have recently been identified. It is, therefore, an attractive strategy to apply PRAME specific immunotherapy on patients with PRAME positive leukemia in MRD condition.</p>

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Expression of mRNAs for telomeric repeat binding factor (TRF)-1 and TRF2 in atypical adenomatous hyperplasia and adenocarcinoma of the lung

Nakanishi Kuniaki, Kawai Toshiaki, Kumaki Fumiyuki, Hiroi Sadayuki, Mukai Makio, Ikeda Eiji, Koering CElaine, Gilson Eric

Clinical cancer research 9   1105-1111 2003.03

Research paper (scientific journal), Joint Work

Suppression of autoreactive T-cell response to glycoprotein IIb/IIIa by blockade of CD40/CD154 interaction

Kuwana Masataka, Kawakami Yutaka, Ikeda Yasuo

Blood 101 ( 2 ) 621 - 623 2003.01

ISSN  0006-4971

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<p>The potential immunosuppressive effect of an anti-CD154 monoclonal antibody (mAb) on the pathogenic autoreactive T-cell response was evaluated using an in vitro culture system with glycoprotein IIb/IIIa (GPIIb/IIIa)-reactive T cells from patients with immune thrombocytopenic purpura (ITP). The anti-CD154 mAb did not inhibit T-cell proliferation, but suppressed anti-GPIIb/IIIa antibody production, in bulk peripheral blood mononuclear cell cultures stimulated with GPIIb/IIIa. Repeated antigenic stimulation of GPIIb/IIIa-reactive CD4⁺ T-cell lines in the presence of anti-CD154 mAb resulted in the loss of proliferative capacity and helper function for promoting anti-GPIIb/IIIa antibody production. These anergic T-cell lines showed a cytokine profile of low interferon γ and high interleukin 10 and suppressed anti-GPIIb/IIIa antibody production. Our results indicate that blockade of the CD40/CD154 interaction induces generation of autoantigen-specific anergic CD4⁺ T cells with regulatory function and could be a therapeutic option for suppressing pathogenic autoimmune responses in patients with ITP.</p>

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Cellular immunotherapy of cancer

Kawakami Yutaka

[Rinshō ketsueki] The Japanese journal of clinical hematology 44 ( 6 ) 358 - 367 2003

ISSN  0485-1439

Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells

Sumimoto Hidetoshi, Tsuji Takashi, Miyoshi Hiroyuki, Hagihara Masao, Takada-Yamazaki Rie, Okamoto Shin Ichiro, Ikeda Yasuo, Takahashi Tsuneo, Kawakami Yutaka

Journal of Immunological Methods 271 ( 1-2 ) 153 - 165 2002.12

ISSN  0022-1759

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<p>Since dendritic cells (DC) play pivotal roles in both innate and adaptive immunity, DC can be a good target for immuno-gene therapy. However, the optimal generation method for gene-modified DC has not yet been well exploited. CD34+ cells from cord blood (CB), bone marrow (BM), or peripheral blood (PB) were expanded in a medium containing stem cell factor (SCF), flt 3 ligand (Flt3L) and thrombopoietin (TPO) with or without HESS-5, a murine BM stromal cell line, for 2 weeks (the first expansion step), then differentiated to DC in a medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF), flt 3 ligand (Flt3L), stem cell factor (SCF), tumor necrosis factor-α (TNF-α), IL-4, and lipopolysaccharide (LPS) for 9 days (the second differentiation step). DC progenitors were transduced with human immunodeficiency virus (HIV) vectors at different time points during the second step. Use of HESS-5 during the first step resulted in more DC generation than without it (cell expansion: CB, 10,461 vs. 354-fold; BM, 962 vs. 225-fold; peripheral blood mononuclear cell (PBMC), 8,506 vs. 240-fold; %DC: CB, 83.4% vs. 76.9%; BM, 83.6 vs. 69.8%; PBMC, 85.9 vs. 60.5%). Gene transduction to the in vitro expanded DC progenitors at day 3 during the second step, resulted in better final yield of the gene-modified DC than that to those at day 0 or day 6 (as much as 44% of DC expressed green fluorescence protein (GFP) as a transgene) and the transduction efficiency correlated with endocytic ability and percent of S phase. DC transduced with an HIV vector encoding a melanoma antigen, MART-1, were adequately recognized by specific anti-MART-1 CTL. The two-step culture method with HESS-5 is useful for rapid expansion of DC progenitors and subsequent lentiviral gene transduction to DC.</p>

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Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells

Sumimoto Hidetoshi, Tsuji Takashi, Miyoshi Hiroyuki, Hagihara Masao, Takada-Yamazaki Rie, Okamoto Shinichiro, Ikeda Yasuo, Takahashi Tsuneo, Kawakami Yutaka

Journal of immunological methods 271 ( 1-2 ) 153-165 2002.12

Research paper (scientific journal), Joint Work

Identification of an immunodominant epitope on RNA polymerase III recognized by systemic sclerosis sera

Kuwana Masataka, Kimura Kyoko, Kawakami Yutaka

Arthritis and Rheumatism 46 ( 10 ) 2742 - 2747 2002.10

ISSN  0004-3591

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<p>Objective. To characterize an immunodominant epitope on RNA polymerase III (RNAP III) recognized by systemic sclerosis (SSc) sera and to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of serum anti-RNAP I/III antibodies. Methods. RNAP III-specific subunits RPC62 and RPC155 were generated in a bacterial expression system as a series of recombinant fragments. Reactivities to these recombinant fragments were examined by immunoblots and/or ELISA in 16 SSc sera containing anti-RNAP I/III antibodies, 89 SSc sera lacking anti-RNAP I/III antibodies, 61 systemic lupus erythematosus (SLE) sera, and 61 healthy control sera. Results. Anti-RNAP I/III-positive SSc sera recognized several distinct epitopes on RPC62 and RPC155 in various combinations, but the fragment encoding amino acids at positions 732-1166 of RPC155 was recognized by all 11 anti-RNAP I/III-positive SSc sera tested. Carboxyl- and amino-terminal deletion studies showed that at least 130 amino acids at positions 891-1020 of RPC155 were necessary for the antibody binding, but strong reactivity required an additional amino-terminal extension. When a purified recombinant fragment containing the immunodominant epitope was used as the antigen source in an ELISA, elevated antibody reactivity was detected in all 16 anti-RNAP I/III-positive SSc sera, but in no anti-RNAP I/III-negative SSc, SLE, or healthy control sera, representing a sensitivity of 100% and a specificity of 100%. Conclusion. A major epitope commonly recognized by SSc sera containing anti-RNAP I/III antibodies was identified on RPC155. The ELISA using a recombinant fragment expressing the immunodominant epitope should be a valuable tool for routine screening for anti-RNAP I/III antibodies in clinical diagnostic laboratories.</p>

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Differences in autoantibody response to Th/To between systemic sclerosis and other autoimmune diseases

Kuwana M., Kimura K., Hirakata M., Kawakami Y., Ikeda Y.

Annals of the Rheumatic Diseases 61 ( 9 ) 842 - 846 2002.09

ISSN  0003-4967

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<p>Objective: To examine differences in autoantibody response and immunogenetic background between patients with systemic sclerosis (SSc) and those with other autoimmune diseases who had serum anti-Th/To antibodies. Methods: Serum samples from 1048 Japanese patients with various autoimmune diseases were screened for anti-Th/To antibodies using RNA and protein immunoprecipitation assays. The reactivity to RNase P subunits was examined by immunoprecipitating ³⁵S labelled recombinant Rpp38, Rpp30, and hPop1 produced by in vitro transcription and translation. HLA-DRB1, DQB1, and DPB1 alleles were identified using a polymerase chain reaction followed by a restriction fragment length polymorphism assay. Results: Serum anti-Th/To antibodies were detected in 14 of 303 patients with SSc and seven of 745 patients without SSc (4.6% v 0.9%; p=0.0003). Similar percentages of patients with and without SSc showed immunoreactivity to Rpp38 and Rpp30, but more patients with SSc than patients without SSc showed a reactivity to hPop1 (93% v 14%; p=0.002). In patients with anti-Th/To antibodies DRB1*1502 or *0802 was detected more often, and the DRB1*0405-DQB1*0401 haplotype less often in patients with SSc than in patients without SSc (79% v 14%, p=0.02, and 7% v 71%, p=0.01, respectively). Conclusions: Anti-Th/To antibodies were detected in a small proportion of autoimmune patients lacking clinical features related to SSc. A close relationship between disease expression and anti-hPop1 reactivity as well as HLA class II alleles in anti-Th/To positive patients suggests that the process of anti-Th/To antibody production may be different between patients with and those without SSc.</p>

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Differences in autoantibody response to Th/To between systemic sclerosis and other autoimmune diseases

Kuwana Masataka, Kimura K, Hirakata M, Kawakami Yutaka, Ikeda Y

Annals of rheumatic disease 61 ( 9 ) 842-846 2002.09

Research paper (scientific journal), Joint Work

Identification of tumor antigens recognized by T cells and their use for analysis of anti-tumor responses

Fujita Tomonobu, Kawakami Yutaka

Biotherapy 16 ( 5 ) 441 - 448 2002.09

ISSN  0914-2223

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<p>One of the problems in the development of immunotherapy in the past was poor evaluation of anti-tumor immune responses. Consequently, it was difficult to improve the immunotherapy. Various human tumor antigens recognized by T cells have recently been identified using various methods, including cDNA expression cloning with tumor reactive T cells and antibodies, as well as various cDNA subtraction methods in combination with in vitro T cell induction. The identification of tumor antigenic peptides recognized by T cells allows us to monitor anti-tumor T cell response quantitatively and qualitatively during immunotherapy as well as to develop more efficient immunotherapy with a sufficient amount of antigens in more immunogenic forms.</p>

Correspondence re

Fodor János, Kawakami Yutaka, Kiniwa Yukiko

Cancer Research 62 ( 16 ) 4836 - 4837 2002.08

ISSN  0008-5472

Autoantibody to c-Mpl (thrombopoietin receptor) in systemic lupus erythematosus

Kuwana Masataka, Okazaki Yuka, Kajihara Mikio, Kaburaki Junichi, Miyazaki Hiroshi, Kawakami Yutaka, Ikeda Yasuo

Arthritis and Rheumatism 46 ( 8 ) 2148 - 2159 2002.08

ISSN  0004-3591

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<p>Objective. To examine the prevalence, clinical associations, and pathogenic role of autoantibodies to c-Mpl, the thrombopoietin (TPO) receptor, in patients with systemic lupus erythematosus (SLE). Methods. Sera from 69 SLE patients, 84 patients with idiopathic thrombocytopenic purpura (ITP), and 60 healthy individuals were screened for anti-c-Mpl antibodies by enzyme-linked immunosorbent assay using recombinant c-Mpl as an antigen. Clinical findings, autoantibody profiles, and serum TPO levels were compared between SLE patients with and without anti-c-Mpl antibodies. A pathogenic role for the anti-c-Mpl antibody was evaluated by examining its inhibitory effect on TPO-dependent cell proliferation and megakaryocyte colony formation. Results. Serum anti-c-Mpl antibody was detected in 8 SLE patients (11.6%) and 7 ITP patients (8.3%), but in none of the healthy controls. Anti-c-Mpl antibody was associated with thrombocytopenia (P = 0.0002) and a decrease in bone marrow megakaryocytes (P = 0.02) in SLE patients. Serum TPO levels in thrombocytopenic SLE patients with anti-c-Mpl antibodies were significantly elevated compared with levels in those without the antibodies (P = 0.007). IgG fractions purified from anti-c-Mpl antibody-positive sera bound to c-Mpl expressed on the cell surface and inhibited TPO-dependent cell proliferation and megakaryocyte colony formation. Conclusion. Autoantibody to c-Mpl is present in a subset of SLE patients with thrombocytopenia and megakaryocytic hypoplasia. It is likely that the impaired thrombopoiesis in these patients is mediated by the anti-c-Mpl antibody, which functionally blocks an interaction between TPO and c-Mpl.</p>

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Autoantibody to c-Mpl (thrombopoietin receptor) in systemic lupus erythematosus: Relationship to thrombocytopenia with megakaryocytic hypoplasia

Kuwana Masataka, Okazaki Yuka, Kajihara Mikio, Kaburaki junichi, Miyazaki Hiroshi, Kawakami Yutaka, Ikeda Yasuo

Arthritis & rheumatism 46 ( 8 ) 2148-2159 2002.08

Research paper (scientific journal), Joint Work

A simple and reliable behavioral analysis of locomotor function after spinal cord injury in mice

Mikami Yuji, Toda Masahiro, Watanabe Masahiko, Nakamura Masaya, Toyama Yoshiaki, Kawakami Yutaka

Journal of neurosurgery 97 ( 1Suppl ) 142-147 2002.07

Research paper (scientific journal), Joint Work

Development of HLA-A alleles typing in Japanese using biotinylated dideoxynucleotide and TOF-MS sequencing

Dan Katsuaki, Kawamura Miki, Miyashita Kiichi, Kawakami Yutaka

Tissue antigens 59 ( Suppl. 2 ) 111-111 2002.05

Research paper (scientific journal), Joint Work

Development of HLA-A alleles typing in Japanese using biotinylated dideoxynucleotide and TOF- MAS sequencing

Dan Katsuaki, Kawamura Miki, Miyashita Kiichi, Kawakami Yutaka

Tissue antigens 59 ( Suppl. 2 ) 111 2002.05

Research paper (scientific journal), Joint Work

Restricted T-cell receptor β-chain usage by T cells autoreactive to β₂-glycoprotein I in patients with antiphospholipid syndrome

Yoshida Kazue, Arai Takahide, Kaburaki Junichi, Ikeda Yasuo, Kawakami Yutaka, Kuwana Masataka

Blood 99 ( 7 ) 2499 - 2504 2002.04

ISSN  0006-4971

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<p>We recently identified CD4⁺ T cells that are autoreactive to β₂-glycoprotein I (β₂GPI) and that promote antiphospholipid antibody production in patients with antiphospholipid syndrome (APS). In this study, T-cell receptor (TCR) β chains of β₂GPI-reactive T cells were examined in 8 β₂GPI-responders, including 5 patients with APS and 3 healthy subjects, using polymerase chain reaction and single-strand conformation polymorphism (PCRSSCP) analysis combined with in vitro stimulation of peripheral blood T cells with recombinant β₂GPI. The TCR Vβ segments that expanded oligoclonally after stimulation with β₂GPI varied among responders, but the Vβ7 and Vβ8 segments were commonly detected in 6 and 4 β2GPI-responders, respectively. Analysis of the complementarity-determining region 3 sequence of β₂GPI-reactive T cells revealed limited diversity, and all Vβ7⁺ TCRs had an amino acid motif of TGxxN/Q or minor variations. The Vβ8⁺ TCRs had another motif, PxAxxD/E. Surprisingly, an identical Vβ7⁺ TCRβ chain was used by β₂GPI-reactive T cells in 3 patients with APS. There was no apparent difference in the TCRβ usage between APS patients and healthy responders. Some of the Vβ7⁺ TCRs with the TGxxN/Q motif detected by PCR-SSCP analysis were also used by β₂GPI-specific CD4⁺ T-cell clones responsive to an immunodominant epitope containing the major phospholipid-binding site. Depletion of Vβ7+ or Vβ8⁺ T cells from the peripheral blood mononuclear cell cultures significantly inhibited in vitro anti-β₂GPI antibody production in response to β2GPI. Our results indicate preferential usage of TCRβ chains by β₂GPI-reactive T cells. These TCRβ chains can be reasonable targets for TCR-based immunotherapy for patients with APS.</p>

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Spleen is a primary site for activation of platelet-reactive T and B cells in patients with immune thrombocytopenic purpura

Kuwana Masataka, Okazaki Yuka, Kaburaki Junichi, Kawakami Yutaka, Ikeda Yasuo

Journal of Immunology 168 ( 7 ) 3675 - 3682 2002.04

ISSN  0022-1767

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<p>We have recently reported that in patients with chronic immune thrombocytopenic purpura (IMTP), circulating T and B cells that are responsive to gpIIb-IIIa can induce anti-platelet autoantibody production. In this study, the frequencies and activation status of gpIIb-IIIa-reactive T and B cells were evaluated in the peripheral blood and spleen obtained from nine IMTP patients undergoing splenectomy. There was no difference in gpIIb-IIIa-reactive T cell frequencies between peripheral blood and spleen (6.4 ± 2.6 vs 5.2 ± 2.4 per 10⁵ T cells), as determined by limiting dilution analysis, but activated T cells responsive to gpIIb-IIIa showing accelerated proliferation kinetics and those expressing CD154 were more frequent in spleen than in peripheral blood. The frequencies of anti-gpIIb-IIIa Ab-producing B cells, as determined by ELISPOT assay, were also similar in peripheral blood and spleen (61.2 ± 24.0 vs 77.7 ± 45.3 per 10⁵ B cells); however, an anti-gpIIb-IIIa Ab was spontaneously produced by splenocytes in vitro, but scarcely secreted by PBMCs. CD19^-/surface Ig^-/CD38⁺/CD138⁺ plasma cells secreting anti-gpIIb-IIIa Ab were exclusively detected in the spleen. In serial analysis, the frequencies of circulating gpIIb-IIIa-reactive T and B cells were decreased after splenectomy in patients with a complete response, but were unchanged in nonresponders. These findings indicate that an interaction between gpIIb-IIIa-reactive T and B cells inducing anti-platelet Ab production in IMTP patients occurs primarily in the spleen and that the significant number of gpIIb-IIIa-reactive T and B cells activated in the spleen are released into the circulation as memory cells.</p>

Restricted T cell receptor _-chain usage by T cells autoreactive to _2-glycoprotein I in patients with antiphospholipid syndrome

Yoshida Kazue, Araki Takashi, Kaburaki J, Ikeda Yasuo, Kawakami Yutaka, Kuwana Masataka

Blood 99 ( 7 ) 2499-2504 2002.04

Research paper (scientific journal), Joint Work

ミニ移植 ―Con―

Yamazaki Rie, Kawakami Yutaka

血液・免疫・腫瘍 7 ( 2 ) 202-206 2002.04

Research paper (scientific journal), Joint Work

Spleen is a primary site for activation of platelet-reactive T and B cells in patients with immune thrombocytopenic purpura¹

Kuwana Masataka, Okazaki Yuka, Kaburaki Junichi, Kawakami Yutaka, Ikeda Yasuo

The journal of immunology 168 ( 7 ) 3675-3682 2002.04

Research paper (scientific journal), Joint Work

In situcancer vaccination with a replication-conditional HSV for the treatment of liver metastasis of colon cancer

Endo Takashi, Toda Masahiro, Watanabe Masahiko, Iizuka Yukihiko, Kubota Tetsuro, Kitajima Masaki, Kawakami Yutaka

Cancer Gene Therapy 9 ( 2 ) 142-148 2002.02

Research paper (scientific journal), Joint Work

In situ cancer vaccination with a replication-conditional HSV for the treatment of liver metastasis of colon cancer

Endo Takashi, Toda Masahiro, Watanabe Masahiko, Iizuka Yukihiko, Kubota Tetsuro, Kitajima Masaki, Kawakami Yutaka

Cancer gene therapy 9 ( 2 ) 142-148 2002.02

Research paper (scientific journal), Joint Work

Immuno-viral therapy of brain tumors by combination of viral therapy with cancer vaccination using a replication-conditional HSV

Toda Masahiro, Iizuka Yukihiko, Kawase Takeshi, Uyemura Keiichi, Kawakami Yutaka

Cancer Gene Therapy 9 ( 4 ) 356 - 364 2002

ISSN  0929-1903

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<p>Here we developed an effective therapeutic approach using a replication-conditional mutant of herpes simplex virus (HSV), G207, for the treatment of metastatic tumors in the immunologically privileged central nervous system. An experimental model of brain metastasis was developed using BALB/c mice that harbored both intracranial (i.c.) and subcutaneous (s.c.) mouse CT26 colon adenocarcinoma tumors. Intratumoral injections of G207 into s.c. tumors elicited cytotoxic T-cell responses not only to HSV but also to a tumor antigen; however, only a limited antitumor effect was observed on metastatic brain tumors. To improve this antitumor effect, G207 was also injected into the brain tumor. After intratumoral injections of G207 into both i.c. and s.c. CT26 tumors, a significant antitumor effect was observed in the metastatic brain tumors. This therapeutic efficacy was absent in athymic mice, indicating that the antitumor effect could be mediated by T cells. Cytotoxic T-cell responses to HSV and the tumor antigen were induced by injections of G207 into i.c. and s.c. CT26 tumors. These results suggest that HSV-infected brain tumors may be efficiently eliminated by the induced anti-HSV T cells as well as by antitumor T cells. Therefore, this strategy of immuno-viral therapy, involving direct viral oncolytic activities and inducing antitumor and antiviral immune responses, may be useful for the treatment of tumors in the immunologically privileged central nervous system.</p>

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Dry eye associated with chronic graft-versus-host disease

Ogawa Y., Kuwana M., Yamazaki K., Mashima Y., Okamoto S., Tsubota K., Oguchi Y., Kawakami Y.

Advances in Experimental Medicine and Biology 506 B   1041 - 1045 2002

ISSN  0065-2598

In situ cancer vaccination with a replication-conditional HSV for the treatment of liver metastasis of colon cancer

Endo Takashi, Toda Masahiro, Watanabe Masahiko, Iizuka Yukihiko, Kubota Tetsuro, Kitajima Masaki, Kawakami Yutaka

Cancer Gene Therapy 9 ( 2 ) 142 - 148 2002

ISSN  0929-1903

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<p>In this study, we investigated the therapeutic efficacy of a replication-conditional mutant HSV, G207, for the treatment of liver metastasis of colon carcinoma. Three liver metastasis models in syngeneic BALB/c mice were developed: (i) splenic injection, (ii) splenic and subcutaneous (s.c.) injection, and (iii) orthotopic implantation of CT26 colon carcinoma. In the splenic injection model, G207 was injected into the established splenic tumor on day 7. In the splenic and s.c. injection model, G207 were injected into the established s.c. tumor on days 5 and 8. In the orthotopic implantation model, a piece of CT26 tumor tissue was transplanted onto the wall of the cecum and G207 was injected in the established cecum tumor on day 7. On day 21 or 28, animals were sacrificed and liver metastases were evaluated. In all three models in immunocompetent mice, liver metastases were significantly reduced by intratumoral inoculation with G207 compared to the control. In athymic mice, however, there was no significant therapeutic effect of intratumoral inoculation with G207 on liver metastases. Tumor-specific cytotoxic T-lymphocyte responses were induced in mice treated with G207 in the orthotopic implantation model. These results suggest that intratumoral inoculation of G207, as an in situ cancer vaccine, can be an effective approach against liver metastasis of colon cancer and the efficacy involves tumor-specific T-cell responses.</p>

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A simple and reliable behavioral analysis of locomotor function after spinal cord injury in mice

Mikami Yuji, Toda Masahiro, Watanabe Masahiko, Nakamura Masaya, Toyama Yoshiaki, Kawakami Yutaka

Journal of Neurosurgery 97 ( 1 SUPPL. ) 142 - 147 2002

ISSN  0022-3085

　View Summary

<p>To establish a simple and reliable method to assess the behavioral function after spinal cord injury (SCI) in mice, the authors used an automated animal movement analysis system, SCANET. Two different SCI lesions were created in adult female BALB/c and C57BL/6 mice by transecting both the posterior columns and the left lateral and anterior funiculi or only the left lateral and anterior funiculi at T-8. Control mice underwent laminectomy only. The SCANET system consists of a cage equipped with two crossing sensor frames arranged at different heights, by which small (MI) and large (M2) horizontal movements and the vertical movement involved in rearing (RG) can be monitored. The authors assessed locomotor function by determining the M1, M2, and RG scores; to this end, they used the SCANET system and a previously established behavior test, the 21-point open-field Basso-Beattie-Bresnahan (BBB) Locomotor Rating Scale. The results indicated that the RG scores were significantly and consistently different between the spinal cord-injured and control mice, irrespective of the mouse strain or injury model, but that MI and M2 scores were not. Moreover, there was a statistically positive correlation between the RG score and the BBB Scale score. For the assessment of locomotor function after SCI, use of the SCANET sytem in behavioral analysis is simple and the method is highly reproducible. The analysis of vertical movement is useful for assessing the recovery of limb function in mice following thoracic hemisection.</p>

Tumor antigens isolated from a patient with vitiligo and T-cell-infiltrated melanoma

Kiniwa Y., Fujita T., Akada M., Ito K., Shofuda T., Suzuki Y., Yamamoto A., Saida T., Kawakami Y.

Cancer Research 61 ( 21 ) 7900 - 7907 2001.11

ISSN  0008-5472

　View Summary

<p>Serological identification of tumor antigens by cDNA expression cloning is a technique used to isolate cDNAs encoding tumor antigens that are recognized by IgG antibodies in sera from cancer patients. It is also useful for the isolation of tumor antigens recognized by T cells. We applied this method to identify melanoma antigens recognized by the serum from a patient with a good prognosis who had T-cell-infiltrated melanoma and vitiligo. By screening a λ phage cDNA library constructed from a highly pigmented melanoma cell line, SKme123, with the patient's serum, 50 positive cDNA clones consisting of 26 distinct antigens were isolated. Of these, 20 encoded known proteins, and 6 encoded previously uncharacterized ones. The most frequently isolated clone, which we named KU-MEL-1, was unknown previously but was homologous to partial cDNA sequences registered in the expressed sequence tag database. Reverse transcription-PCR and Northern blot analysis demonstrated that KU-MEL-1 was strongly expressed in most melanoma cell lines, melanoma tissue samples, and cultured melanocytes and weakly expressed in cell lines derived from other types of tumors, as well as in some normal tissues, including testis. Western blot analysis with polyclonal murine antibody generated by immunization with the recombinant KU-MEL-1 protein demonstrated that the KU-MEL-1 protein was preferentially expressed in melanoma cells and melanocytes IgG antibodies against KU-MEL-1 were detected in the sera from 9 of 26 melanoma patients and from some patients with other cancers including brain tumor, esophageal cancer, colon cancer, and chronic myelogenous leukemia, but were not detected in sera from 30 healthy individuals. Although the IgG specific for KU-MEL-1 was not detected in sera from 12 vitiligo patients, it was detected in sera from 7 of 11 patients with Vogt-Koyanagi-Harada disease that is thought to be an autoimmune disease against melanocytes. These results suggest that KU-MEL-1 may be a useful target for the development of diagnostic and therapeutic methods for patients with various cancers, particularly with melanoma as well as patients with autoimmune diseases against melanocytes.</p>

Autoantibodies to the amino-terminal fragment of β-fodrin expressed in glandular epithelial cells in patients with Sjögren's syndrome

Kuwana M., Okano T., Ogawa Y., Kaburaki J., Kawakami Y.

Journal of Immunology 167 ( 9 ) 5449 - 5456 2001.11

ISSN  0022-1767

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<p>Sjögrens's syndrome (SS) is an autoimmune disease characterized by destruction of lacrimal and salivary glands, but the mechanisms underlying the disease process are unclear. By immunoscreening a HepG2 cDNA library with serum from an SS patient we isolated a cDNA encoding amino-terminal 616 aa of β-fodrin, a membrane skeleton protein associated with ion channels and pumps. Serum Ab to the amino-terminal fragment of β-fodrin was frequently detected in SS patients compared with rheumatic disease patients without SS or healthy controls (70 vs 12 or 4%; p &lt; 0.00001). All the anti-β-fodrin-positive sera recognized the amino-terminal fragment with no homology to α-fodrin. Anti-β-fodrin Abs in patients' sera as well as mouse polyclonal sera raised against the amino-terminal β-fodrin fragment did not react with intact β-fodrin, but recognized the 65-kDa amino-terminal fragment generated through cleavage by caspase-3 or granzyme B. When expression of intact and fragmented β-fodrin in lacrimal glands was assessed by immunohistochemistry, the antigenic amino-terminal fragment was distributed diffusely in acinar epithelial cell cytoplasm, whereas the carboxyl-terminal fragment and/or intact β-fodrin were localized in peripheral cytoplasm, especially at the basal membrane, in SS patients. In contrast, intact β-fodrin was detected primarily at the apical membrane of epithelia, and the amino-terminal fragment was scarcely detected in control patients with chronic graft-vs-host disease. These findings suggest that cleavage and altered distribution of β-fodrin in glandular epithelial cells may induce impaired secretory function and perpetuate an autoimmune response to β-fodrin, leading to autoantibody production and glandular destruction in SS.</p>

Tumor antigens isolated from a patient with vitiligo and T-cell-infiltrated melanoma

Kiniwa Yukiko, Fujita Tomonobu, Akada M, Ito Keiich, Shofuda Tomoko, Suzuki Yuriko, Yamamoto Akifumi, Saida Toshiaki, Kawakami Yutaka

Cancer Research 61 ( 21 ) 7900-7907 2001.11

Research paper (scientific journal), Joint Work

Graft versus leukemia (GVL) 効果の免疫制御

Yamazaki Rie，Kawakami Yutaka

血液フロンティア 11 ( 11 ) 1399-1408 2001.11

Research paper (scientific journal), Joint Work

Recent topics on basic research for idiopathic thrombocytopenic purpura

Kuwana M., Kawakami Y., Ikeda Y.

Rinsho byori. The Japanese journal of clinical pathology 49 ( 10 ) 992 - 995 2001.10

ISSN  0047-1860

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<p>Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease characterized by increased platelet clearance caused by anti-platelet autoantibodies, which bind to circulating platelets resulting in destruction. by the reticuloendothelial system. We have recently developed enzyme-linked immunospot assay to detect circulating B cells secreting anti-platelet antibody. An increase in anti-platelet antibody-producing B cells in peripheral blood was specifically detected in ITP patients, but in none of thrombocytopenic patients without ITP or healthy donors. While earlier studies reported the presence of platelet-reactive T cells in ITP patients, we have recently found that GPIIb-IIIa is one of major target antigens recognized by platelet-reactive CD4+ T cells. Since GPIIb-IIIa-reactive CD4+ T cells had helper activity promoting production of anti-platelet antibody, these autoreactive T cells are involved in production of pathogenic anti-platelet autoantibody in ITP patients. Suppression of GPIIb-IIIa-reactive CD4+ T cells may be of therapeutic use in treating refractory patients.</p>

特発性血小板減少性紫斑病

Kuwana Masataka，Kawakami Yutaka，Ikeda Yasuo

臨床病理 49 ( 10 ) 992-995 2001.10

Research paper (scientific journal), Joint Work

Increase of oligodendrocyte progenitor cells after spinal cord injury

Ishii Ken, Toda Masahiro, Nakai Yoko, Asou Hiroaki, Watanabe Masahiko, Nakamura Masaya, Yato Yoshiyuki, Fujimura Yoshikazu, Kawakami Yutaka, Toyama Yoshiaki, Uyemura Keiichi

Journal of Neuroscience Research 65 ( 6 ) 500 - 507 2001.09

ISSN  0360-4012

　View Summary

<p>The reaction of oligodendrocyte progenitor cells (OPCs) after spinal cord injury (SCI) is poorly understood. In this study, we examined oligodendroglial reactions after contusion SCI in adult rats by immunohistochemistry. OPCs were identified by staining with monoclonal antibodies (mAbs) A2B5 and O4. Each of the A2B5-, O4-positive OPCs and galactocerebroside-positive oligodendrocytes dramatically increased in the lesion of the dorsal posterior funiculus. Bromodeoxyuridine (BrdU) incorporation studies showed that most O4-positive cells in the lesion were labeled with BrdU, suggesting that these OPCs were proliferative. In contrast, the expression of myelin basic protein was decreased in the lesion compared with controls that received laminectomy only. From the injured cord, OPCs were isolated by immunopanning with mAb A2B5. We observed an increased number of OPCs from the injured spinal cords compared with those isolated from controls and unoperated animals. After several days in culture, the OPCs from the lesion expressed galactocerebroside. These results suggest that OPCs are induced and can differentiate following SCI in the adult rat.</p>

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Autoreactive CD4⁺ T-cell clones to β₂-glycoprotein I in patients with antiphospholipid syndrome

Arai Takahide, Yoshida Kazue, Kaburaki Junichi, Inoko Hidetoshi, Ikeda Yasuo, Kawakami Yutaka, Kuwana Masataka

Blood 98 ( 6 ) 1889 - 1896 2001.09

ISSN  0006-4971

　View Summary

<p>Autoreactive CD4⁺ T cells to β₂-glycoprotein I (β₂GPI) that promote antiphospholipid antibody production were recently identified in patients with antiphospholipid syndrome (APS). To further examine antigen recognition profiles and T-cell helper activity in β₂GPI-reactive T cells, 14 CD4⁺ T-cell clones specific to β₂GPI were generated from 3 patients with APS by repeated stimulation of peripheral blood T cells with recombinant β₂GPI. At least 4 distinct T-cell epitopes were identified, but the majority of the β₂GPI-specific T-cell clones responded to a peptide encompassing amino acid residues 276 to 290 of β₂GPI (KVSFFCKNKEKKCSY; single-letter amino acid codes) that contains the major phospholipid-binding site in the context of the DRB4*0103 allele. Ten of 12 β₂GPI-specific T-cell clones were able to stimulate autologous peripheral blood B cells to promote anti-β₂GPI antibody production in the presence of recombinant β₂GPI. T-cell helper activity was exclusively found in T-cell clones capable of producing interleukin 6 (IL-6). In vitro anti-β₂GPI antibody production induced by T-cell clones was inhibited by anti-IL-6 or anti-CD40 ligand monoclonal antibody. In addition, exogenous IL-6 augmented anti-β₂GPI antibody production in cultures of the T-cell clone lacking IL-6 expression. These results indicate that β₂GPI-specific CD4⁺ T cells in patients with APS preferentially recognize the antigenic peptide containing the major phospholipid-binding site and have the capacity to stimulate B cells to produce antiβ₂GPI antibodies through IL-6 expression and CD40-CD40 ligand engagement. These findings are potentially useful for clarifying the pathogenesis of APS and for developing therapeutic strategies that suppress pathogenic antiphospholipid antibody production in these patients.</p>

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Autoreactive CD4⁺ T cell clones to b₂-glycoprotein I in patients with antiphospholipid syndrome: preferential recognition of the major phospholipid-binding site

Arai Takahide, Yoshida Kazue, Kaburaki Junichi, Inoko Hidetoshi, Ikeda Yasuo, Kawakami Yutaka, Kuwana Masataka

Blood 98 ( 6 ) 1889-1896 2001.09

Research paper (scientific journal), Joint Work

Increase of oligodendrocyte progentior cells after spinal cord injury

Ishii Ken, Toda Masahiro, Nakai Yoko, Asou Hiroaki, Watanabe Masahiko, Nakamura Masaya, Yato Yoshiyuki, Fujimura Yoshikazu, Kawakami Yutaka, Toyama Yoshiaki, Uyemura Keiichi

Journal of Neuroscience Research 65 ( 6 ) 500-507 2001.09

Research paper (scientific journal), Joint Work

Induction of antigen-specific human CD4⁺ T cell anergy by peripheral blood DC2 precursors

Kuwana Masataka, Kaburaki Junichi, Wright Timothy M, Kawakami Yutaka, Ikeda Yasuo

European Journal of Immunology 31 ( 9 ) 2547-2557 2001.09

Research paper (scientific journal), Joint Work

Heterogeneous immunogenetic background in Japanese adults with myasthenia gravis

Suzuki S., Kuwana M., Yasuoka H., Tanaka K., Fukuuchi Y., Kawakami Y.

Journal of the Neurological Sciences 189 ( 1-2 ) 59 - 64 2001.08

ISSN  0022-510X

　View Summary

<p>The aim of this study was to elucidate the roles of human leukocyte antigen (HLA) class II genes in disease susceptibility in Japanese adult patients with myasthenia gravis (MG). A number of studies have shown that MG is correlated with DR3 in Caucasians. In Japanese, infantile MG is associated with DR9, but the HLA class II alleles associated with adult MG remains unclear. HLA-DRB1 and DQB1 alleles were determined by genotyping in 75 Japanese adult patients with MG and in 115 race-matched healthy adults. No statistically significant difference was observed in the overall prevalences of DRB1 and DQB1 alleles between MG patients and healthy controls, even when the patients and controls were stratified on the basis of their gender. MG patients with DQB1*0604 were younger and those with DQB1*0402 were older at disease onset than those without (P=0.03 and 0.008, respectively). Concomitant autoimmune thyroid disease was associated with DRB1*0803 (P=0.0009, corrected P=0.04). In addition, anti-acetylcholine receptor antibody levels were significantly higher in patients with DQB1*0604 than in those without (P=0.045). These findings indicate that immunogenetic backgrounds in Japanese adult MG patients are heterogeneous and are apparently different from those in Caucasian patients.</p>

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Immunodominant epitopes on glycoprotein IIb-IIIa recognized by autoreactive T cells in patients with immune thrombocytopenic purpura

Kuwana Masataka, Kaburaki Junichi, Kitasato Hidero, Kato Miyako, Kawai Shinichi, Kawakami Yutaka, Ikeda Yasuo

Blood 98 ( 1 ) 130 - 139 2001.07

ISSN  0006-4971

　View Summary

<p>It was recently reported that autoreactive CD4⁺ T cells to glycoprotein IIb-IIIa (GPIIb-IIIa) mediate antiplatelet autoantibody production in patients with immune thrombocytopenic purpura (ITP). To further examine the antigenic specificity of the GPIIb-IIIa-reactive T cells, 6 recombinant fragments encoding different portions of GPIIbα or GPIIIa were generated and tested for their ability to stimulate antigen-specific T-cell proliferation and antiGPIIb-IIIa antibody production in vitro. T cells from the peripheral blood of 25 patients with ITP and 10 healthy donors proliferated in response to recombinant GPIIb-IIIa fragments in various combinations. The amino-terminal portions of both GPIIbα and GPIIIa (IIbα18-259 and IIIa22-262) were frequently recognized (60% and 64%, respectively) compared with other fragments (4%-28%) in patients with ITP, but this tendency was not detected in healthy donors. In subsequent analyses in patients with ITP, T-cell reactivities to IIbα18-259 and IIIa22-262 were consistently detected, whereas those to other fragments were sometimes lost. In vitro antigenic stimulation of peripheral blood mononuclear cells with IIbα18-259 or IIIa22-262 promoted the synthesis of anti-GPIIb-IIIa antibodies in patients with ITP, but not in healthy donors. Of 15 CD4⁺ T-cell lines specific for platelet-derived GPIIb-IIIa generated from 5 patients with ITP, 13 lines recognized IIbα18-259, IIIa22-262, or both. T-cell lines reactive to IIbα18-259 or IIIa22-262 promoted the production of anti-GPIIb-IIIa antibodies that were capable of binding to normal platelet surfaces. These results indicate that the immunodominant epitopes recognized by pathogenic CD4⁺ T cells in patients with ITP are located within the amino-terminal portions of both GPIIbα and GPIIIa.</p>

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Immunodominant epitopes on glycoprotein IIb-IIIa recognized by autoreactive T cells in patients with immune thrombocytopenic purpura

Kuwana Masataka, Kaburaki Junichi, Kitasato Hidero, Kato Miyako, Kawai Shinichi, Kawakami Yutaka, Ikeda Yasuo

Blood 98 ( 1 ) 130-139 2001.07

Research paper (scientific journal), Joint Work

Expression of the neural RNA-binding protein Musashi1 in human gliomas

Toda Masahiro, Iizuka Yukihiko, Yu Wangjie, Imai Takao, Ikeda Eiji, Yoshida Kazunari, Kawase Takeshi, Kawakami Yutaka, Okano Hideyuki, Uyemura Keiichi

Glia 34 ( 1 ) 1-7 2001.04

Research paper (scientific journal), Joint Work

Quantitative monitoring of the PRAME gene for the detection of minimal residual disease in leukaemia

Matsushita Maiko, Ikeda Hideyuki, Kizaki Masahiro, Okamoto Shinichiro, Ogasawara Masahiro, Ikeda Yasuo, Kawakami Yutaka

British Journal of Heamatology 112 ( 4 ) 916-926 2001.03

Research paper (scientific journal), Joint Work

Quantitative monitoring of the PRAME gene for the detection of minimal residual disease in leukaemia

Matsushita Maiko, Ikeda Hideyuki, Kizaki Masahiro, Okamoto Shinichiro, Ogasawara Masahiro, Ikeda Yasuo, Kawakami Yutaka

British Journal of Haematology 112 ( 4 ) 916-926 2001.03

Research paper (scientific journal), Joint Work

Quantitative monitoring of the PRAME gene for the detection of minimal residual disease in leukaemia

Matsushita Maiko, Ikeda Hideyuki, Kizaki Masahiro, Okamoto Shinichiro, Ogasawara Masahiro, Ikeda Yasuo, Kawakami Yutaka

British Journal of Haematology 112 ( 4 ) 916-926 2001.03

Research paper (scientific journal), Joint Work, Accepted

Isolation of a new melanoma antigen, MART-2, containing a mutated epitope recognized by autologous tumor-infiltrating T lymphocytes

Kawakami Y., Wang X., Shofuda T., Sumimoto H., Tupesis J. P., Fitzgerald E., Rosenberg S. A.

Journal of Immunology 166 ( 4 ) 2871 - 2877 2001.02

ISSN  0022-1767

　View Summary

<p>Using cDNA expression cloning, a cDNA encoding a novel human melanoma Ag, MART-2 (melanoma Ag recognized by T cells-2), recognized by HLA-A1-restricted CD8⁺ T cells from tumor-infiltrating lymphocytes (TIL1362) was isolated from an autologous melanoma cell line, 1362 mel. Homologous sequences to the cDNA had been registered in the EST database. This gene encoded an uncharacterized protein expressed ubiquitously in most normal and cancer cells. A mutation (A to G transition) was found in the cDNA obtained from the1362 mel melanoma cell line in the sequences encoding the phosphate binding loop (P-loop) that resulted in loss of the ability to bind GTP. Transfection of NIH-3T3 with the mutated MART-2 did not result in the development of significant foci. By screening 36 various cancer cell lines using single-strand conformation polymorphism, a possible mutation in the P-loop of MART-2 was found in one squamous cell lung cancer cell line, EBC1. The T cell epitope for TIL1362, FLEG-NEVGKTY, was identified to be encoded by the mutated sequence of the MART-2 Ag. The mutation substituted glycine in the normal peptide with glutamic acid at the third amino acid of the epitope, which is an important primary anchor amino acid for HLA-A1 peptide binding. The normal peptide, FLGGNEVGKTY, was not recognized by TIL1362, suggesting that this T cell response was specific for the autologous tumor. Although transforming activity was not detected in the NIH-3T3 assay, MART-2 with the mutation in the P-loop may be involved in the generation of melanoma through a loss of GTP binding activity.</p>

A significant role of stromal fibroblasts in rapidly progressive dry eye in patients with chronic GVHD

Ogawa Yoko, Yamazaki Kazuto, Kuwana Masataka, Mashima Yukihiko, Nakamura Yu, Ishida Susumu, Toda Ikuko, Oguchi Yoshihisa, Tsubota Kazuo, Okamoto Shinichiro, Kawakami Yutaka

Investigative Ophthalmology & Visual Science 42 ( 1 ) 111-119 2001.01

Research paper (scientific journal), Joint Work

A significant role of stromal fibroblasts in rapidly progressive dry eye in patients with chronic GVHD

Ogawa Yoko, Yamazaki Kazuto, Kuwana Masataka, Mashima Yukihiko, Nakamura Yu, Ishida Susumu, Toda Ikuko, Oguchi Yoshihisa, Tsubota Kazuo, Okamoto Shinichiro, Kawakami Yutaka

Investigative Ophthalmology & Visual Science 42 ( 1 ) 111-119 2001.01

Research paper (scientific journal), Joint Work

Adenovirus-mediated p16 gene transfer changes the sensitivity to taxanes and vinca alkaloids of human ovarian cancer cells

Kawakami Y., Hama S., Hiura M., Nogawa T., Chiba T., Yokoyama T., Takashima S., Tajiri H., Eguchi K., Nagai N., Shigemasa K., Ohama K., Kurisu K., Heike Y.

Anticancer Research 21 ( 4 A ) 2537 - 2546 2001

ISSN  0250-7005

　View Summary

<p>Background: Deletions and point mutations of the p16 gene are detectable in more than 50% of ovarian cancer cells. In this study, we examined the effect of p16 gene transduction on the growth of ovarian cancer cells and on the effect of anti-cancer agents. Materials and Methods: p16-null human ovarian cancer cell lines, SKOV-3 and OVCAR-5, were used in this study. We transduced the full-length human p16 gene using recombinant adenovirus (AxCA-hp16). Results: The spontaneous growth of these cells was significantly inhibited by hp16 transduction. MTT assay revealed that AxCA-hp16 infection induced chemoresistance in both cell lines. Flow cytometric analysis revealed that only hp16-transduced SKOV-3, were arrested at the G1-phase for 3 days whereas those infected with AxCA-mock and OVCAR-5 infected with both recombinant viruses did not. Western blot analysis showed increased microtubule-associated proteins 4 (MAP4) in both cell lines. Conclusion: These results suggest that in SKOV-3 cells, G1-arrest induced by p16-transduction prevents paclitaxel- and vindesine- induced cell death, and in OVCAR-5 cells, the other unknown mechanisms play a role of chemoresistance.</p>

Induction of antigen-specific human CD4⁺ T cell anergy by peripheral blood DC2 precursors

Kuwana Masataka, Kaburaki Junichi, Wright Timothy M., Kawakami Yutaka, Ikeda Yasuo

European Journal of Immunology 31 ( 9 ) 2547 - 2557 2001

ISSN  0014-2980

　View Summary

<p>Dendritic cells (DC) are antigen (Ag)-presenting cells that are essential for initiation of T cell-dependent immunity, and distinct DC subsets are known to direct different classes of immune responses. DC2 precursors (pDC2) or plasmacytoid DC were recently identified as a Th2-skewing and IFN-α-producing human DC subset. Here, we demonstrate that pDC2 enriched from human peripheral blood have a capacity to induce an anergic state in human Ag-specific CD4⁺ T cell lines. Tetanus toxoid-specific T cell lines incubated with tetanus toxoid-pulsed autologous pDC2 failed to proliferate in secondary cultures with optimal Ag stimulation. T cell anergy induction required TCR engagement with Ag/MHC complex presented on pDC2. T cells rendered anergic lost IL-2 production but produced IFN-γ and IL-10 upon stimulation. The pDC2-induced unresponsiveness was completely or partially reversible when a high concentration of exogenous IL-2 was added in the secondary cultures. Autoreactive CD4⁺ T cell clones specific for topoisomerase I derived from a patient with scleroderma were also rendered anergic after co-culture with topoisomerase I-pulsed autologous pDC2, resulting in failure to proliferate or provide help to B cells. These results suggest that pDC2 are involved in maintenance of peripheral T cell tolerance and have potential for use in the suppression of pathogenic T cell responses in autoimmune diseases and organ transplantation.</p>

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A significant role of stromal fibroblasts in rapidly progressive dry eye in patients with chronic GVHD

Ogawa Y., Yamazaki K., Kuwana M., Mashima Y., Nakamura Y., Ishida S., Toda I., Oguchi Y., Tsubota K., Okamoto S., Kawakami Y.

Investigative Ophthalmology and Visual Science 42 ( 1 ) 111 - 119 2001

ISSN  0146-0404

　View Summary

<p>PURPOSE. To elucidate histopathologic features of the lacrimal gland in chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. METHODS. Lacrimal gland specimens from five patients who had dry eye as part of the symptoms of chronic GVHD were examined by immunohistochemistry and transmission electron microscopy. Lacrimal gland specimens from five patients with Sjögren's syndrome (SS) were used as control samples. RESULTS. Lymphocytes, predominantly T cells, were found primarily in the periductal areas of the lacrimal gland from patients with chronic GVHD, whereas B cells were the dominant infiltrating cells in the acinar areas of the lacrimal gland from patients with SS. Notable findings in the lacrimal gland from patients with chronic GVHD were marked fibrosis of the glandular interstitium and an increase in the number of CD34⁺ stromal fibroblasts. These findings were more prominent in patients with severe dry eye than in those with mild dry eye. Electron microscopic observations of the lacrimal gland from patients with chronic GVHD revealed that stromal fibroblasts were attached to various inflammatory cells, especially T cells, through primitive or rudimentary contacts. In addition, the presence of a well-developed rough endoplasmic reticulum in the fibroblasts and newly synthesized collagen fibrils in the extracellular matrix indicated an active production of extracellular matrix components. Electron micrographs revealed multilayered and thickened basal laminae of blood vessels, ducts. and lobules in the lacrimal gland of patients with chronic GVHD; however, these observations were infrequently observed in the lacrimal glands of patients with SS. CONCLUSIONS. The results suggest substantial differences in the lacrimal gland histopathology of patients with chronic GVHD and SS. In addition, it is likely that stromal fibroblasts are actively involved in the pathogenic process of chronic GVHD in the lacrimal gland by producing excessive extracellular matrix components.</p>

Lack of evidence for an increased microchimerism in the circulation of patients with Sjögren's syndrome

Toda I., Kuwana M., Tsubota K., Kawakami Y.

Annals of the Rheumatic Diseases 60 ( 3 ) 248 - 253 2001

ISSN  0003-4967

　View Summary

<p>Objective - To examine the hypothesis that fetal microchimerism plays a part in the pathogenic process of Sjögren's syndrome (SS). Methods - Genomic DNA samples were extracted from peripheral blood whole nucleated cells and the CD34+ cell enriched fraction of patients with SS and healthy women who had male offspring as well as nulliparous women. A Y chromosome-specific sequence was detected as a marker for fetal cells by a nested polymerase chain reaction (PCR) and by DNA hybridisation combined with PCR using specific primers and probes. All procedures were performed with great care to avoid the contamination of male DNA. Results - A nested PCR and DNA hybridisation combined with PCR was established that can detect a single male cell out of 1.67×10⁵ female cells. It was not possible to increase the sensitivity further because the amount of template DNA held in the PCR was limited. When these methods were used, no fetal cells were detected in any samples from patients with SS, though they were detected in whole nucleated cells from two healthy women who had delivered sons previously. Conclusions - The findings indicate that circulating fetal cells in patients with SS are uncommon (&lt;1 in 1.67×10⁵), if they exist. With the conventional PCR based methods that were used, it is difficult to evaluate the quantitative difference in circulating fetal cells between patients with SS and healthy women.</p>

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Quantitative monitoring of the PRAME gene for the detection of minimal residual disease in leukaemia

Matsushita Maiko, Ikeda Hideyuki, Kizaki Masahiro, Okamoto Shinichiro, Ogasawara Masahiro, Ikeda Yasuo, Kawakami Yutaka

British Journal of Haematology 112 ( 4 ) 916 - 926 2001

ISSN  0007-1048

　View Summary

<p>PRAME (Preferentially expressed antigen of melanoma) has been previously identified as a melanoma antigen recognized by cytotoxic T cells (CTLs) and found to be expressed in a variety of cancer cells including leukaemic cells. We have screened 98 Japanese patients with leukaemia and lymphoma for expression of the PRAME gene using semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Forty-one patients (42%) showed high levels of PRAME expression. Eight of these patients were then monitored using real-time PCR for a period of 10-37 months. Significant reductions in the PRAME expression were observed in all patients after chemotherapy. An increased expression was detected in the two patients who relapsed, one of which was before cytological diagnosis. These changes were correlated with those of other known genetic markers, such as the bcr-abl gene. Therefore, quantitative monitoring of the PRAME gene using real-time PCR method may be useful for detecting minimal residual disease and to predict subsequent relapse, especially in patients without known genetic markers. In addition, a PRAME-positive leukaemia cell line and fresh leukaemic cells were found to be susceptible to lysis by PRAME-specific CTLs established from a patient with melanoma, suggesting that the PRAME peptide can also be a target leukaemia antigen for T cells.</p>

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Clinical significance of MART-1 and HLA-A2 expression and CD8+ T cell infiltration in melanocytic lesions in HLA-A2 phenotype patients

Kageshita Toshiro, Kawakami Yutaka, Ono Tomomichi

Journal of Dermatological Science 25 ( 1 ) 36 - 44 2001

ISSN  0923-1811

　View Summary

<p>MART-1 is a good candidate antigen for immunotherapy against HLA-A2 patients with melanoma, since it is a highly immunogenic antigen recognized by HLA-A2 and HLA-B45 restricted CD8+ cytotoxic T cells and expressed in the majority of melanoma lesions. In the present study the expression of MART-1 and HLA-A2 on melanocytic cells and CD8+ T cell infiltration was immunohistochemically analyzed. MART-1 was expressed in most melanocytic lesions, while HLA-A2 was down-regulated with melanoma disease progression. Furthermore, concomitant down-regulation of MART-1 and HLA-A2 in melanoma cells was corrrelated with poor prognosis. These findings suggest both MART-1 and HLA-A2 expression in melanoma lesions should be analyzed for selection of patients eligible for MART-1 based immunotherapy and monitoring for emergence of melanoma cells resistant to T cell therapy.</p>

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Expression of the neural RNA- binding protein Musashi1 in human gliomas

Toda Masahiro, Iizuka Yukihiko, Yu Wangjie, Imai Takao, Ikeda Eiji, Yoshida Kazunari, Kawase Takeshi, Kawakami Yutaka, Okano Hideyuki, Uyemura Keiichi

GLIA 34 ( 1 ) 1 - 7 2001

ISSN  0894-1491

　View Summary

<p>Tumor cells arising from a particular tissue may exhibit the same gene expression patterns as their precursor cells. To test this proposition, we have analyzed the expression of a neural RNA-binding protein, Musashi1, in primary human central nervous system (CNS) tumors. In rodents, Musashi1 is expressed predominantly in proliferating multipotent neural precursor cells, but not in newly generated postmitotic neurons. The expression of Musashi1 is downregulated with the successive progression of neurogenesis. In normal adult human tissues, we detected low levels of Musashi1 expression in brain and testis by RT-PCR analysis. In an RNA panel of 32 cancer tissues and cell lines, elevated expression of Musashi1 was seen in all five malignant gliomas studied, in contrast to the slight expression seen in other tumor cells, including those in several melanomas and a prostate cancer. Western blot analysis showed strong Musashi1 expression in malignant gliomas compared with nonneoplastic brain tissue. Glioblastomas, the most malignant form of glioma, showed higher Musashi1 expression than less malignant gliomas by immunohistochemical analysis. Tumors with strong Musashi1 expression tended to have high proliferative activity. Thus, the expression of Musashi1 correlated with the grade of the malignancy and proliferative activity in gliomas. These results suggest that primary CNS tumors may share gene expression patterns with primitive, undifferentiated CNS cells and that Musashi1 may be a useful marker for the diagnosis of CNS tumors.</p>

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特発性血小板減少性紫斑病における病因的自己反応性T細胞の同定

Kuwana Masataka，Kawakami Yutaka，Ikeda Yasuo

日本臨床免疫学会雑誌 23 ( 6 ) 555-557 2000.12

Research paper (scientific journal), Joint Work

SEREX抗原によるグリオーマの血清診断

Toda Masahiro，Iiduka Yukihiko，Kawase Takeshi，Kawakami Yutaka

神経免疫研究 13   44-46 2000.12

Research paper (scientific journal), Joint Work

HLA class II alleles in Japanese patients with immune thrombocytopenic purpura: associations with anti-platelet glycoprotein antibodies and responses to splenectomy

Kuwana Masataka, Kaburaki Junichi, Pandey Janardan P, Murata Mitsuru, Kawakami Yutaka, Inoko Hidetoshi, Ikeda Yasuo

Tissue Antigens 56 ( 4 ) 337-343 2000.10

Research paper (scientific journal), Joint Work

New cancer therapy by immunomanipulation

Kawakami Yutaka

Cornea 19 ( 3 SUPPL. 1 )  2000.05

ISSN  0277-3740

　View Summary

<p>Purpose and Methods. T cells play an important role in in vivo rejection of human melanoma. Human melanoma antigens recognized by autologous T cells were identified. These antigens are classified as tissue (melanocyte)- specific proteins, cancer-testis antigens (proteins expressed in normal testis and various cancers), tumor-specific peptides derived from mutations in tumor cells, and others. Results. A variety of mechanisms generating T cell epitopes on tumor cells were discovered. Various clinical observations, including tumor regression observed in adoptive transfer of gp 100-reactive T cells suggest that these identified melanoma peptides may function as tumor rejection antigens. Immunodominant common epitopes that could expand melanoma-reactive cytotoxic T lymphocytes (CTLs) in vitro were found in the MART-1 and gp 100 antigens. New immunization protocols - including immunization with peptides, recombinant viruses, plasmid DNAs, and dendritic cells pulsed with peptides as well as adoptive transfer of in vitro-generated CTLs by stimulation with antigenic peptides - were developed (phase I clinical trials have been performed in the Surgery Branch of the National Cancer Institute, Bethesda, MD, U.S.A.). Immunization with the gp100(209(210M)) peptide that was modified to have high HLA-A2 binding affinity, along with incomplete Freund's adjuvant and interleukin (IL)-2, resulted in a 42% response rate in patients with melanoma. Conclusion. These immunotherapies need further improvement due to the mechanisms of tumor escape from T cell responses.</p>

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Longitudinal analysis of autoantibody response to topoisomerase I in systemic sclerosis

Kuwana Masataka, Kaburaki Junichi, Mimori Tsuneyo, Kawakami Yutaka, Tojo Takeshi

Arthritis and Rheumatism 43 ( 5 ) 1074 - 1084 2000.05

ISSN  0004-3591

　View Summary

<p>Objective. To examine serial changes in serum anti-topoisomerase I (anti-topo I) antibody levels in patients with systemic sclerosis (SSc), as well as associations with clinical features and the in vivo activation status of circulating topo I-reactive T and B cells. Methods. Serum anti-topo I antibody levels were serially measured at different time points in 28 SSc patients who were positive for anti-topo I antibody at their first visit (range of followup 6-29 years). The patients were subgrouped according to the disappearance (group 1) or persistence (group 2) of anti-topo I antibody. Clinical findings as well as T and B cell responses to topo I were compared between these 2 groups. Results. Serum anti-topo I antibody disappeared during the period of followup in 6 patients (group 1), but persisted in 22 patients (group 2). Loss of anti-topo I antibody occurred within 10 years after the first visit and independently of treatment. Group I patients had less extensive skin and lung involvement and better survival rates than did group 2 patients. Complete loss of anti-topo I antibody followed a reduction in isotype expression and epitope reactivities. The kinetics of in vitro T cell proliferation induced by topo I were delayed and circulating topo I- reactive T Cells were less frequently detected in group I versus group 2 patients, suggesting that the disappearance of anti-topo I antibody was due to loss of activation of topo I-reactive T cells. In vitro production of anti-topo I antibody in peripheral blood mononuclear cell cultures in response to antigenic stimulation in both group 1 and group 2 patients indicated persistence of anti-topo I antibody-producing 'memory' B cells even after the loss of serum anti-topo I antibody. Conclusion. Our results indicate that there is a distinct subset of anti-topo I-positive SSc patients who lose anti-topo I antibody during the disease course and have a favorable outcome. In vivo production of anti-topo I autoantibody may require antigenic stimulation that activates topo I-reactive T and B cells.</p>

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T cells that are autoreactive to β₂-glycoprotein I in patients with antiphospholipid syndrome and healthy individuals

Hattori Noriko, Kuwana Masataka, Kaburaki Junichi, Mimori Tsuneyo, Ikeda Yasuo, Kawakami Yutaka

Arthritis and Rheumatism 43 ( 1 ) 65 - 75 2000.01

ISSN  0004-3591

　View Summary

<p>Objective. To identify the T cells responsive to β₂-glycoprotein I (β₂GPI) that mediate antiphospholipid antibody production in patients with antiphospholipid syndrome (APS). Methods. In vitro proliferative responses and anti-β₂GPI antibody production induced by β₂GPI were examined in peripheral blood mononuclear cell (PBMC) cultures from 12 APS patients, 13 systemic lupus erythematosus patients without APS, and 12 healthy donors. Results. Peripheral blood T cells from all subjects failed to respond to β₂GPI in its native form. In contrast, reduced β₂GPI was able to stimulate T cells not only from all 12 patients with anti-β₂GPI antibodies, but also from 10 of 25 individuals without anti-β₂GPI antibodies. The specificity of the responses to β₂GPI was confirmed by activation of the reduced β₂GPI-primed T cells by recombinant β₂GPI in secondary cultures. Characterization of the T cell response induced by β₂GPI revealed that the response was associated with the presence of the DR53-associated alleles, the responding T cells were CD4+ and restricted by HLA class II, and antigenic peptides were located in domains IV and/or V. Anti-β₂GPI antibody production was induced specifically in anti-β₂GPI antibody-positive patients, in PBMC cultures with reduced β₂GPI. Anti-β₂GPI antibodies produced in vitro recognized β₂GPI immobilized with cardiolipin or β₂GPI coated on 'high-binding' polystyrene plates. Conclusion. These results strongly suggest that CD4+ and HLA class II-restricted T cells responsive to β₂GPI are involved in the production of antiphospholipid antibodies in APS patients.</p>

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T cells that are autoreactive to β₂-glycoprotein I in patients with antiphospholipid syndrome and healthy individuals

Hattori Noriko, Kuwana Masataka, Kaburaki Junichi, Mimori Tsuneyo, Ikeda Yasuo, Kawakami Yutaka

Arthritis and Rheumatism 43 ( 1 ) 65-75 2000.01

Research paper (scientific journal), Joint Work

Recognition of shared melanoma antigens in association with major hla-a alleles by tumor infiltrating t lymphocytes from 123 patients with melanoma

Kawakami Yutaka, Dang Nita, Wang Xiang, Tupesis Janis, Robbins Paul F., Wang Rong Fu, Wunderlich John R., Yannelli John R., Rosenberg Steven A.

Journal of Immunotherapy 23 ( 1 ) 50817 2000

ISSN  1524-9557

In situ cancer vaccination with a mutant HSV for the treatment of liver metastasis of colon cancer

Endo T, Toda M, Watanabe M, Iizuka Y, Kubota T, Kitajima M, Kawakami Y

Proceedings of the American Association for Cancer Research 41   798 2000

Research paper (scientific journal), Joint Work

Multiple in situ cancer vaccinations with HSV and IL-12 synergize to facilitate tumor regression

Suzuki A, Toda M, Iizuka Y, Kitagawa Y, Kawakami Y

Proceedings of the American Association for Cancer Research 41   469 2000

Research paper (scientific journal), Joint Work

Characterization of tumor antigens recognized by IgG antibody in patients with glioma using SEREX

Takahashi S, Toda M, Iizuka Y, Yoshida K, Kawase T, Kawakami Y

Proceedings of the American Association for Cancer Research 41   880 2000

Research paper (scientific journal), Joint Work

Identification and characterization of human brain tumor antigens: a potential target for specific immunotherapy and diagnostic marker

Toda M, Iizuka Y, Takahashi S, Fujita T, Ikeda E, Yoshida K, Kawase T, Okano H, Uyemura K, Kawakami Y

Proceedings of the American Association for Cancer Research 41   710 2000

Research paper (scientific journal), Joint Work

Cancer immunotherapy with dendritic cells engineered to express tumor antigens

Sumimoto H., Kawakami Y.

Biotherapy 14 ( 12 ) 1197 - 1204 2000

ISSN  0914-2223

　View Summary

<p>Dendritic cells engineered to express tumor-associated antigens can be used to prime and potentiate cytotoxic T cell responses against cancer cells. Numerous delivery systems for introducing genes to dendritic cells have been examined, which include (I) Viral vector systems such as Moloney leukemia virus vector (MLV), human immunodeficiency virus (HIV) vector, and adenovirus vector, vaccinia virus vector, canarypox virus vector and herpes simplex virus (HSV) vector, and (II) Non-viral vector systems such as cationic liposome, particle bombardment (gene gun) and electroporation methods. In this review, we adress several issues regarding these gene delivery systems in terms of genetic modification of dendritic cells. Since every method has advantages and/or disadvantages, the optimal methods for presenting antigen epitopes to dendritic cells need to be clarified. Several animal models by using dendritic cells transduced with model tumor antigens have shown in vivo antitumor effects, suggesting the potential clinical usefulness of this strategy.</p>

HLA class II alleles in Japanese patients with immune thrombocytopenic purpura. Associations with anti-platelet glycoprotein autoantibodies and responses to splenectomy

Kuwana M., Kaburaki J., Pandey J. P., Murata M., Kawakami Y., Inoko H., Ikeda Y.

Tissue Antigens 56 ( 4 ) 337 - 343 2000

ISSN  0001-2815

　View Summary

<p>HLA class II alleles and immunoglobulin allotypes were determined in 83 Japanese patients with immune thrombocytopenic purpura (ITP) and 114 race-matched healthy controls. Distribution of DRB and DQB1 alleles as well as G1M(f, z), G2M(n+, n-), and KM(1, (1,2), 3) were not different between ITP patients and healthy controls, while DPB1*0201 was marginally increased in ITP patients vs. healthy controls (51% vs. 28%, Pc = 0.04, OR = 2.6 [1.4-4.8]). In contrast, strong associations between anti-glycoprotein autoantibodies and HLA class II genes were found as follows: Anti-GPIIb-IIIa antibody with DRB1*0405 and DQB1*0401; and anti-GPIb-IX antibody with DRB1*0803 and DQB1*0601. When factors influencing therapeutic responses to splenectomy were examined, a poor response was correlated with the presence of DRB1*0405, DQB1*0401 and anti-GPIIb-IIIa antibody (P = 0.01, 0.002, and 0.03, respectively). Our results indicate that HLA class II genes influence the production of anti-glycoprotein antibody specificities rather than the development of ITP. In addition, HLA class II genotyping could be useful in predicting therapeutic responses to splenectomy in Japanese patients with ITP.</p>

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Identification and characterization of pathogenic autoreactive T cells in patients with idiopathic thrombocytopenic purpura

Kuwana M., Kawakami Y., Ikeda Y.

Nihon Rinshō Men'eki Gakkai kaishi = Japanese journal of clinical immunology 23 ( 6 ) 555 - 557 2000

ISSN  0911-4300

Recognition of shared melanoma antigens in association with major HLA-A alleles by tumor infiltrating T lymphocytes from 123 patients with melanoma

Kawakami Yutaka, Dang Nita, Wang Xiang, Tupesis Janis, Robbins Paul F., Wang Rong Fu, Wunderlich John R., Yannelli John R., Rosenberg Steven A.

Journal of Immunotherapy 23 ( 1 ) 17 - 27 2000

ISSN  1053-8550

　View Summary

<p>A total of 123 tumor-infiltrating T lymphocyte (TIL) cultures established from patients with HLA-A1, -A2, -A3, -A24, or -A31 metastatic melanoma in the Surgery Branch, National Cancer Institute, were screened for recognition of shared melanoma antigens including five melanosomal proteins (tyrosinase, MART-1/melan-A, gp100, TRP1, TRP2) as well as peptides derived from MAGE-1 and MAGE-3. Examination of the specificity of these T cells indicated that 16% of HLA-A1 TIL, 57% of HLA-A2 TIL, 7% of HLA-A3 TIL, 13% of HLA-A24 TIL, and 27% of HLA-A31 TIL recognized shared melanoma antigens restricted by major histocompatibility complex class I. Melanosomal proteins were frequently recognized by these TIL, and MART- 1_27-35, gp100_{154- 162}, gp100_209-217, and gp100_280-288 represent highly immunogenic epitopes that were recognized by a high percentage of HLA-A2 restricted melanoma reactive TIL. Recognition of gp100 by HLA-A2 restricted TIL significantly correlated with clinical response to adoptive immunotherapy with TIL in 21 HLA-A2 melanoma patients (p = 0.024). Four HLA-A1, two HLA-A2, two HLA-A3, one HLA-A24, and two HLA-A31 restricted shared antigen-specific TIL did not recognize the previously identified antigens tested in this study, and may be useful for the identification of new melanoma antigens. The observation that TILs isolated from patients with metastatic melanoma recognized melanosomal proteins in the context of predominant HLA-A alleles implies that it may be possible to develop immunotherapies for patients with melanoma expressing diverse HLA types.</p>

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T cell immune responses against melanoma and melanocytes in cancer and autoimmunity

Kawakami Yutaka, Suzuki Yuriko, Shofuda Tomoko, Kiniwa Yukiko, Inozume Takashi, Dan Katsuaki, Sakurai Toshiharu, Fujita Tomonobu

Pigment Cell Research 13 ( SUPPL. 8 ) 163 - 169 2000

ISSN  0893-5785

　View Summary

<p>T cell responses specific for melanoma cells and melanocytes appear to be involved in the rejection of melanoma tumors, as well as in the development of autoimmune reactions in patients with Vogt-Koyanagi-Harada disease (VKH), sympathetic ophthalmia, or autoimmune vitiligo. Some of the target antigens for those T cells have been isolated using cDNA expression cloning with melanoma reactive T cells derived from tumor infiltrating lymphocytes (TIL) of patients with melanoma. These include melanocyte specific proteins, such as tyrosinase, TRP1, TRP2, gp100, and MART-1, cancer-testis antigens, and mutated peptidcs derived from genetic alterations in melanoma cells. Some of the melanoma reactive T cells appear to respond to cryptic or subdominant self epitopes in melanosomal proteins. Modification of those epitopes to increase their immunogenicity by replacement of amino acids at primary anchor residues for peptide/MHC binding, allowed an improvement in immunotherapy for patients with melanoma. Targets for autoreactive T cells against melanocytes in those autoimmune disorders remain to be identified. Isolation of novel target antigens is important for understanding these pathological T cell responses, as well as for developing new diagnostic and treatment methods for these diseases. A variety of techniques, including cDNA expression cloning with T cells, serological analysis of recombinant cDNA expression libraries (SEREX), cDNA subtraction with representational differential analysis (RDA), and serial analysis of gene expression (SAGE) are now being applied to identify novel melanoma/melanocyte antigens recognized by T cells and antibodies.</p>

Immuno-gene therapy for cancer

Kawakami Y.

Biotherapy 14 ( 4 ) 345 - 350 2000

ISSN  0914-2223

　View Summary

<p>A variety of molecules involved in the immune system have recently been isolated. These molecules have allowed the design of immunological interventions of immune responses to human cancer, or so called immuno-gene therapy. Clinical trials have been performed in Europe and the U.S. using various protocols, including gene-modified T cells; highly immunogenic cancer cells transfected with genes encoding cytokines, co-stimulatory molecules, and foreign antigens; immunization with tumor antigen genes as forms of plasmids and recombinant viruses; and immunization with dendritic cells transduced with tumor antigen genes. However, the anti-tumor effects observed in these reports have been limited. Improvement of various points will be necessary to establish immuno-gene therapy for cancer on the basis of fundamental research on immunoregulation against cancer, including isolation of tumor antigens and analysis of tumor escape mechanisms.</p>

転移性脳腫瘍に対する癌ワクチン療法の試み

Toda Masahiro, Iizuka Yukihiko, Yazaki Takahito, Uemura Keiichi, Kawase Takeshi, Kawakami Yutaka

Neurologia medico-chirurgica 10 ( abstr ) 80-80 1999.10

Research paper (scientific journal), Joint Work

In situ canver vaccination using a mutant HSV-1 for the treatment of metastatic brain tumor

Toda Masahiro, Iizuka Yukihiko, Yazaki Takahito, Kawase Takeshi, Uyemura Keiichi, Kawakami Yutaka

Neuro-Oncology 1 ( suppl ) S105-S105 1999.10

Research paper (scientific journal), Joint Work

造血器腫瘍における腫瘍抗原と免疫療法

Matsushita Maiko, Ikeda Hideyuki, Kawakami Yutaka

現代医療 31 ( 5 ) 1155-1160 1999.05

Research paper (scientific journal), Joint Work

転移性腫瘍に対する免疫療法・免疫遺伝子治療の現状と展望

Kitagawa Yukou, Kawakami Yutaka, Kitajima Masaki

臨床外科 54 ( 2 ) 203-208 1999.02

Research paper (scientific journal), Joint Work

Key points for development of immunotherapy for cancer using T cell defined tumor antigens

Kawakami Y.

Biotherapy 13 ( 5 ) 429 - 433 1999

ISSN  0914-2223

　View Summary

<p>New immunotherapies using cancer antigens recognized by T-cells have recently been developed and a variety of phase I clinical trals has been conducted in the US and Europe. An anti-tumor effect has already been observed in some protocols. However, many questions still remain to establish effective immunotherapies for cancer. Key points which were raised in the research on immunotherapies against melanoma are discussed in this article.</p>

PRAME protein expressed in leukemia cells as a target molecule for immunotherapy.

池田英之，松下麻衣子，河上裕

Rinsho Ketsueki 40 ( 6 ) 484-486 1999

Research paper (scientific journal), Joint Work

Identification of human melanoma antigens recognized by tumor infiltrating T lymphocytes and their use for immunotherapy

Kawakami Y.

Gann Monographs on Cancer Research 48   179 - 189 1999

ISSN  0072-0151

　View Summary

<p>Adoptive transfer of cultured tumor infiltrating T-lymphocytes (TIL) with interleukin 2 (IL2) resulted in tumor regression in melanoma patients, indicating the important role of T-cells in mediating in vivo melanoma regression. TIL secrete cytokines and lyse tumor cells in vitro by recognizing autologous melanoma. Using cDNA expression cloning methods with these TIL, cDNAs encoding melanoma antigens including 5 melanosomal proteins (MART-1, gp100, tyrosinase, TRP1, and TRP2), p15 and β-catenin, were identified. Non-mutated and mutated T cell epitopes have also been identified. A variety of mechanisms to generate these T cell epitopes on melanoma has been identified including peptides derived from regular open reading frames (ORFs), an alternative ORF, introns, or sequences with a mutation. Some melanoma epitopes had relatively low major histocompatibility complex (MHC) binding affinities, suggesting that these were subdominant self epitopes. Several MART-1 and gp100 epitopes were recognized by TIL from many patients, suggesting that these were immunodominant common epitopes. By in vitro stimulation with these identified epitopes, melanoma reactive cytotoxic T lymphocytes (CTL) could be efficiently induced from peripheral blood lymphocytes (PBL) or TIL of melanoma patients. Since adoptive transfer of the TIL reacting to these melanoma antigens into autologous patients with IL2 resulted in tumor regression, these peptides may function as tumor rejection antigens. Tumor regression correlated with vitiligo development after IL2 based immunotherapies in melanoma indicating that these epitopes may also be present on the surface of melanocytes in vivo and that autoreactive T-cells may be involved in the tumor regression. A variety of Phase I clinical trials including immunization with the MART-1 and gp100 peptides, or recombinant viruses containing the MART-1 or gp100 gene, with or without administration of cytokines such as IL2, granulocyte macrophagecolony stimulating factor (GM-CSF), IL12, is being conducted in the Surgery Branch of the National Cancer Institute (NCI), and tumor regression has been observed in some patients.</p>

Identification of new melanoma epitopes on melanosomal proteins recognized by tumor infiltrating T lymphocytes restricted by HLA-A1, -A2, and -A3 alleles

Kawakami Yutaka, Robbins Paul F., Wang Xiang, Tupesis Janis P., Parkhurst Maria R., Kang Xiaoqiang, Sakaguchi Kazuyasu, Appella Ettore, Rosenberg Steven A.

Journal of Immunology 161 ( 12 ) 6985 - 6992 1998.12

ISSN  0022-1767

　View Summary

<p>To isolate melanoma Ags recognized by T cells, cDNA libraries made from melanoma cell lines were screened with four CTLs derived from tumor infiltrating lymphocytes (TIL) that were able to recognize melanoma cells in a HLA-A1, -A2, or -A3 restricted manner. Although cDNAs encoding the previously identified melanoma Ags, tyrosinase and gp100, were isolated, these TIL were found to recognize previously unidentified peptides. An HLA- A1-restricted CTL, TIL1388, was found to recognize a tyrosinase peptide (SSDYVIPIGTY), and an HLA-A3-restricted CTL, TIL1351, recognized a gp100 peptide (LIYRRRLMK). CTL clones isolated from the HLA-A2-restricted TIL1383 recognized a gp100 peptide (RLMKQDFSV). HLA-A2-restricted CTL, TIL1200, recognized a gp100 peptide (RLPRIFCSC). Replacement of either cysteine residue with α-amino butyric acid in the gp100 peptide, RLPRIFCSC, enhanced CTL recognition, suggesting that the peptide epitope naturally presented on the tumor cell surface may contain reduced cysteine residues. Oxidation of these cysteines might have occurred during the course of the synthesis or pulsing of the peptide in culture. These modifications may have important implications for the development of efficient peptide-based vaccines. These newly identified peptide epitopes can extend the ability to perform immunotherapy using synthetic peptides to a broader population of patients, especially those expressing HLA-A1 or HLA-A3 for whom only a few melanoma epitopes have previously been identified.</p>

Identification of a shared HLA-A*0201-restricted T-cell epitope from the melanoma antigen tyrosinase-related protein 2 (TRP2)

Parkhurst Maria R., Fitzgerald Ellen B., Southwood Scott, Sette Alessandro, Rosenberg Steven A., Kawakami Yutaka

Cancer Research 58 ( 21 ) 4895 - 4901 1998.11

ISSN  0008-5472

　View Summary

<p>Tyrosinase-related protein 2 (TRP2) is a melanosomal enzyme expressed in most mammalian melanocytes and melanomas. This protein has been identified as a melanoma antigen recognized by tumor reactive CTLs derived from tumor infiltrating lymphocytes in the context of HLA-A31 and HLA-A33. The frequencies of these HLA-A alleles among melanoma patients in the United States is low (~6% for HLA-A31 and ~2% for HLA-A33) compared with that of HLA-A*0201 (~46%). Therefore, to extend significantly the use of TRP2- based immunotherapies for the treatment of patients with melanoma, we searched for new HLA-A*0201-restricted epitopes from this protein by screening TRP2-derived peptides for the induction of melanoma-reactive CTL. Fifty-one peptides were selected from TRP2 based on a permissive HLA-A*0201 binding motif, and the 21 peptides with the highest experimentally determined binding affinities were used to stimulate peripheral blood lymphocytes from HLA-A*0201⁺ melanoma patients in vitro. One peptide, TRP2(180188) (SVYDFFVWL), induced CTLs from three of four patients that specifically recognized peptide-pulsed T2 cells, COS-7 cells expressing HLA-A*0201 and TRP2, and HLA-A2⁺ TRP2⁺ melanomas. TRP2(180188) is identical to a previously identified TRP2 epitope recognized by murine melanoma-reactive CTLs in the context of H-2K^b. These results suggest that TRP2 may be useful for the development of murine tumor immunotherapy models and for the treatment of melanoma patients who are diverse in HLA expression.</p>

Immunotherapy using T cell defined tumor antigens for melanoma

Kawakami Yutaka

Microbiology and Immunology 42 ( 12 ) 803 - 813 1998

ISSN  0385-5600

Immunotherapy using human melanoma epitopes recognized by T cells

Kawakami Y.

Biotherapy 12 ( 5 ) 542 - 548 1998

ISSN  0914-2223

　View Summary

<p>Tumor reactive T cells can be induced from peripheral blood or tumor infiltrating lymphocytes of patients with melanoma. Based on the analysis of the previous immunotherapies, T cells appeared to be involved in vivo tumor regression. Genes encoding human melanoma antigens and their epitope peptides recognized by T cells, have been identified using cDNA expression cloning techniques. Mechanisms for generation of T cell epitopes on growing melanoma cells have been identified. A variety of Phase I clinical studies using MART- 1 and gp100 antigens are being performed in the Surgery Branch, National Cancer Institute, NIH, and some patients have responded to these immunotherapy protocols.</p>

Identification of the genes encoding cancer antigens

Rosenberg S. A., Kawakami Y., Robbins P. F., Wang R. F.

Advances in Cancer Research 70   145 - 177 1998

ISSN  0065-230X

Development of new immunotherapies using melanoma antigens recognized by T cells

Kawakami Y.

Biotherapy 12 ( 9 ) 1187 - 1194 1998

ISSN  0914-2223

　View Summary

<p>Melanoma is a relatively immunogenic cancer and a good model to evaluate the possibilities of immunotherapies. Recent progress of molecular biology and immunology has allowed us to understand immune responses to human cancer at the molecular level. Although antigens for CD4⁺ T cells have not well been identified, various melanoma antigens recognized by CD8⁺ T cells have been identified, including tissue specific proteins (melanosomal proteins), proteins expressed in testis and a variety of cancers (Cancer-Testis antigens) and tumor specific mutated proteins. The immunological features of these antigens have been characterized for development of new immunotherapies. Based on these findings, antigen specific immunotherapies have been developed. Some of the phase I clinical trials with the identified melanoma antigens demonstrated anti-tumor effects. Immunization with the modified gp100 epitope that had high MHC binding affinity, in conjunction with incomplete freund adjuvant and high-dose IL-2, resulted in a 42 % response rate (CR+PR). Immunization with dendritic cells pulsed with tumor lysates or epitope peptides resulted in a 31 % response rate in metastatic melanoma. These immunotherapies need to be improved through modifications and better understanding of tumor escape mechanisms.</p>

The use of melanosomal proteins in the immunotherapy of melanoma

Kawakami Yutaka, Robbins Paul F., Wang Rong Fu, Parkhurst Maria, Kang Xiaoqiang, Rosenberg Steven A.

Journal of Immunotherapy 21 ( 4 ) 237 - 246 1998

ISSN  1053-8550

　View Summary

<p>Clinical observations in the interleukin (IL) 2-based immunotherapies suggest that T cells play a central role in the rejection of melanoma. Using cDNA expression cloning, we have isolated genes encoding melanoma antigens recognized by tumor-infiltrating T lymphocytes. These antigens are categorized as (a) melanocyte-specific melanosomal proteins (MART-1/melan A, gp100, tyrosinase, TRP-1, and TRP-2), (b) tumor-specific mutated proteins (β-catenin), and (c) others (p15). A variety of mechanisms has been identified for the generation of T cell epitopes on tumor cells. Some of the HLA-A2 binding epitopes from the melanosomal antigens appear to be subdominant self-determinants with relatively low major histocompatibility complex binding affinity. The effectiveness of adoptive transfer into patients of cytotoxic T lymphocytes recognizing the melanosomal antigens, the significant correlation between vitiligo development and clinical response in patients receiving IL-2-based immunotherapies, and the sporadic tumor regressions observed in some patients following immunization with the MART-1 or gp100 peptides in incomplete Freund's adjuvant or recombinant viruses expressing the MART-1 antigen suggest that these epitopes may represent tumor rejection antigens. Phase I immunization trials using peptides or recombinant viruses containing genes encoding the melanosomal antigens MART- 1 or gp100, with or without co-administration of cytokines such as IL-2, IL-12, or granulocyte-macrophage colony-stimulating factor, are being conducted in the Surgery Branch of the National Cancer Institute. These studies may demonstrate the feasibility of using melanosomal proteins for the immunotherapy of patients with melanoma.</p>

Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma

Rosenberg Steven A., Yang James C., Schwartzentruber Douglas J., Hwu Patrick, Marincola Francesco M., Topalian Suzanne L., Restifo Nicholas P., Dudley Mark E., Schwarz Susan L., Spiess Paul J., Wunderlich John R., Parkhurst Maria R., Kawakami Yutaka, Seipp Claudia A., Einhorn Jan H., White Donald E.

Nature Medicine 4 ( 3 ) 321 - 327 1998

ISSN  1078-8956

　View Summary

<p>The cloning of the genes encoding cancer antigens has opened new possibilities for the treatment of patients with cancer. In this study, immunodominant peptides from the gp100 melanoma-associated antigen were identified, and a synthetic peptide, designed to increase binding to HLA-A2 molecules, was used as a cancer vaccine to treat patients with metastatic melanoma. On the basis of immunologic assays, 91% of patients could be successfully immunized with this synthetic peptide, and 13 of 31 patients (42%) receiving the peptide vaccine plus IL-2 had objective cancer responses, and four additional patients had mixed or minor responses. Synthetic peptide vaccines based on the genes encoding cancer antigens hold promise for the development of novel cancer immunotherapies.</p>

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T細胞が認識するヒトメラノーマ抗原の単離同定と免疫遺伝子治療への臨床応用

Kawakami Yutaka

Biother Today 4 ( 2 ) 62-67 1997.12

Research paper (scientific journal), Single Work

Mart1 is located on mouse Chromosome 19 and is excluded as a candidate for ep and ru

Wright A., Kawakami Y., Pavan W.

Mammalian Genome 8 ( 5 ) 377 - 378 1997.05

ISSN  0938-8990

• Access to Document (DOI)

Production of recombinant MART-1 proteins and specific anti-MART-1 polyclonal and monoclonal antibodies

Kawakami Yutaka, Battles Jane K., Kobayashi Takeshi, Ennis Willis, Wang Xiang, Tupesis Janis P., Marincola Francesco M., Robbins Paul F., Hearing Vincent J., Gonda Matthew A., Rosenberg Steven A.

Journal of Immunological Methods 202 ( 1 ) 13 - 25 1997.03

ISSN  0022-1759

　View Summary

<p>Recombinant human MART-1 protein was produced by bacterial and baculoviral-insect cell expression systems. By immunization with bacterial MBP-MART-1 fusion prorein or MBP cleaved MART-1 protein, a rabbit polyclonal and two murine monoclonal antibodies specific for MART-1 were produced. These antibodies specifically detected MART-1 in immuno-precipitation, Western blotting, flow cytometric assays and in immunohistochemical analysis of tissue sections. They also stained cytoplasmic components in melanocytes and most melanoma cells in frozen or paraffin embedded tissue sections, indicating that these antibodies may be useful for the diagnosis of melanoma. One of the monoclonal antibodies M2-7 C10 recognized only human MART-1, but the other monoclonal antibody M2-9 E3 recognized both human and murine MART-1. The size of the human MART-1 molecule detected by SDS-PAGE with these antibodies was approximately 18 kDa, suggesting possible posttranslational modifications in the MART-1 protein. Subcellular fractionation studies suggested that MART-1 was present in melanosomes and endoplasmic reticulum, although known melanogenic enzymatic activities were not detected in the MART-1 protein. These reagents may be useful for biological studies on melanocytes and melanoma cells as well as for the development and monitoring of immunotherapy for patients with melanoma.</p>

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Identification of Subdominant CTL Epitopes of the GP100 Melanoma-Associated Tumor Antigen by Primary in Vitro Immunization with Peptide-Pulsed Dendritic Cells

Tsai Van, Southwood Scott, Sidney John, Sakaguchi Kazuyasu, Kawakami Yutaka, Appella Ettore, Sette Alessandro, Celis Esteban

Journal of Immunology 158 ( 4 ) 1796 - 1802 1997.02

ISSN  0022-1767

　View Summary

<p>The gp100 melanoma-associated tumor Ag was selected as a model system to study the diversity of human antitumor cytotoxic T cell responses. First, peptides corresponding to dominant gp100 HLA-A2.1-restricted CTL epitopes were tested using lymphocytes from normal volunteers and an in vitro priming protocol that uses peptide-pulsed dendritic cells as APCs and IL-7 and IL-10 as immune-enhancing cytokines. High CTL activity toward both peptide-pulsed target cells and gp100⁺ melanoma cells was obtained with four out of five peptides tested. Second, HLA-A2.1-binding peptides from gp100 that do no appear to represent CTL epitopes in melanoma patients were also tested for their capacity to induce CTL using the in vitro priming protocol. Three of six peptides tested induced CTL in lymphocytes from normal volunteers. One of these peptides was also immunogenic for lymphocytes derived from a melanoma patient in remission. Because these three CTL epitopes were not recognized in the natural immune response in melanoma patients but do appear as immunogens when peptides are used to induce the T cell response, they may be considered as typical "subdominant" epitopes. The results are discussed in the context of the usefulness of this approach to detail the immunologic potential of a given tumor-associated Ag and its relevance for the design of effective immune-based therapies.</p>

Human tumor antigens recognized by T-cells

Kawakami Yutaka, Rosenberg Steven A.

Immunologic Research 16 ( 4 ) 313 - 339 1997

ISSN  0257-277X

　View Summary

<p>T-cells play an important role in in vivo tumor rejection in many animal tumor models and in human melanoma. Many human tumor antigens recognized by autologous T-cells have now been identified. These are found to be nonmutated and mutated peptides derived from various self proteins as well as viral proteins. A variety of mechanisms involved in generating these T-cell epitopes on growing cancers have also been identified. However, the role of these identified antigens remains to be evaluated. Passive or active immunotherapies using these identified tumor antigens are being conducted in many institutions. The results obtained from these clinical trials may give us better insight into the role of T-cell responses to each antigen in tumor rejection as well as the development of new antigen-specific immunotherapies for patients with cancer.</p>

Induction of melanoma reactive T cells by stimulator cells expressing melanoma epitope-major histocompatibility complex class I fusion proteins

Kang X., Robbins P. F., Fitzgerald E. B., Wang R. F., Rosenberg S. A., Kawakami Y.

Cancer Research 57 ( 2 ) 202 - 205 1997

ISSN  0008-5472

　View Summary

<p>Several epitopes in the human melanoma antigens recognized by HLA-A2- restricted CTLs have a relatively low MHC-binding affinity and as a result may be expressed at very low densities on the cell surface, indicating that these epitopes may not be efficient immunogens. To express these epitopes at higher densities on the surface of antigen-presenting cells and therefore improve their immunogenicity, a DNA construct in which a cDNA fragment encoding the melanoma epitope MART-1_27-35 or gp100_280-288 was inserted between sequences encoding the leader and the HLA-A* 0201 protein. Cells transfected with these epitope-HLA fusion constructs were recognized by HLA-A2-restricted melanoma-reactive CTLs specific for the MART-1 or gp100 epitope. In addition, tumor-reactive CTLs could be induced from PBMCs of patients with metastatic melanoma by in vitro stimulation with HMY-C1R B- cell lines expressing the MART-1 or gp100 epitope-HLA-A* 0201 fusion protein. These epitope-HLA fusion constructs may be useful for the development of immunotherapies for patients with melanoma.</p>

The Intronic Region of an Incompletely Spliced gp100 Gene Transcript Encodes an Epitope Recognized by Melanoma-Reactive Tumor-Infiltrating Lymphocytes

Robbins Paul F., El-Gamil Mona, Li Yong F., Fitzgerald Ellen B., Kawakami Yutaka, Rosenberg Steven A.

Journal of Immunology 159 ( 1 ) 303 - 308 1997

ISSN  0022-1767

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<p>Recent studies have characterized a number of the Ags that are recognized by melanoma-reactive T cells. Although the majority of tumor Ags appear to represent nonmutated gene products, a variety of epitopes have been shown to arise from either mutated or alternatively processed transcripts. Here, we report that the screening of a cDNA library with a HLA-A24-restricted melanoma-reactive T cell cloid derived from tumor infiltrating lymphocytes resulted in the isolation of a variant of the gp100 gene that had retained the entire fourth intron of this gene, termed gp100-in4. The gp100-in4 transcript could be detected by reverse transcriptase-PCR but could not be detected in Northern blots conducted with melanoma RNA, indicating that it represents a relatively rare transcript. Read-through of this transcript into the region corresponding to the fourth intron gave rise to an additional 35 amino acids not found in the normal gp100 glycoprotein, and a peptide within this region conforming to the HLA-A24 consensus motif (VYFFLPDHL) was shown to be recognized by the T cell cloid. The sequence of the intron was identical with that of a previously isolated genomic gp100 clone, and T cells that recognized the gp100-in4 gene product were found to recognize HLA-A24-matched allogeneic melanoma cell lines and melanocytes, demonstrating that this represents a nonmutated epitooe. These results further extend the types of Ags that can be recognized by melanoma-reactive T cells to aberrant transcripts of melanosomal genes.</p>

Differential expression of MART-1 in primary and metastatic melanoma lesions

Kageshita Toshiro, Kawakami Yutaka, Hirai Shunji, Ono Tomomichi

Journal of Immunotherapy 20 ( 6 ) 460 - 465 1997

ISSN  1053-8550

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<p>Twenty-eight primary and 29 metastatic melanoma lesions and 18 pigmented nevi lesions were analyzed by using the immunoperoxidase reaction with anti- MART-1 and anti-gp100 monoclonal antibodies (mAbs). The MART-1 was expressed in 28, 29, and 18, and gp100 was expressed in 27, 28, and eight of these lesions, respectively. Intensity and percentage of stained cells with anti- MART-1 mAb were stronger and higher than those with anti-gp100 mAb. MART-1 was expressed homogeneously in primary melanoma and pigmented nevi, whereas it was heterogeneously expressed in metastatic melanoma lesions. The level of expression of MART-1 in primary melanoma lesions did not correlate with any clinicopathologic parameters. These results suggest that anti-MART-1 mAb is a useful tool for immunohistochemical analysis of melanocytic lesions and also is useful for patient's selection and monitoring of antigen-loss variants in clinical trials with the MART-1-based immunotherapy.</p>

Immunobiology of human melanoma antigens MART-1 and gp100 and their use for immuno-gene therapy

Kawakami Yutaka, Rosenberg Steven A.

International Reviews of Immunology 14 ( 2-3 ) 173 - 192 1997

ISSN  0883-0185

Melanosomal proteins as melanoma-specific immune targets

Sakai C., Kawakami Y., Law L. W., Furumura M., Hearing V. J.

Melanoma Research 7 ( 2 ) 83 - 95 1997

ISSN  0960-8931

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<p>Pigmentation of our skin, hair and eyes is essential for photoprotection, embryological development, detoxification end protective/cosmetic coloration. A number of proteins important to the production of melanin within melanosomes have now been identified including enzymatic and structural proteins encoded at the murine albino, brown, pinkeyed-dilution, MART1, siaty end silver loci. Interestingly, many of those melanosomal proteins (including epitopes derived from tyrosinase, TRP1/gp75, silver/gp100 end MART1/melan-A) function in vivo as targets of humoral and cellular autoimmune responses directed specifically against normal or transformed melanocytes. These findings have provided new impetus to research on immune responses to melanoma end, perhaps more importantly, examining why they are insufficient to provide protection against tumour growth end what type of immune therapy can be designed to correct that. The melanosome must now be considered beyond its function in pigmentation, end assumes the role of a valuable source for specific immune targets for malignant melenoma.</p>

Identification of TRP-2 as a human tumor antigen recognized by cytotoxic T lymphocytes

Wang Rong Fu, Appella Ettore, Kawakami Yutaka, Kang Xiaoqiang, Rosenberg Steven A.

Journal of Experimental Medicine 184 ( 6 ) 2207 - 2216 1996.12

ISSN  0022-1007

　View Summary

<p>The infusion of TIL586 along with interleukin-2 into the autologous patient with metastatic melanoma resulted in the objective regression of tumor. A gene encoding a tumor antigen recognized by TIL586 was previously isolated and shown to encode gp75 or TRP-1. Here we report that TRP-2 was identified as a second tumor antigen recognized by a HLA-A31-restricted CTL clone derived from the TIL586 cell line. The peptide LLPGGRPYR epitope was subsequently identified from the coding region of TRP-2 based on studies of the recognition of truncated TRP-2 cDNAs and the HLA-A31 binding motif. This epitope peptide was capable of sensitizing target cells for lysis by a CTL clone at 1 nM peptide concentration. Although some modified peptides could be recognized by the CTL clone, none were found to be better recognized by T cells than the parental peptide. Like other melanoma differentiation antigens, TRP-2 was only expressed in melanoma, melanocytes, and retina, but not in other human tissues tested.</p>

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Human tumor antigens recognized by T cells

Robbins Paul F., Kawakami Yutaka

Current Opinion in Immunology 8 ( 5 ) 628 - 636 1996.10

ISSN  0952-7915

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<p>The ability of tumor-reactive T cells to mediate in vivo tumor regression has been demonstrated in murine tumor models and by the clinical responses to adoptive immunotherapy with tumor-infiltrating lymphocytes isolated from human melanomas. Investigations carried out in the past several years have resulted in the isolation of a number of the genes encoding antigens recognized by melanoma-reactive T cells. The ability of these products to serve as tumor regression antigens has now begun to be evaluated in clinical vaccine trials.</p>

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Improved Induction of Melanoma-Reactive CTL with Peptides from the Melanoma Antigen gp100 Modified at HLA-A*0201-Binding Residues

Parkhurst Maria R., Salgaller Michael L., Southwood Scott, Robbins Paul F., Sette Alessandro, Rosenberg Steven A., Kawakami Yutaka

Journal of Immunology 157 ( 6 ) 2539 - 2548 1996.09

ISSN  0022-1767

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<p>Recognition of the melanoma Ag gp100 by tumor-infiltrating lymphocytes (TIL) in vitro has been correlated with tumor regression in patients with metastatic melanoma treated with the adoptive transfer of TIL plus IL-2. Three common gp100 epitopes have been identified that are recognized in the context of HLA-A2 by TIL from different patients: G9₁₅₄ (KTWGQYWQV), G9₂₀₉ (ITDQVPFSV), and G9₂₈₀ (YLEPGPVTA). Upon stimulation with these peptides, melanoma-reactive CTL could be induced in vitro from PBL of some HLA-A2⁺ melanoma patients. However, numerous restimulations were required, and specific reactivity could not be generated in many patients. Therefore, to enhance the immunogenicity of gp100 peptides, amino acid substitutions were introduced into G9₁₅₄, G9₂₀₉, and G9₂₈₀ at HLA-A*0201-binding anchor positions, but not at TCR contact residues, to increase peptide class I MHC-binding affinity. Several modified gp100 peptides bound with greater affinity to HLA-A*0201 than unmodified peptides and were recognized by TIL specific for the natural epitopes. These peptides were used to sensitize PBL from HLA-A2⁺ melanoma patients in vitro using peptide-pulsed autologous PBMC as stimulators. After five weekly restimulations with either the native G9₂₀₉ or G9₂₈₀ peptide, melanoma-reactive CTL could only be induced from two of seven patients. However, amino acid substitutions in these peptides enabled the induction of melanoma-reactive CTL from all seven patients. These results suggest that modified gp100 peptides may be more immunogenic than the native epitopes, and may be useful in immunotherapy protocols for patients with melanoma.</p>

T-cell recognition of self peptides as tumor rejection antigens

Kawakami Yutaka, Rosenberg Steven A.

Immunologic Research 15 ( 3 ) 179 - 190 1996.09

ISSN  0257-277X

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<p>Human melanoma antigens and their epitopes recognized by T cells have recently been identified. HLA-A2 binding epitopes of melanoma antigens MART-1 and gp100 were characterized and suspected to be subdominant/cryptic self determinants. Together with other findings of tumor-specific mutated self peptides as tumor antigens recognized by T cells, the nature of the antitumor immune response to human melanoma has been revealed at a molecular level. These findings have implications not only for understanding of the immune response to self peptides in normal and pathologic conditions, but also for the development of immunotherapies for cancer and autoimmune diseases.</p>

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Human melanoma antigens recognized by T lymphocytes

Kawakami Yutaka, Robbins Paul F., Rosenberg Steven A.

Keio Journal of Medicine 45 ( 2 ) 100 - 108 1996.06

ISSN  0022-9717

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<p>Human melanoma antigens and their epitopes recognized by T cells have been identified using a variety of methods. These antigens are classified as 1) melanocyte specific melanosomal proteins (MART-1, gp100, tyrosinase and TRP-1), 2) proteins expressed in testis and a variety of cancers (MAGE-1, MAGE-3, BAGE and GAGE), 3) tumor specific mutated proteins (β-catenin, MUM-1 and CDK4), and 4) others (p15). Some of the HLA-A2 binding non-mutated melanoma epitopes contained non-dominant anchor amino acids and have relatively low HLA-A2 binding affinity, suggesting that these epitopes were likely to be subdominant or cryptic self determinants. The significant correlation observed between vitiligo development and IL2 based immunotherapy suggested that autoreactive T cells specific for these self peptides were involved in melanoma regression in vivo. In addition, since adoptive transfer into patients of CTL recognizing these epitopes resulted in tumor regression, these epitopes may be tumor rejection antigens. Melanoma reactive CTL were efficiently induced from PBL of patients by in vitro stimulation wilh PBMC pulsed with these melanoma epitopes and may be useful in adoptive transfer protocols for the treatment of patients with metastatic melanoma. An immunization trial using the MART-1 and gp100 peptides in conjunction with incomplete Freund's adjuvant is in progress. These identified antigens may be useful for the development of new immunotherapies for the treatment of melanoma patients as well as for understanding the mechanisms of anti-tumor immune responses and autoimmune disorders against melanocytes.</p>

Utilization of an alternative open reading frame of a normal gene in generating a novel human cancer antigen

Wang Rong Fu, Parkhurst Maria R., Kawakami Yutaka, Robbins Paul F., Rosenberg Steven A.

Journal of Experimental Medicine 183 ( 3 ) 1137 - 1140 1996.03

ISSN  0022-1007

　View Summary

<p>Tumor infiltrating lymphocytes (TILs) derived from tumor-bearing patients recognize tumor-associated antigens presented by major histocompatibility complex (MHC) class I molecules The infusion of TIL586 along with interleukin (IL) 2 into an autologous patient with metastatic melanoma resulted in the objective regression of tumor. A gene encoding a tumor antigen recognized by TIL586 was recently isolated and shown to encode gp75. Here we report that an antigenic peptide, MSLQRQFLR, recognized by TIL586 was not derived from the normal gp75 protein. Instead, this nonamer peptide resulted from translation of an alternative open reading frame of the same gene. Thus, the gp75 gene encodes two completely different polypeptides, gp75 as an antigen recognized by immunoglobulin G antibodies in sera from a patient with cancer, and a 24-amino acid product as a tumor rejection antigen recognized by T cells. This represents the first demonstration that a human tumor rejection antigen can be generated from a normal cellular gene using an open reading frame other than that used to encode the normal protein. These findings revealed a novel mechanism for generating tumor antigens which may be useful as vaccines to induce tumor-specific cell-mediated immunity against cancer.</p>

A mutated β-catenin gene encodes a melanoma-specific antigen recognized by tumor infiltrating lymphocytes

Robbins Paul F., El-Gamil Mona, Li Yong F., Kawakami Yutaka, Loftus Douglas, Appella Ettore, Rosenberg Steven A.

Journal of Experimental Medicine 183 ( 3 ) 1185 - 1192 1996.03

ISSN  0022-1007

　View Summary

<p>A number of antigens recognized by tumor-reactive T cells have recently been identified. The antigens identified in mouse model systems appear, with one exception, to represent thee products o mutated genes. In contrast, most of the antigens recognized by human tumor-reactive C cells reported to date appear to represent the products of non-mutated genes. Here we report the isolation o a cDNA clone encoding β-catenin, which was shown to be recognized by the tumor-infiltrating lymphocyte (TIL) 1290, a HLA-A24 restricted melanoma-specific CTL line from patient 888. Thee cDNA clone, which was isolated from thee autologous melanoma cDNA library, differed by a single base pair from the published β-catenin sequence, resulting in a change from a serine to a phenylalanine residue at position 37. Normal tissues from this patient did not express the altered sequence, nor did 12 allogeneic melanomas, indicating that this represented a unique mutation in this patient's melanoma. A peptide corresponding to the sequence between amino acids 29 and 37 of the mutant gene product was identified as the T cell epitope recognized by TIL 1290. The observation that HLA-A24 binding peptides contain an aromatic or hydrophobic residue at position 9 suggested that the change at position 37 may have generated a peptide (SYLDSGIHF) which was capable of binding to HLA-A24, and a competitive binding assay confirmed this hypothesis. The β-catenin protein has been shown previously to be involved in cell adhesion mediated through the cadherin family of cell surface adhesion molecules. The high frequency of mutations found in members of cellular adhesion complexes in a variety of cancers suggests that these molecules may play a role in development of the malignant phenotype.</p>

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Loss of functional beta₂-microglobulin in metastatic melanomas from five patients receiving immunotherapy

Restifo Nicholas P., Marincola Francesco M., Kawakami Yutaka, Taubenberger Jeff, Yannelli John R., Rosenberg Steven A.

Journal of the National Cancer Institute 88 ( 2 ) 100 - 108 1996.01

ISSN  0027-8874

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<p>Background: In a subset of patients with metastatic melanoma, T lymphocytes bearing the cell-surface marker CD8 (CD8⁺ T cells) can cause the regression of even large tumors. These antitumor CD8⁺ T cells recognize peptide antigens presented on the surface of tumor cells by major histocompatibility complex (MHC) class I molecules. The MHC class I molecule is a heterodimer composed of an integral membrane glycoprotein designated the α chain and a non-covalently associated, soluble protein called beta₂- microglobulin (β₂m). Loss of β₂m generally eliminates antigen recognition by antitumor CD8⁺ T cells. Purpose: We studied the loss of β₂m as a potential means of tumor escape from immune recognition in a cohort of patients receiving immunotherapy. Methods: We successfully grew 13 independent tumor cell cultures from tumor specimens obtained from 13 patients in a cohort of 411 consecutive patients undergoing immunotherapy for metastatic melanoma and for whom tumor specimens were available. These cell lines, as well as another melanoma cell line (called 1074mel) that had been derived from tumor obtained from a patient in a cytokine-gene therapy study, were characterized in vitro cytofluorometrically for MHC class I expression and by northern and western blot analyses for messenger RNA (mRNA) and protein expression, respectively, and ex vivo by immunohistochemistry. Results: After one melanoma cell line (107mel) was found not to express functional β₂m by cytofluorometric analysis, four (31%) of the 13 newly established melanoma cell lines were found to have an absolute lack of functional MHC class I expression. Northern blot analysis of RNA extracted from the five cell lines exhibiting no functional MHC class I expression showed that these cells contained normal levels of α-chain mRNA but variable levels of β₂m mRNA. In addition, no immunoreactive β₂m protein was detected by western blot analysis. When human β₂m was transiently expressed with the use of a recombinant vaccinia virus, cell-surface MHC class I expression was reconstituted and the ability of these five cell lines to present endogenous antigens was restored. Immunohistochemical staining of tumor sections revealed a lack of immunoreactive MHC class I in vivo, supporting the notion that the in vitro observations were not artifactual. Furthermore, archival tumor sections obtained from patients prior to immunotherapy were available from three patients and were found to be β₂m positive. This result was consistent with the hypothesis that loss of β₂m resulted from immunotherapy. Conclusions: These data suggest that the loss of β₂m may be a mechanism whereby tumor cells can acquire immunoresistance. This study represents the first characterization of a molecular route of escape of tumors from immune recognition in a cohort of patients being treated with immunotherapy.</p>

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Antigen-specific tumor vaccines

Zhai Yifan, Yang James C., Kawakami Yutaka, Spiess Paul, Wadsworth Samuel C., Cardoza Lisa M., Couture Larry A., Smith Alan E., Rosenberg Steven A.

Journal of Immunology 156 ( 2 ) 700 - 710 1996.01

ISSN  0022-1767

　View Summary

<p>The human melanoma tumor Ags, MART1 and gp100, are specifically recognized by HLA-A2-restricted CD8⁺ CTLs derived from melanoma patients and appear to be involved in tumor regression. In order to develop immunizing vectors for the treatment of patients with metastatic melanoma, replication-defective recombinant adenoviruses, Ad2CMV-MART1 and Ad2CMV-gp100, which encode these tumor Ags, have been generated. Infection of non-Ag expressing HLA-A2⁺ cell lines A375 and MDA-231 with the vectors resulted in recognition by Ag- specific CTLs as demonstrated by specific target cell lysis and release of cytokines, including IFN-γ, TNF-α, and granulocyte-macrophage-CSF. Sodium butyrate and TNF-α can further augment adenovirus-mediated transgene expression and increase recognition by specific CTLs. Although adenovirus- infected cells expressed the E3/19K protein at detectable levels, significant reduction of surface MHC class I expression was observed in only 3 of 10 tumor cell lines infected with either Ad2CMV-MART1 or Ad2CMV-gp100. Because of the suspected homology between the human MART1 and gp100 genes and their murine counterparts, we immunized C57BL/6 mice with these recombinant adenoviruses and demonstrated that immunization with Ad2CMV-gp100 could protect mice from murine melanoma B16 challenge administered intradermally. Depletion of CD8⁺ but not CD4⁺ T cells in vivo from Ad2CMV-gp100-vaccinated mice eliminated the protective effect. The anti-gp100 T cells induced by Ad2CMV-gp100 vaccination appeared to be responsible for the protection. Thus, these recombinant adenoviruses encoding tumor Ags may be useful as vaccines to induce specific T cell immunity for cancer therapy.</p>

Analysis of expression of the melanoma-associated antigens MART-1 and gp100 in metastatic melanoma cell lines and in in situ lesions

Marincola Francesco M., Hijazi Yasmine M., Fetsch Patricia, Salgaller Michael L., Rivoltini Licia, Cormier Janice, Simonis Toni B., Duray Paul H., Herlyn Meenhard, Kawakami Yutaka, Rosenberg Steven A.

Journal of Immunotherapy 19 ( 3 ) 192 - 205 1996

ISSN  1053-8550

　View Summary

<p>MART-1 and gp100 melanoma associated antigens (MAA) are expressed by cells of the melanocytic lineage and are recognized by the majority of HLA- A2 restricted tumor-infiltrating lymphocytes. Heterogeneity of expression of MAA in tumor deposits may affect the natural history or response to therapy of patients with melanoma. In this study, we evaluated the expression of these MAA with a new monoclonal antibody (mAb) directed against MART-1 (M2- 7C10) and the commercially available HMB45 mAb directed against gp100. Expression was tested in vitro by intracellular fluorescence analysis and in vivo by immunophenotyping of tissue specimens. Nine melanoma cell lines and 25 tissue specimens from metastatic melanoma were analyzed. One cell line did not express MART-1 or gp100. The expression of both antigens was more heterogeneous and significantly reduced (p &lt; 0.01) in melanoma cell lines compared with melanocytes, suggesting progressive loss of expression of MAA by neoplastic cells. None of the nonmelanoma cancer lines tested stained for MART-1 or gp100. Analysis of melanoma lesions by immunohistochemistry showed significant heterogeneity of expression of both MART-1 and gp100 MAA either as a percentage of cells expressing MAA or as intensity of expression. Ten of 25 frozen sections expressed MART-1 in &lt;50% of the cells. In 6 of 25 lesions, immunoreactivity for MART-1 was totally absent. Fine needle aspiration of metastatic lesions seemed to yield information accurately about amount and heterogeneity of expression of MAA in tumor lesions in vivo. Heterogeneity of expression of MAA may be one of several mechanisms leading to tumor escape from immune recognition, and pretreatment evaluation of tumor lesion for expression of these antigens may help in selecting patients best suited to antigen-specific vaccine therapies.</p>

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Identification of tumor-regression antigens in melanoma.

Kawakami Y., Robbins P. F., Wang R. F., Rosenberg S. A.

Important advances in oncology    3 - 21 1996

ISSN  0883-5896

Recognition of Multiple Epitopes in the Human Melanoma Antigen gplOO by Peripheral Blood Lymphocytes Stimulated in Vitro with Synthetic Peptides

Salgaller Michael L., Afshar Alireza, Marincola Francesco M., Rivoltini Licia, Kawakami Yutaka, Rosenberg Steven A.

Cancer Research 55 ( 21 ) 4972 - 4979 1995.11

ISSN  0008-5472

　View Summary

<p>gplOO is a melanocyte lineage-specific antigen recognized by tumor-infiltrating lymphocytes whose adoptive transfer has been associated with tumor regression in patients with metastatic melanoma. The peripheral blood mononuclear cells of five melanoma patients were sensitized in vitro with synthetic peptides to elicit antigen-specific cytotoxic T lymphocyte (CTL) lines against four gplOO epitopes. These epitope-specific CTL lines were generated following weekly in vitro stimulation with the synthetic decamer G10476 (V-L-Y-R-Y-G-S-F-S-V) or the nonamers G9280 (Y-L-E-P-G-P-V-T-A), G9154 (K-T-W-G-Q-Y-W-Q-V), or G9209 (I-T-D-Q-V-P-F-S-V) pulsed onto autologous irradiated peripheral blood mononuclear cells. These lines grew as long as 4 months in culture in low-dose interleukin 2 (30 IU/ml) and exhibited antigen-specific, MHC class I-restricted lysis of peptide-pulsed T2 cells and HLA-A2+, gplOO+ established melanoma cell lines. G10476- and G9280-specific CTLs demonstrated specific release of granulocyte-macrophage-colony-stimnlating factor and tumor necrosis factor a in response to T2 cells pulsed with relevant peptide, as well as to gplOO+ melanoma cell lines. These results demonstrate that several peptides derived from the gplOO protein are presented on the surface of melanoma cells and are sufficiently immunogenic to generate, in vitro, potent CTLs capable of cytolysis and the secretion of cytokines. Therefore, for HLA-A2+ melanoma patients, these and possibly other gplOO peptides could represent good candidates for antigen-specific immunotherapy either singly or in a multivalent regimen.</p>

Molecular characterization of defective antigen processing in human prostate cancer

Sanda Martin G., Restifo Nicholas P., Walsh Jonathan C., Kawakami Yutaka, Nelson William G., Pardoll Drew M., Simons Jonathan W.

Journal of the National Cancer Institute 87 ( 4 ) 280 - 285 1995.02

ISSN  0027-8874

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<p>Background:Gene-modified tumor cell vaccines have shown efficacy in animal models of malignancy, including prostate cancer. Class I major histocompatibility complex (MHC) assembly and function in the cellular targets of such therapies is pivotal in determining the efficacy of specific cytokine-secreting tumor vaccines.Purpose To help guide development of genetically engineered vaccine therapy for human prostate cancer, potential immune resistance pathways were evaluated by analysis of class I MHC assembly in prostate cancer cells. Method: Class I MHC assembly in metastasis-derived human prostate cancer cell lines (LNCaP, PPC-1, DU-145, PC-3, and TSU) and a normal prostate-derived cell line (TP-2) were characterized by phenotypic, molecular, and functional assays. Assembled class I MHC and antigen was measured by flow cytometry; mRNA levels of assembly components (class I MHC heavy chain,; β2-microglobulin, and the antigen transporter gene product TAP-2) were determined; and antigen processing was measured with a chimeric reconstituted system using vaccinia vectors. Restoration of antigen processing was attempted by interferon gamma stimulation and by transfection with mouse class I MHC heavy-chain cDNA. Results: Assembled class I MHC was underexpressed in two (LNCaP and PPC-1) of five prostate cancer cell lines compared with normal prostate-derived controls. PPC-1 cells underexpressed TAP-2 mRNA despite abundant class I MHC and β2-microglobulin message. Induction of TAP-2 by interferon gamma indicated that coding sequences for TAP-2 message were present in PPC-1. Resistance to cytotoxic T lymphocytes (CTL) lysis showed a functional defect in antigen transport by PPC-1 cells; reversal of the molecular defect with interferon gamma led to restoration of functional antigen processing. In contrast, LNCaP cells had competent antigen transport but deficient class I MHC heavy-chain function despite abundant class I MHC RNA; though refractory to stimulation by interferon gamma, this defect responded to transfection of class I MHC heavy-chain cDNA. Conclusions: Metastatic prostate cancer cells can escape T-cell recognition via divergent mechanisms of defective class I MHC assembly. The specific under-expression of TAP-2 gene product in PPC-1 cells contrasts with prior studies of TAP gene underexpression in lung cancer (which concurrently underexpressed class I MHC heavy chain) and provides evidence for a regulatory pathway controlling TAP-2 gene expression in human cancers that may not affect class I MHC heavy-chain expression- Implications:In clinical application of gene therapy for prostate cancer, these findings provide a rationale for focusing on strategies that can circumvent sole reliance on class I MHC-mediated tumor cell recognition by CTL. [J Natl Cancer Inst 87: 280-285, 1995]</p>

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Identification of a gene encoding a melanoma tumor antigen recognized by HLA-A31-restricted tumor-infiltrating lymphocytes

Wang Rong Fu, Robbins Paul F., Kawakami Yutaka, Kang Xiao Qiang, Rosenberg Steven A.

Journal of Experimental Medicine 181 ( 2 ) 799 - 804 1995.02

ISSN  0022-1007

　View Summary

<p>The availability of antitumor cytotoxic T lymphocytes which can be generated from either peripheral blood lymphocytes after stimulation in vitro or tumor infiltrating lymphocytes (TIL) has made it possible to identify a number of melanoma antigens presented by major histocompatibility complex class I molecules. The present and previous studies indicated that TIL586 recognized an antigen expressed on most melanoma and normal melanocytes in the context of the HLA-A31 molecule. We report here the cloning of a cDNA that directs the expression of the shared melanoma antigen recognized by this TIL. The DNA sequence analysis revealed that the cDNA was almost identical to the gene encoding tyrosinase-related protein 1 or glycoprotein gp75 which was originally identified by serum antibodies in a patient with melanoma. The gene was found to be expressed only in melanoma, normal melanocyte cell lines, and retina, but not in other normal tissues tested. The gp75 antigen presented by HLA-A31 may therefore constitute a useful immune target for specific treatment of patients with melanoma, since both antibody- and T cell mediated immune responses can be generated against this antigen.</p>

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Induction of tumor-reactive CTL from peripheral blood and tumor-infiltrating lymphocytes of melanoma patients by in vitro stimulation with an immunodominant peptide of the human melanoma antigen MART-1

Rivoltini Licia, Kawakami Yutaka, Sakaguchi Kazuyasu, Southwood Scott, Sette Alessandro, Robbins Paul F., Marincola Francesco M., Salgaller Michael L., Yannelli John R., Appella Ettore, Rosenberg Steven A.

Journal of Immunology 154 ( 5 ) 2257 - 2265 1995

ISSN  0022-1767

　View Summary

<p>MART-1 is an Ag expressed on melanomas and melanocytes, and is recognized by the majority of HLA-A2-restricted tumor-specific tumor-infiltrating lymphocytes (TIL) from melanoma patients. In the present study we have analyzed 10 potential 9-mer epitopes containing the HLA-A2.1 binding motifs for their ability to induce melanoma-specific T cell lines. Antimelanoma CTL could be generated only with MART-1_27-35 peptide, which has been previously shown to be recognized by a majority of HLA-A2-restricted TIL. Anti-MART-1_35-43-specific CTL could also be induced, but these T cells did not recognize melanoma cells. MART-1_27-35-specific CTL could be effectively generated from a total of 11 of 12 PBL and from 3 of 3 TIL derived from HLA-A2⁺ melanoma patients, as well as from 2 of 4 PBL from HLA-A2⁺ healthy donors by in vitro stimulation with autologous PBMC pulsed with the synthetic MART-1_27-35 peptide. These CTL lines specifically lysed and release cytokines (TNF-α, IFN-γ, and GM-CSF) in response to T2 cells pulsed with MART-1_27-35, as well as to HLA-A2⁺ MART-1⁺ melanoma cells. CTL generated with MART-1_27-35 also lysed uncultured HLA-A2⁺ melanoma cells derived from tumor biopsies, indicating that this MART-1 epitope is likely to be expressed in association with HLA-A2 on the surface of tumor cells in vivo. CTL lines generated with MART-1_27-35 mediated 25- to 100-fold higher lytic activity than MART-1 -reactive CTL grown from TIL in the presence of high dose IL-2. These results demonstrate that MART-1_27-35 peptide may represent an ideal candidate for Ag-specific immunotherapy in melanoma patients.</p>

Melanoma tumor-infiltrating lymphocytes derived from four distinct anatomic sites obtained from a single patient

Yannelli J. R., McConnell S., Parker L., Nishimura M., Robbins P., Yang J., El Gamil M., Kawakami Y.

Journal of Immunotherapy 18 ( 4 ) 263 - 271 1995

ISSN  1053-8550

　View Summary

<p>Tumor-infiltrating lymphocytes (TILs) were grown from four distinct anatomic sites from a patient with metastatic melanoma. The metastatic sites included a tumor-involved lymph node, a subcutaneous lesion obtained from the chest wall, a portion of bowel, and adrenal gland. TILs grown from each anatomic site over the course of 20 days in the presence of 6,000 IU/ml recombinant interleukin-2 exhibited comparable growth rates. Between days 30 and 45, the TILs were a mixture of CD3⁺CD4⁺ and CD3⁺CD8⁺ lymphocytes expressing the αβ form of the T-cell receptor. TILs derived from each anatomic site specifically lysed autologous tumor obtained from all four anatomic sites. In fine specificity analysis, the TILs exhibited human leukocyte antigen (HLA-A2)-restricted lysis of fresh tumor targets and cultured melanoma cell lines. Each TIL recognized a product of the MART-1 gene, and specifically, the monomer peptide MART-1_27-35. Thus lymphocytes reactive with the MART-1 melanoma antigen appeared to be widely distributed in diverse metastases in this patient. This information, along with previous data on the reactivity of multiple patients to this antigen, attests to its dominance in the immune reactivity of humans to melanoma.</p>

Characterization of the functional specificity of a cloned T-cell receptor heterodimer recognizing the MART-1 melanoma antigen

Cole D. J., Weil D. P., Shilyansky J., Custer M., Kawakami Y., Rosenberg S. A., Nishimura M. I.

Cancer Research 55 ( 4 ) 748 - 752 1995

ISSN  0008-5472

　View Summary

<p>T cells can play a central role in the immune response to cancer, with tumor-specific T-lymphocyte reactivity provided by the T-cell receptor (TCR) α and β chain heterodimer. This study is the first report of the definitive identification and characterization of a functional tumor-associated, antigen-specific TCR by reconstitution in an alternate cell line. Jurkat T cells were transfected with the cDNAs encoding the full-length α and β T- cell receptor chains from the HLA-A2 restricted, melanoma-reactive T-cell clone, clone 5. Expression of the transfected TCR was evaluated by immunofluorescence after down-modulation of the endogenous receptor with Jurkat T-cell receptor β chain-specific mAb. Jurkat clone 5 TCR⁺ cells recognized MART-1 peptides presented by T2 cells in a pattern and sensitivity equivalent to native MART-1-reactive T-cells. Recognition of HLA-A2+ melanoma cell lines by the Jurkat clone 5 TCR⁺ cells, however, did not occur without the addition of exogenous MART-1 peptide. The cloning and expression of functional TCR genes which are capable of specifically recognizing MART-1 antigen provides reagents which could be used for the study of the mechanisms of T-cell/tumor antigen interactions and creates immortalized reagents which can facilitate studies requiring detection of the MART-1 antigen. The tumor reactivity provided by these genes could also have application in novel immunotherapeutic strategies for treating patients with melanoma, including redirection of tumor-infiltrating lymphocyte specificity and bone marrow stem cell therapy.</p>

Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression

Kawakami Yutaka, Eliyahu Siona, Jennings Christopher, Sakaguchi Kazuyasu, Kang Xiaoqiang, Southwood Scott, Robbins Paul F., Sette Alessandro, Appella Ettore, Rosenberg Steven A.

Journal of Immunology 154 ( 8 ) 3969 - 3974 1995

ISSN  0022-1767

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<p>Four often HLA-A2-restricted melanoma specific CTL that were derived from tumor-infiltrating lymphocytes (TIL) and administered to patients recognized the gp100 melanoma Ag and nine of ten recognized the MART-1 Ag. Adoptive transfer of the four gp100-reactive CTL, but not the other TIL, resulted in tumor regression when infused into autologous patients along with IL-2. Tumor regression was thus correlated with the recognition of gp100 by the administered T cells (p = 0.0048). To identify the epitopes recognized by these four gp100-reactive CTL, 169 peptides containing HLA-A2.1 binding motifs were synthesized and screened for their recognition by TIL using cytotoxicity and IFN-γ release assays. Five gp100 epitopes (two for TIL620, three for TIL660, one for TIL1143, and two for TIL1200) were recognized by CTL derived from different patients. Five of eight HLA-A2 binding melanoma epitopes (five gpl 00, one MART-1/Melan-A, two tyrosinase) had intermediate binding affinity to HLA-A2.1. These gp100 epitopes may be responsible for mediating tumor rejection in vivo and thus may be useful for the devel opment of immunotherapies for patients with melanoma.</p>

Identification of a tyrosinase epitope recognized by HLA-A24-restricted, tumor-infiltrating lymphocytes

Kang X., Kawakami Y., El-Gamil M., Wang R., Sakaguchi K., Yannelli J. R., Appella E., Rosenberg S. A., Robbins P. F.

Journal of Immunology 155 ( 3 ) 1343 - 1348 1995

ISSN  0022-1767

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<p>A number of Ags recognized by class I-restricted, melanoma-specific T cells have recently been identified. In this report we demonstrated that tumor-infiltrating lymphocytes (TIL) from melanoma patient 1413 recognize a tumor Ag, tyrosinase, in the context of HLA-A24. This Ag had previously been shown to be recognized by an HLA-A24-restricted TIL, TIL 888, as well as HLA- A2-restricted, melanoma-specific T cells isolated from two additional patients. The peptide epitope recognized by TIL 1413 was then identified through the use of sequential deletions of the tyrosinase cDNA, as well as through prediction of HLA-A24 binding peptides based on a previously identified motif. Two peptides, a 9-amino acid peptide (AFLPWHRLF) and an overlapping 10-amino acid peptide (AFLPWHRLFL) containing an additional leucine at the carboxyl terminus, were both recognized by TIL 1413. Anti- peptide-specific CTL could be induced by repeated stimulation of peripheral blood lymphocytes from melanoma patient 1413, and this CTL line specifically recognized both HLA-A24⁺ B cell lines pulsed with the peptide and HLA-A24⁺ tyrosinase⁺ melanoma cells. This peptide thus represents a reagent that may be used to generate melanoma-specific T cells for adoptive immunotherapy, as well as in peptide vaccines for HLA-A24⁺ melanoma patients.</p>

Generation of tumor-specific CTLs from melanoma patients by using peripheral blood stimulated with allogeneic melanoma tumor cell lines

Stevens E. J., Jacknin L., Robbins P. F., Kawakami Y., El Gamil M., Rosenberg S. A., Yannelli J. R.

Journal of Immunology 154 ( 2 ) 762 - 771 1995

ISSN  0022-1767

　View Summary

<p>PBLs were isolated from 13 patients with metastatic melanoma. Mixed lymphocyte tumor cell cultures (ML TCs) were established (15 times) by using irradiated HLA-matched (one class I locus) allogeneic melanoma tumor cell lines (13 times) or autologous melanoma tumor cell lines (two times) in medium containing 120 IU/ml IL-2 and 100 IU/ml IL-4. PBLs grew to levels that could be assessed for functional reactivity 9 of 15 times. In seven of nine cases, CD3⁺CD8⁺ CTLs grew from MLTCs that were tumor specific; five were restricted by HLA-A2 and two were restricted by HLA-A24. Four of the tumor- specific CTL lines lysed autologous fresh tumor cells. Tumor-specific CTLs from two of three patients had cytolytic activity identical with tumor- infiltrating lymphocytes (TIL) derived from tumor biopsies removed earlier and grown in high concentrations (6000 IU/ml) of IL-2. Three of the HLA-A2- restricted tumor-specific CTLs were shown to recognize 293 cells transfected with HLA-A2.1 cDNA and the gene encoding the melanoma Ag, MART-1. In addition, these CTLs recognized the T2 cell line pulsed exogenously with the peptide MART-1_27-35, which is the nine-amino acid immunodominant epitope of the MART-1 Ag recognized on melanoma tumor cells by nearly all HLA-A2- restricted TIL. Thus, we have demonstrated the ability to generate tumor- specific CTLs from PBLs that are similar in their reactivity to TIL. This technique obviates the need for autologous tumor tissue and suggests that PBLs contain sufficient CTL precursors for use in generating antitumor CTLs for cellular immunotherapy trials.</p>

Identification of a novel peptide derived from the melanocyte-specific gp100 antigen as the dominant epitope recognized by an HLA-A2.1-restricted anti-melanoma CTL line

Bakker A. B H, Schreurs M. W J, Tafazzul G., De Boer A. J., Kawakami Y., Adema G. J., Figdor C. G.

International Journal of Cancer 62 ( 1 ) 97 - 102 1995

ISSN  0020-7136

　View Summary

<p>Cytotoxic T lymphocytes (CTL) reactive with human melanoma tumor cells occasionally display cross-reactivity with normal melanocytes. Previously, we identified the melanocyte lineage-specific antigen gp100 that is expressed by both melanoma cells and normal melanocytes, as a target antigen for tumor-infiltrating lymphocytes derived from a melanoma patient (TIL 1200). Here, we demonstrate that the oligoclonal HLA-A2.1-restricted TIL 1200 line is reactive with 2 distinct peptides derived from the gp100 protein. Apart from the peptide corresponding to gp100 amino acids 457-466, we identified the gp100 peptide 154-162 as a second epitope recognized by TIL 1200. A 100-fold lower concentration of this novel gp100 peptide was required for target-cell sensitization compared to peptide 457-466, indicating that the 154-162 peptide is the dominant gp100 epitope for TIL 1200. Together with the recently described gp100 280-288 epitope, 3 distinct CTL epitopes have now been identified in gp100, all presented in the context of HLA-A2.1. Therefore, gp100 is an attractive target antigen in the development of immune-therapeutic protocols against melanoma.</p>

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Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression

Kawakami Y., Eliyahu S., Jennings C., Sakaguchi K., Kang X., Southwood S., Robbins P. F., Sette A., Appella E., Rosenberg S. A.

Journal of Immunology 154 ( 8 ) 3961 - 3968 1995

ISSN  0022-1767

　View Summary

<p>Four of ten HLA-A2-restricted melanoma specific CTL that were derived from tumor-infiltrating lymphocytes (TIL) and administered to patients recognized the gp100 melanoma Ag and nine of ten recognized the MART-1 Ag. Adoptive transfer of the four gp100-reactive CTL, but not the other TIL, resulted in tumor regression when infused into autologous patients along with IL-2. Tumor regression was thus correlated with the recognition of gp100 by the administered T cells (p = 0.0048). To identify the epitopes recognized by these four gp100-reactive CTL, 169 peptides containing HLA-A2.1 binding motifs were synthesized and screened for their recognition by TIL using cytotoxicity and IFN-γ release assays. Five gp100 epitopes (two for TIL620, three for TIL660, one for TIL1143, and two for TIL1200) were recognized by CTL derived from different patients. Five of eight HLA-A2 binding melanoma epitopes (five gp100, one MART-1/Melan-A, two tyrosinase) had intermediate binding affinity to HLA-A2.1. These gp100 epitopes may be responsible for mediating tumor rejection in vivo and thus may be useful for the development of immunotherapies for patients with melanoma.</p>

Quantitative correlation between HLA class I allele expression and recognition of melanoma cells by antigen-specific cytotoxic T lymphocytes

Rivoltini L., Barracchini K. C., Viggiano V., Kawakami Y., Smith A., Mixon A., Restifo N. P., Topalian S. L., Simonis T. B., Rosenberg S. A., Marincola F. M.

Cancer Research 55 ( 14 ) 3149 - 3157 1995

ISSN  0008-5472

　View Summary

<p>MHC class I antigen expression is necessary for CD8+ T-cell-mediated recognition of tumors. Recently, several mechanisms leading to loss or decreased expression of MHC antigens on the tumor cell surface have been described that may account for tumor escape from immune recognition. It is yet unknown whether tumor recognition by CTL occurs at a threshold amount of MHC molecules or correlates with the level of HLA-allele expression. In this study, a model was developed in which clones derived from the 624-MEL melanoma cell line and expressing varying amounts of HLA-A2 molecules were lysed in a standard ⁵¹Cr release assay by an HLA-A2-restricted CTL clone (A42) or a bulk culture of tumor-infiltrating lymphocytes. The A42 clone and the tumor-infiltrating lymphocyte culture were characterized previously as specifically recognizing the melanoma antigen MART-1_27-35 peptide. A marked heterogeneity in the susceptibility to lysis by A42 was observed in tumor clones and was not due to heterogeneous expression of MART-1 by the clones or loss of accessory molecules involved in the lymphocyte-target interaction. Lysis by A42 and by the tumor-infiltrating lymphocyte culture significantly correlated with the level of HLA-A2 expression, evaluated as mean channel number of fluorescence by flow cytometry (P &lt; 0.001). Transfection of an HLA-A2-negative clone (624.28) with the HLA-A2.1 gene produced a panel of clones expressing different levels of HLA-A2, the lysis of which was highly correlated with the expression of HLA-A2 (P &lt; 0.001). The addition of exogenous MART-1_27-35 peptide enhanced lysis of clones expressing intermediate amounts of HLA-A2 but did not affect clones with high expression. These data suggest that the number of HLA molecules present on the surface of tumor cells can quantitatively affect their lysis by CTL in situations with borderline amounts of peptide and/or MHC.</p>

Cloning of a new gene encoding an antigen recognized by melanoma-specific HLA-A24-restricted tumor-infiltrating lymphocytes

Robbins P. F., El-Gamil M., Li Y. F., Topalian S. L., Rivoltini L., Sakaguchi K., Appella E., Kawakami Y., Rosenberg S. A.

Journal of Immunology 154 ( 11 ) 5944 - 5950 1995

ISSN  0022-1767

　View Summary

<p>The role of tumor-specific T cells in mediating the regression of metastatic melanoma has been suggested by the clinical response of patients to treatment with tumor infiltrating lymphocytes (TIL). A number of Ags recognized by class I-restricted melanoma-specific T cells have recently been isolated, raising the hope that this will lead to the development of improved therapies. In this study, we report the cloning of a tumor Ag recognized by T cells from melanoma patient 888. Previously, we reported that TIL 888, grown from the tumor of this patient, recognized tyrosinase in an HLA-A24- restricted fashion. This line, when infused into the autologous patient, resulted in complete regression of multiple metastases. Three years later, a second TIL line, TIL 1290, was isolated from a recurrent pelvic tumor. Infusion of a mixture of TIL 888 and TIL 1290 cell lines into the patient resulted in complete regression of a residual abdominal mass and the patient remains disease-free 2 yr later. The TIL 1290 cell line, which recognized melanoma in an HLA-A24-restricted manner, failed to recognize tyrosinase. TIL 1290 was then used to screen an 888 melanoma cDNA library, and air Ag was isolated that did not correspond to any found in sequence databases. This gene, termed p15, was found to be expressed in a variety of normal tissues, and a peptide epitope recognized by TIL 1290 was found to represent the product of an nonmutated gene. Screening of additional cDNA pools resulted in the isolation of a second clone which stimulated TIL 1290. This clone also appeared to represent a transcript of the p15 gene, indicating that this gene may encode the predominant Ag recognized by TIL 1290.</p>

Identification of MART-1-specific T-cell receptors

Cole David J., Weil Daniel P., Shamamian Peter, Rivoltini Licia, Kawakami Yutaka, Topalian Suzanne, Jennings Christopher, Eliyahu Siona, Rosenberg Steven A., Nishimura Michael I.

Cancer Research 54 ( 20 ) 5265 - 5268 1994.10

ISSN  0008-5472

　View Summary

<p>Tumor-specific cytotoxic T lymphocytes (CTLs) can mediate tumor regression in patients with metastatic melanoma and play a central role in the immune response to cancer. The recent identification of shared melanoma antigens has raised the possibility of a limited melanoma-specific T-cell receptor (TCR) repertoire, but subsequent studies have been controversial and difficult to interpret without knowing which tumor-associated antigens (TAAs) are being recognized by specific TCRs. However, the recent cloning of several melanoma TAAs now allows for the identification of the specifically recognized TAA and its epitope. We evaluated the TCR of two clonal CD8⁺ CTL lines, A42 and 1E2, from two HLA-A2⁺ patients with metastatic melanoma. Both CTL lines were MART-1 specific, and both demonstrate reactivity to the same epitope when presented in an HLA-A2.1 context. The TCR genes of the two clones were sequenced. All of the productively rearranged A42 TCR β chain genes were Vβ7/Dβ2.1/Jβ2.7/Cβ2; the TCR α chain genes were Vα21/Jα42/Cα. The 1E2 TCR β chain genes were Vβ3/Dβ1.1/Jβ1.1/Cβ1, and TCR α chains were Vα25/Jα54/Cα. This study is the first report of TCR sequences specific for a melanoma epitope. These TCR clones may be useful for the development of more effective immunotherapies and in studies of the mechanism of T-cell recognition of tumor antigen. They also provide direct evidence that the immune system can provide more than one TCR capable of recognizing a TAA epitope.</p>

Human CD4⁺ T cells specifically recognize a shared melanoma-associated antigen encoded by the tyrosinase gene

Topalian Suzanne L., Rivoltini Licia, Mancini Marie, Markus Nancy R., Robbins Paul F., Kawakami Yutaka, Rosenberg Steven A.

Proceedings of the National Academy of Sciences of the United States of America 91 ( 20 ) 9461 - 9465 1994.09

ISSN  0027-8424

　View Summary

<p>Although commonly expressed human melanoma-associated antigens recognized by CD8⁺ cytolytic T cells have been described, little is known about CD4⁺ T-cell recognition of melanoma-associated antigens. Epstein-Barr virus- transformed B cells were used to present antigens derived from whole cell lysates of autologous and allogeneic melanomas for recognition by melanoma- specific CD4⁺ T-cell lines and clones cultured from tumor-infiltrating lymphocytes. HLA-DR-restricted antigens were detected in the lysates on the basis of specific release of cytokines from the responding T cells. Antigen sharing was demonstrated in the majority of melanomas tested, as well as in cultured normal melanocytes, but not in other normal tissues or nonmelanoma tumors. T-cell clones manifested a single recognition pattern, suggesting the presence of an immunodominant epitope. This epitope was identified as a product of the tyrosinase gene, which has also been shown to encode class I- restricted epitopes recognized by CD8⁺ T cells from melanoma patients. Identification of commonly expressed tumor-associated protein molecules containing epitopes presented by both class I and class II major histocompatibility molecules may provide optimal reagents for cancer immunization strategies.</p>

Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection

Kawakami Yutaka, Eliyahu Siona, Delgado Cynthia H., Robbins Paul F., Sakaguchi Kazuyasu, Appella Ettore, Yannelli John R., Adema Gosse J., Miki Toru, Rosenberg Steven A.

Proceedings of the National Academy of Sciences of the United States of America 91 ( 14 ) 6458 - 6462 1994.07

ISSN  0027-8424

　View Summary

<p>The cultured T-cell line TIL1200, established from the tumor-infiltrating lymphocytes (TILs) of a patient with advanced metastatic melanoma, recognized an antigen on most HLA-A2⁺ melanomas and on all HLA-A2⁺ cultured neonatal melanocytes in an HLA-A2 restricted manner but not on other types of tissues or cell lines tested. A cDNA encoding an antigen recognized by TIL1200 was isolated by screening an HLA-A2⁺ breast cancer cell line transfected with an expression cDNA library prepared from an HLA-A2⁺ melanoma cell line. The nucleotide and amino acid sequences of this cDNA were almost identical to the genes encoding glycoprotein gp100 or Pme117 previously registered in the GenBank. Expression of this gene was restricted to melanoma and melanocyte cell lines and retina but was not expressed on other fresh or cultured normal tissues or other types of tumor tested. The cell line transfected with this cDNA also expressed antigen recognized by the melanoma-specific antibody HMB45 that bound to gp100. A synthetic 10-amino acid peptide derived from gp100 was recognized by TIL1200 in the context of HLA-A2.1. Since the administration of TIL1200 plus interleukin 2 resulted in regression of metastatic cancer in the autologous patient, gp100 is a possible tumor rejection antigen and may be useful for the development of immunotherapies for patients with melanoma.</p>

Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes

Kawakami Yutaka, Eliyahu Siona, Sakaguchi Kazuyasu, Robbins Paul F., Rivoltini Licia, Yannelli John R., Appella Ettore, Rosenberg Steven A.

Journal of Experimental Medicine 180 ( 1 ) 347 - 352 1994.07

ISSN  0022-1007

　View Summary

<p>Four melanoma proteins, MART-1, gp100, tyrosinase, and tyrosinase-related protein-1 (gp75) were evaluated for recognition by HLA-A2-restricted melanoma-specific cytotoxic T lymphocytes (CTLs) derived from the tumor- infiltrating lymphocytes (TIL) of 10 different patients. 9 of 10 TIL recognized MART-1, 4 recognized gp100 (including 3 that also recognized MART- 1), but none of the TIL recognized tyrosinase or gp75. Based on the known HLA-A2.1 peptide binding motifs, 23 peptides from MART-1 were synthesized in an attempt to identify the epitopes recognized by TIL. Three peptides were recognized by TIL when pulsed on T2 target cells. One of the 9-mer peptides, AAGIGILTV, was most effective in sensitizing the T2 cells for TIL lysis. This peptide was recognized by 9 of 10 HLA-A2-restricted melanoma-specific CTLs. Therefore, this peptide appears to be a very common immunogenic epitope for HLA-A2-restricted melanoma-specific TIL and may be useful for the development of immunotherapeutic strategies.</p>

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Recognition of tyrosinase by tumor-infiltrating lymphocytes from a patient responding to immunotherapy

Robbins Paul F., El-Gamil Mona, Kawakami Yutaka, Rosenberg Steven A.

Cancer Research 54 ( 12 ) 3124 - 3126 1994.06

ISSN  0008-5472

　View Summary

<p>The observation that allogeneic melanoma cells matched for particular HLA class I alleles stimulate T-cells isolated from patients suggest that widely shared antigens exist on these tumors. A transient expression system was developed for screening a melanoma complementary DNA library using the highly transfectable human kidney cell line 293. Using this system, large numbers of complementary DNA clones can be rapidly screened for the expression of antigens which stimulate T-cells. Tumor-infiltrating lymphocytes from patient 888, which recognized melanoma in the context of HLA-A24, were used to screen a complementary DNA library made from the autologous melanoma. Our results demonstrate that these tumor-infiltrating lymphocytes recognize tyrosinase, a gene previously shown to be recognized by T-cells only in the context of HLA- A2. These data demonstrate that a single antigen can be recognized in the context of two different class 1 HLA alleles. In addition, this study suggests that recognition of tyrosinase by antigen-specific T-cells may be involved in tumor rejection.</p>

Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor

Kawakami Yutaka, Eliyahu Siona, Delgado Cynthia H., Robbins Paul F., Rivoltini Licia, Topalian Suzanne L., Miki Toru, Rosenberg Steven A.

Proceedings of the National Academy of Sciences of the United States of America 91 ( 9 ) 3515 - 3519 1994.04

ISSN  0027-8424

　View Summary

<p>By cDNA expression cloning we have isolated a gene encoding a shared human melanoma antigen recognized by HLA-A2 restricted autologous and allogeneic tumor-infiltrating lymphocytes (TILs) from patients with metastatic melanoma. By using both transient and stable expression systems, transfection of this gene into non-antigen-expressing HLA-A2⁺ cell lines resulted in recognition by the antigen-specific TILs. The sequence of this cDNA revealed a previously undescribed putative transmembrane protein whose expression was restricted to melanoma and melanocyte cell lines and human retina but no other fresh or cultured normal tissues tested or other tumor histologies. Thus, we have identified a gene encoding a melanocyte lineage-specific protein (MART-1; melanoma antigen recognized by T cells 1) that is a widely shared melanoma antigen recognized by the T lymphocytes of patients with established malignancy. Identification of this gene opens possibilities for the development of immunotherapies for patients with melanoma.</p>

T-cell receptor usage by melanoma-specific clonal and highly oligoclonal tumor-infiltrating lymphocyte lines

Shilyansky Joel, Nishimura Michael I., Yannelli John R., Kawakami Yutaka, Jacknin Laura S., Charmley Patrick, Rosenberg Steven A.

Proceedings of the National Academy of Sciences of the United States of America 91 ( 7 ) 2829 - 2833 1994.03

ISSN  0027-8424

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<p>Tumor-infiltrating lymphocytes (TIL) obtained from human melanomas can specifically lyse autologous tumor in vitro and mediate tumor regression in vivo. To develop more effective therapeutic reagents and to further understand the T-cell response to tumors, the diversity of T-cell receptors (TCRs) involved in melanoma antigen recognition has been studied. The TCR variable (V) genes, joining (J) segments, and N diversity regions used by five clonal lines and one highly oligoclonal, melanoma-specific, CD8⁺ TIL line were examined utilizing PCR amplification with V gene subfamily-specific primers and anchor PCR. The TIL lysed multiple allogeneic melanomas expressing matched surface major histocompatibility complex class I molecules. TCR analysis confirmed the clonal nature of the TIL lines; however, the TCR repertoire was diverse. Even among the three HLA-A2 restricted TIL (TIL 1200, TIL F2-2, and TIL 5), no common V gene usage was found. Comparison of the third complementarity-determining regions of the TCRs from the HLA-A2 restricted TIL revealed no homology. Results presented here identify T-cell clonotypes that recognize epitopes on highly prevalent, shared melanoma tumor-associated antigens presented in the context of HLA- 855, HLA-A1, and HLA-A2. These T cells and the antigens they recognize represent important components for the design of new immunotherapies for patients with advanced melanoma.</p>

Melanocyte lineage-specific antigen gp100 is recognized by melanoma-derived tumor-infiltrating lymphocytes

Bakker Alexander B H, Schreurs Marco W J, De Boer Annemiek J., Kawakami Yutaka, Rosenberg Steven A., Adema Gosse J., Figdor Carl G.

Journal of Experimental Medicine 179 ( 3 ) 1005 - 1009 1994.03

ISSN  0022-1007

　View Summary

<p>We recently isolated a cDNA clone that encodes the melanocyte lineage-specific antigen glycoprotein (gp)100. Antibodies directed against gp100 are an important tool in the diagnosis of human melanoma. Since the gp100 antigen is highly expressed in melanocytic cells, we investigated whether this antigen might serve as a target for antimelanoma cytotoxic T lymphocytes (CTL). Here, we demonstrate that cytotoxic tumor-infiltrating lymphocytes (TIL) derived from a melanoma patient (TIL 1200) are directed against gp100. HLA-A2.1⁺ melanoma cells are lysed by TIL from this patient. In addition, murine double transfectants, expressing both HLA-A2.1 and gp100, are lysed by TIL 1200, whereas transfectants expressing only HLA-A2.1 are not susceptible to lysis. Furthermore, the HLA-A2.1⁺ melanoma cell line BLM, which lacks gp100 expression and is resistant to lysis, becomes susceptible after transfection of gp100 cDNA. Finally, HLA-A2.1⁺ normal melanocytes are lysed by TIL 1200. These data demonstrate that the melanocyte differentiation antigen gp100 can be recognized in the context of HLA-A2.1 by CTL from a melanoma patient. Gp100 may therefore constitute a useful target for specific immunotherapy against melanoma, provided that no unacceptable cytotoxicity towards normal tissue is observed.</p>

Recognition of neuroectodermal tumors by melanoma-specific cytotoxic T lymphocytes

Shamamian Peter, Mancini Marie, Kawakami Yutaka, Restifo Nicholas P., Rosenberg Steven A., Topalian Suzanne L.

Cancer Immunology Immunotherapy 39 ( 2 ) 73 - 83 1994.03

ISSN  0340-7004

　View Summary

<p>Melanomas from different patients have been shown to express shared tumor antigens, which can be recognized in the context of the appropriate MHC class 1 molecules by cytolytic T cells. To determine if T-cell-defined melanoma antigens are expressed on other tumors of neuroectodermal origin, four melanoma-specific cytotoxic T lymphocyte (CTL) cultures derived from tumor-infiltrating lymphocytes (TIL) were tested for lysis of a panel of 23 HLA-A2⁺ neuroectodermal tumor cell lines of various histologies, including retinoblastoma (1), neuroblastoma (8), neuroepithelioma (6), astrocytoma (2), neuroglioma (1), and Ewing's sarcoma (5). Low expression of MHC class I and/or ICAM-1 molecules was found on 22 of 23 neuroectodermal tumor lines, and could be enhanced by treatment with interferon γ (IFNγ). Following IFNγ treatment, three Ewing's sarcoma lines were lysed by at least one melanoma TIL culture, and levels of lysis were comparable to melanoma lysis by these TIL. Lysis could be inhibited by monoclonal antibodies directed against MHC class I molecules and against CD3, indicating specific immune recognition of tumor-associated antigens. None of the other neuroectodermal tumors tested were lysed by TIL, but they could be lysed by non-MHC-restricted lymphokine-activated killer cells. This demonstration of immunological cross-reactivity between melanomas and Ewing's sarcomas, two tumors of distinct histological types with a common embryonic origin, has implications for the developmental nature of these CTL-defined tumor antigens. It also raises the possibility that specific antitumor immunotherapies, such as vaccines, may be reactive against more than one form of cancer.</p>

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T-cell receptor repertoire in tumor-infiltrating lymphocytes. Analysis of melanoma-specific long-term lines

Nishimura M. I., Kawakami Y., Charmley P., O'Neil B., Shilyansky J., Yannelli J. R., Rosenberg S. A., Hood L.

Journal of Immunotherapy 16 ( 2 ) 85 - 94 1994

ISSN  1053-8550

　View Summary

<p>Cytotoxic T-lymphocytes (CTLs) can be isolated from human melanoma biopsies that specifically lyse autologous melanoma in vitro and can be effective therapeutic agents for patients with advanced disease. Recent evidence indicates that HLA-A2-restricted, melanoma-specific tumor- infiltrating lymphocytes (TILs) recognize melanomas obtained from different HLA-A2⁺ patients, suggesting the presence of one or more common melanoma antigens. Furthermore, T-cell receptor (TCR) repertoire analysis by other groups of TILs from fresh melanoma biopsies suggests that there is limited TCR V gene usage in TILs. One serious limitation in analyzing the TCR repertoire in fresh tumors has been the inability to correlate TCR usage with immune function. Therefore, the TCR repertoire was determined in long-term TIL cultures that specifically lysed autologous melanoma in vitro and in many cases mediated in vivo regression of metastatic cancer in patients with advanced disease. The TCR repertoire in cultured melanoma-specific TILs was diverse, with each TIL containing an average of 9.5 ± 5.7 of the 23 Vα and 11.2 ± 5.9 of the 23 Vβ subfamilies. Despite the large diversity observed, several Vα and Vβ genes (Vα1, Vα2, Vα22, Vβ13, Vβ14, and Vβ18) are very commonly found in melanoma-specific TILs. No statistically significant associations were observed between the presence of a TCR V gene subfamily in TILs and clinical response, HLA haplotype, or age of the culture. Even though the results in this study suggest that certain TCR V gene segments may be involved in immune responses to human melanoma, we were unable to demonstrate functionally that a particular T-cell clonotype recognizes melanoma tumor- associated antigens. Only the analysis of melanoma-specific CTL clones can determine which clonotypes are important in lysis of human melanoma.</p>

Detection of shared MHC-restricted human melanoma antigens after vaccinia virus-mediated transduction of genes coding for HLA

O'Neil Bert H., Kawakami Yutaka, Restifo Nicholas P., Bennink Jack R., Yewdell Jonathan W., Rosenberg Steven A.

Journal of Immunology 151 ( 3 ) 1410 - 1418 1993.08

ISSN  0022-1767

　View Summary

<p>To detect shared human melanoma Ag that are recognized by HLA-A2 restricted, melanoma-specific CTL derived from tumor infiltrating lymphocytes, we have developed a convenient method to insert and express foreign HLA genes capable of presenting Agon target cell lines. Seventeen melanoma cell lines and 11 nonmelanoma cell lines were infected with recombinant vaccinia virus containing the HLA-A2.1 gene. Infection by the vaccinia virus resulted in expression of functional HLA-A2 molecules on the cell surface of virtually 100% of infected cells within a 3.5-h period. The results showed that 11 of 17 (65%) naturally HLA-A2^- melanoma cell lines were specifically lysed by the HLA-A2-restricted, melanoma-specfic TIL after infection with the vaccinia-HLA-A2.1 virus. None of the nine human nonmelanoma cell lines tested (three colon cancer, four breast cancer, or two immortalized nontumor cell lines) or two murine melanoma cell lines were lysed by the HLA-A2-restricted TIL after vaccinia-HLA-A2.1 infection. Coinfection of the vaccinia virus containing the β2-microglobulin gene with the vaccinia-HLA-A2.1 virus increased the surface expression of HLA-A2 and subsequent lysis by melanoma-specific tumor infiltrating lymphocytes. With this new method we could extend previous findings demonstrating that shared melanoma Ag recognized by HLA-A2-restricted tumor infiltrating lymphocytes exist among melanoma cells from different patients regardless of HLA type. These Ag represent excellent candidates for the development of vaccines to induce T cell responses for the immunotherapy of patients with melanoma.</p>

Identification of human cancers deficient in antigen processing

Restifo Nicholas P., Esquivel Fernando, Kawakami Yutaka, Yewdell Jonathan W., Mulé James J., Rosenberg Steven A., Bennink Jack R.

Journal of Experimental Medicine 177 ( 2 ) 265 - 272 1993.02

ISSN  0022-1007

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<p>Intracellular antigens must be processed before presentation to CD8⁺ T cells by major histocompatibility complex (MHC) class I molecules. Using a recombinant vaccinia virus (Vac) to transiently express the K^d molecule, we studied the antigen processing efficiency of 26 different human tumor lines. Three cell lines, all human small cell lung carcinoma, consistently failed to process endogenously synthesized proteins for presentation to K^d-restricted, Vac-specific T cells. Pulse-chase experiments showed that MHC class I molecules were not transported by these cell lines from the endoplasmic reticulum to the cell surface. This finding suggested that peptides were not available for binding to nascent MHC molecules in the endoplasmic reticulum. Northern blot analysis of these cells revealed low to nondetectable levels of mRNAs for MHC-encoded proteasome components LMP-7 and LMP-2, as well as the putative peptide transporters TAP-1 and TAP-2. Treatment of cells with interferon γ enhanced expression of these mRNAs and reversed the observed functional and biochemical deficits. Our findings suggest that downregulation of antigen processing may be one of the strategies used by tumors to escape immune surveillance. Potential therapeutic applications of these findings include enhancing antigen processing at the level of the transcription of MHC-encoded proteasome and transporter genes.</p>

Differential expression of MAGE-1, -2, and -3 messenger RNA in transformed and normal human cell lines

Zakut Rina, Topalian Suzanne L., Kawakami Yutaka, Mancini Marie, Eliyahu Siona, Rosenberg Steven A.

Cancer Research 53 ( 1 ) 5 - 8 1993.01

ISSN  0008-5472

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<p>The MAGE-1 gene codes for a tumor-specific antigen, MZ2-E, that elicited a cytotoxic T-lymphocyte response in the melanoma patient from whom it was derived. We have developed a simplified method, using polymerase chain reaction amplification of exon 3 followed by restriction enzyme pattern analysis, to distinguish expression of the MAGE-1 gene from MAGE-2 and MAGE-3, other members of this gene family. MAGE-1 mRNA was expressed in 53% of 17 melanoma lines, two of seven Epstein-Barr virus-transformed B-cell lines, and 2 of 5 breast cell lines including a line established from normal breast epithelium. MAGE-1 is not likely to be the common melanoma antigen recognized by the other HLA-A1- or HLA-A2-restricted cytotoxic T-lymphocytes examined in this study, but the fact that it is expressed in about 50% of melanoma cell lines makes it a reasonable target for the immunotherapy of patients bearing HLA-A1.</p>

T-cell recognition of human melanoma antigens

Kawakami Y., Nishimura M. I., Restifo N. P., Topalian S. L., O'Neil B. H., Shilyansky J., Yannelli J. R., Rosenberg S. A.

Journal of Immunotherapy 14 ( 2 ) 88 - 93 1993

ISSN  1053-8550

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<p>The adoptive transfer of tumor-infiltrating lymphocytes (TILs) with interleukin-2 (IL-2) has antitumor activity in some patients with metastatic melanoma. We have analyzed molecular mechanisms of TIL recognition of human melanoma. Some cultured TILs specifically lysed autologous and some allogeneic melanomas sharing a variety of class I major histocompatibility complex (MHC) molecules. HLA-A2-restricted melanoma-specific TILs lysed many HLA-A2⁺ melanoma cell lines from different patients but failed to lyse HLA- A2^- melanoma and HLA-A2⁺ nonmelanoma cell lines. However, these TILs were capable of lysing many naturally HLA-A2^- melanomas after introduction of the HLA-A2.1 gene by vaccinia virus. These results indicate that shared melanoma antigens (Ag) are expressed in melanomas regardless of their human leukocyte antigen types. In order to identify these shared melanoma Ags, we have tested some known proteins expressed in melanoma. Expression of tyrosinase or HMB45 Ag correlated with lysis of TILs. We are also attempting to isolate antigenic peptides by high performance liquid chromatography separation and genes encoding melanoma Ag by cDNA expression cloning. The T-cell component of the antimelanoma response was also analyzed by determining the genetic structure of the T-cell receptor (TCR) used by melanoma TILs. However, we did not observe common TCR variable region usage by different melanoma TILs. We could establish melanoma cell clones and lines resistant to TIL lysis due to the absence of or defects in the expression of Ag, MHC, or β₂-microglobulin molecules. These data indicate multiple mechanisms for melanoma escape from T-cell immunosurveillance. These findings have important implications for the development of immunotherapies for melanoma.</p>

Expansion of tumor-infiltrating lymphocytes from human tumors using the T- cell growth factors interleukin-2 and interleukin-4

Kawakami Y., Haas G. P., Lotze M. T.

Journal of Immunotherapy 14 ( 4 ) 336 - 347 1993

ISSN  1053-8550

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<p>We examined the role of two T cell-growth factors, interleukin (IL)-2 and IL-4, in expansion of tumor-infiltrating lymphocytes (TILs) from human tumors. In sarcoma, IL-4 (1,000 U/ml) with IL-2 (10 or 1,000 U/ml) grew TILs better than did IL-2 alone in six of 10 cases during 6 weeks of culture. IL- 4 decreased the relative number of CD56⁺ cells, which correlated with a decrease in cytolysis against Daudi in six of 10 cases. The addition of IL-4 with 1,000 U of IL-2 maintained or increased cytolysis against autologous sarcoma, while decreasing nonspecific cytolysis against Daudi or allogeneic sarcoma in three of eight cases. IL-4 decreased cytolysis against both autologous sarcoma and Daudi in four of 10 cases, suggesting nonspecific activity in these instances. In renal cell cancer (RCC), IL-4 with IL-2 (10 or 1,000 U/ml) augmented TIL growth in six of eight cases, especially during the first 2-3 weeks of culture. IL-4 with 10 U of IL-2 increased cytolysis against both autologous RCC and Daudi in six of eight cases, suggesting possible prior cell activation. In contrast, IL-4 addition with 1,000 U of IL-2 maintained or increased cytolysis against autologous RCC, while decreasing cytolysis against Daudi or allogeneic RCC in four of eight cases. In cases of bladder and of prostate cancer, IL-4 with 1,000 U of IL-2 grew TILs slightly better in five of seven cases for the first 2-3 weeks. Bladder TILs grown with IL-2 and/or IL-4 were CD⁺ T cell predominant (three of five) and rarely lytic for autologous tumor. In colon cancer and hepatoma, TILs grown with IL-2 and/or IL-4 were nonlytic for the autologous tumor. IL-4 in conjunction with IL-2 could therefore augment growth of some TILs especially for the first 2-3 weeks from various human tumors.</p>

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Molecular mechanisms used by tumors to escape immune recognition

Restifo N. P., Kawakami Y., Marincola F., Shamamian P., Taggarse A., Esquivel F., Rosenberg S. A.

Journal of Immunotherapy 14 ( 3 ) 182 - 190 1993

ISSN  1053-8550

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<p>In this article, we explore the hypothesis that tumor cells can escape recognition by CD8⁺ T cells via deficiencies in antigen processing and presentation. Aspects of the molecular and cellular biology of major histocompatibility complex class I are reviewed. Evidence for histology- specific molecular mechanisms in the antigen-processing and -presentation deficiencies observed in some human and murine tumors is presented. Mechanisms identified include down-regulation of antigen processing, loss of functional β₂-microglobulin, and deletion of specific α-chain alleles. Finally, we discuss studies using an antigen-presentation-deficient mouse tumor as a model for the immunogenetherapy of an antigen-presentation deficiency.</p>

Shared human melanoma antigens

Kawakami Yutaka, Zakut Rina, Topalian Suzanne L., Stötter Hans, Rosenberg Steven A.

Journal of Immunology 148 ( 2 ) 638 - 643 1992.01

ISSN  0022-1767

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<p>Three predominantly CD8⁺ CTL lines, TIL 501, TIL 620, and TIL 660, were generated from three HLA-A2⁺ melanoma patients by culturing tumor-infiltrating lymphocytes in 1000 U/ml IL-2. These tumor-infiltrating lymphocytes lysed 12 of 18 HLA-A2⁺ autologous and allogeneic melanomas, but none of 20 HLA-A2-negative melanomas. They also did not lyse the MHC class I negative lymphoma-leukemia cell lines, Daudi, K562, or HLA-A2⁺ non-melanoma cell lines including PHA or Con A-induced lymphoblast, fibroblast, EBV-transformed B cell, Burkitt's B cell lymphoma, and colon cancer cell lines. Autologous and allogeneic melanoma lysis was inhibited by anti-CD3, by anti-MHC class I, and by anti-HLA-A2 mAb, indicating recognition of shared tumor Ag among melanoma cell lines in a TCR-dependent, HLA-A2-restricted manner. Six HLA-A2-negative melanoma cell lines obtained from five HLA-A2-negative patients were co-transfected with the HLA-A2.1 gene and pSV2neo. All 17 cloned transfectants expressing cell surface HLA-A2 molecules, but none of 12 transfectants lacking HLA-A2 expression, were lysed by these three HLA-A2-restricted, melanoma-specific CTL. Lysis of the HLA-A2⁺ transfectants was inhibited by anti-CD3, by anti-MHC class I, and by anti-HLA-A2 mAb, indicating recognition of shared tumor Ag on transfectants in a TCR-dependent, HLA-A2-restricted manner. These results identify the HLA-A2.1 molecule as an Ag-presenting molecule for melanoma Ag. They also suggest that common melanoma Ag are expressed among melanoma patients regardless of HLA type. These findings have implications for the development of melanoma vaccines that would induce antitumor T cell responses.</p>

Recognition of shared melanoma antigens by human tumor-infiltrating lymphocytes

Topalian S. L., Hom S. S., Kawakami Y., Mancini M., Schwartzentruber D. J., Zakut R., Rosenberg S. A.

Journal of Immunotherapy 12 ( 3 ) 203 - 206 1992

ISSN  1053-8550

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<p>We have established that melanomas express shared tumor antigens (Ags) that can be recognized by T cells if presented in the context of self-MHC molecules. Tumor-infiltrating lymphocytes (TILs) from six melanoma patients were tested for lysis of large panels of HLA-matched or unmatched targets representing a variety of tissue types. Lysis was specific for allogeneic melanomas sharing at least one HLA-A, -B, or -C Ag with TILs, and demonstrated commonly expressed tumor Ags. Similar findings were obtained when cytokine secretion by TILs was used to indicate specific Ag recognition. Transfection of the HLA-A2.1 gene into HLA-A2 melanoma lines conferred susceptibility to lysis by HLA-A2 restricted melanoma TILs, demonstrating expression of common tumor Ags among patients of diverse HLA types. These findings have important implications for developing broadly applicable cancer immunotherapies such as vaccines.</p>

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Marijuana components suppress induction and cytolytic function of murine cytotoxic T cells in vitro and in vivo

Klein T. W., Kawakami Y., Newton C., Friedman H.

Journal of Toxicology and Environmental Health 32 ( 4 ) 465 - 477 1991

ISSN  0098-4108

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<p>Killer lymphocytes play a major role in host defense against tumors and infectious diseases. Previously, we reported that delta-9-tetrahydrocannabinol (THC) and Il-hydroxy-delta-9-tetrahydrocannabinol (Il-hydroxy-THC) suppressed the cytolytic activity of cultured natural killer (NK) cells. Also, we showed that the drugs appeared to be affecting a stage in the killing process subsequent to the binding of the killer cell to the target cell. In the present report, we have extended these studies to an examination of the effect of cannabinoids on the activity of cytotoxic T lymphocytes (CTLs). The cytolytic activity of CTLs generated by cocultivation with either allospecific stimulators or TNP-modified-self stimulators were suppressed by both THC and Il-hydroxy-THC treatment. Allospecific CTLs generated in vivo were also inhibited by an in vitro exposure to either THC or Il-hydroxy-THC, and the sensitivity of these cells to drug effects appeared to be greater than the sensitivity of the in vitro generated CTLs. Suppression of cytolytic function by THC and Il-hydroxy-THC ws maximal after a 4-h drug treatment, suggesting that the drug effects were inducible and therefore required a finite period of time to develop maximally. As seen in previous studies involving NK cells, drug treatment of mature CTLs appears to have little effect on the binding capacity of these cells for the target. However, the maximal killing capacity of the cells and the frequency of CTLs were significantly reduced by drug treatment. In addition to suppressing the cytolytic activity of mature effector CTLs, we also show that drug treatment inhibits both the proliferation of lymphocytes responding to an allogeneic stimulus and the maturation of these lymphocytes to mature CTLs. Similarly, CTL activity developing in vivo could be inhibited by THC injection. These results suggest that CTLs are inhibited by cannabinoids by at least two mechanisms. First, the cytolytic activity of mature killers is suppressed at some point beyond the binding to the target cell. Second, the cannabinoids appear to suppress the normal development of these mature effector cells from less mature precursor cells.</p>

Interleukin-3 induces proliferation but not lymphokine activated killer activity from human and murine mononuclear cells.

Jablons D. M., Donohue R., Kawakami Y., Young J. D., Lotze M. T.

European Cytokine Network 1 ( 2 ) 91 - 98 1990.05

ISSN  1148-5493

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<p>Recombinant IL-3 (rIL-3) is a potent colony stimulating factor capable of stimulating early hematopoietic pluripotential progenitor cells and of supporting the differentiation of multiple cells. IL-3 has also been shown to have effects on mature, differentiated circulating cells including eosinophils and T cells. We evaluated the role of exogenous rIL-3 in the generation of cells with LAK activity from murine splenocytes and human bone marrow, spleen, unseparated PBMC and purified null cell preparations. rIL-3 was unable to generate lytic activity from any of these populations by itself and appeared to decrease LAK activity in bone marrow cultures containing high dose IL-2, (bone marrow derived cells (n = 3) with LAK activity for fresh tumor, mean lytic units(LU) 94.6 +/- 63.5 vs 32.8 +/- 44.8 for IL-2 and IL-2 plus IL-3 cultures, respectively p2 less than 0.05). Unlike previous reports testing murine cells, IL-3 priming and subsequent culture in IL-2 of human unseparated bone marrow cells or human or murine splenocytes, failed to generate long-term cultures with lytic activity. IL-3 did, however, induce a dose dependent stimulation of bone marrow and null cell preparations (mean null cell stimulation (3H Thymidine incorporation) with IL-3, 436 +/- 168 cpm vs 9802 +/- 9799 cpm, for 0 vs 10(3) units of IL-3, respectively n = 4, p2 less than 0.05). Furthermore, in bone marrow, unseparated PBMC and null cell cultures, the addition of rIL-3 generated characteristic large blastic appearing cells with prominent basophilic granules.(ABSTRACT TRUNCATED AT 250 WORDS)</p>

Mechanisms of immunologic antitumor therapy

Lotze Michael T., Custer Mary C., Bolton Ellen S., Wiebke Eric A., Kawakami Yutaka, Rosenberg Steven A.

Human Immunology 28 ( 2 ) 198 - 207 1990

ISSN  0198-8859

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<p>The use of interleukin 2-based immunotherapies for cancer has been associated with significant resonses in tumor models in both mouse and humans. Further definition of the elements responsible for response is now possible. It appears that the respnse is associated with T-cell infiltration of the tumor, and transfer of tumor-infiltrating lymphocytes expanded in tissue culture with interleukin 2 is associated with significant antitumor effects. Further expansion of cultured human melanoma tumor-infiltrating lymphocytes with suppression of lymphokine-activated killer activity as well as the modulation of monocyte activity by interleukin 4 suggests that this cytokine may be clinically useful alone or in combination with interleukin 2. Other means of enhancing the activity of interleukin 2-based immunotheraphy are suggested by the finding that tumor cell susceptibility to lysis by natural killer cells is depressed following treatment with interferon γ and tumor necrosis factor, but susceptibility to lysis by tumor-infiltrating lymphocytes is markedly enhanced. Further development of these therapies will require innovative interpretation and application of findings related to the processing and presentation of human tumor antigens and the nature of tumor antigens and careful analysis of they T-cell receptor in antitumor effectors.</p>

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Prevention and reversal of delta-9-tetrahydrocannabinol induced depression of natural killer cell activity by interleukin-2

Specter Steven, Rivenbark Melodie, Newton Catherine, Kawakami Yutaka, Lancz Gerald

International Journal of Immunopharmacology 11 ( 1 ) 63 - 69 1989

ISSN  0192-0561

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<p>The effects of delta-9-tetrahydrocannabinol (THC) on NK cell activity were studied. Previously, we reported that incubation of human peripheral blood mononuclear cells in THC resulted in an inhibition of natural killer (NK) cell activity. The present study examined the mechanism(s) of the decrease in NK cell activity. The inhibition of killing by NK cells was not due to a failure of NK cells to bind to K562 target cells. Furthermore, indomethacin did not abrogate the THC-mediated effect, suggesting that prostaglandins are not involved in the process leading to suppression of NK cell activity. However, NK activity was partially restored if cells, pretreated with THC, were washed to remove excess drug and then incubated overnight in fresh medium before assay. Addition of 1 - 100 U IL-2, either during pretreatment with THC or during overnight incubation, precluded or promoted the reversal of the inhibition of NK cell cytotoxicity. We conclude that the regulatory mechanism(s) involved in depression of NK cell cytotoxicity by THC is significantly influenced by IL-2.</p>

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IL-4 regulates IL-2 induction of lymphokine-activated killer activity from human lymphocytes

Kawakami Y., Custer M. C., Rosenberg S. A., Lotze M. T.

Journal of Immunology 142 ( 10 ) 3452 - 3461 1989

ISSN  0022-1767

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<p>IL-4 is a pluripotent lymphokine acting on various cell types. We investigated the role of human IL-4 on the generation of lymphokine-activated killer (LAK) activity. Human IL-4 alone did not induce LAK activity and inhibited IL-2 induction of LAK activity from unstimulated PBMC, peripheral blood null cells, spleen cells, and lymph node cells in a dose-dependent manner. IL-4 also inhibited several phenomena induced by IL-2 such as cell proliferation, augmentation of NK activity, increase of Leu-19⁺ cells, and expression of IL-2R(p55) on either CD3⁺ or Leu-19⁺ cells. IL-4, however, augmented cell proliferation with other T cell mitogens including PHA, Con A, PMA, or allo-MHC Ag with or without IL-2. In contrast to unstimulated cells, IL-4 alone induced marked cell proliferation and LAK activity as well as Leu-19⁺ cells from in vitro IL-2 preactivated PBMC or null cells, and did not inhibit IL-2 induced cell proliferation, LAK activity, Leu-19⁺ cells and IL-2R(p55) expression, but rather augmented them with low doses of IL-2. Although IL-4 alone induced LAK activity from peripheral blood of some patients previously given IL-2, IL-4 inhibited in vitro LAK generation with IL-2 from these cells in most cases. Therefore, IL-4 appears to directly inhibit the IL-2 activation pathway via IL-2R(p70) and prevent resting LAK precursors from proliferating and differentiating into final effector cells. However, once cells were sufficiently preactivated by IL-2, IL-4 induced LAK activity and did not inhibit IL-2 activation of these cells. These data suggest an immunoregulatory role of IL-4 on human null cells and T cells.</p>

Suppression by cannabinoids of the induction and function of murine cytotoxic T lymphocytes

Y. Kawakami Kawakami, Klein T. W., Newton C. A., Young J., Friedman H.

International Journal of Immunopharmacology 10 ( SUPPL. 1 ) 147 1988

ISSN  0192-0561

Suppression by cannabinoids of a cloned cell line with natural killer cell activity

Kawakami Y., Klein T. W., Newton C., Djeu J. Y., Dennert G., Specter S., Friedman H.

Proceedings of the Society for Experimental Biology and Medicine 187 ( 3 ) 355 - 359 1988

ISSN  0037-9727

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<p>Preincubation of a cloned cell line with natural killer (NK) cell activity, as well as splenic mononuclear cells with either Δ⁹-tetrahydrocannabinol (THC) or 11-hydroxy-Δ⁹-tetrahydrocannabinol (11-OH-THC) suppressed NK cytolytic activity against YAC-1 target cells in a dose-dependent manner. THC was more inhibitory for cloned cells than 11-OH-THC and suppressed the lytic activity of these cells without reducing cell viability in the concentration range of 5 μg/ml (16 μM) to 10 μg/ml (32 μM). THC also inhibited proliferation of cloned NK cells, but this inhibitory effect was reversible in that extensive washing of cells following cannabinoid pretreatment eliminated the suppressive effect. Single-cell analysis revealed that THC did not inhibit the binding of cloned NK cells to target cells and further showed that NK cells freshly isolated from mouse spleen were restricted in killing capacity following binding to target cells. Therefore, THC and 11-OH-THC appear to directly inhibit NK cell cytolytic activity in a postbinding stage.</p>

Suppression by delta-9-tetrahydrocannabinol of interleukin 2-induced lymphocyte proliferation and lymphokine-activated killer cell activity

Kawakami Yutaka, Klein Thomas W., Newton Catherin, Djeu Julie Y., Specter Steven, Friedman Herman

International Journal of Immunopharmacology 10 ( 4 ) 485 - 488 1988

ISSN  0192-0561

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<p>The major psychoactive marijuana component, delta-9-tetrahydrocannabinol (THC), suppressed proliferation of murine spleen cells stimulated with recombinant human interleukin 2 (IL-2) and also suppressed the appearance of the lymphokine-activated killer (LAK) cell phenomenon in IL-2-treated spleen cell preparations. Cell function was depressed in a dose-dependent manner with as little as 2.5 μg/ml THC (8μM). In addition, spleen cells previously stimulated in culture with IL-2 and then incubated with THC for 4 h prior to target cell addition, displayed suppressed cytolytic activity against both YAC-1 and EL-4 tumor targets. Killing of EL 4 cells was suppressd at lower drug doses than the killing of YAC-1 targets. These results suggest that THC can suppress several important functions of IL-2 including clonal expansion of lymphocytes, expansion of killer cell populations and stimulation of killer cell cytotoxic activity.</p>

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Interleukin 4 promotes the growth of tumor-infiltrating lymphocytes cytotoxic for human autologous melanoma

Kawakami Y., Rosenberg S. A., Lotze M. T.

Journal of Experimental Medicine 168 ( 6 ) 2183 - 2191 1988

ISSN  0022-1007

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<p>Addition of IL-4 (1,000 U/ml) to either high or low concentrations of IL-2 augmented tumor-infiltrating (TIL) growth from human melanoma. Weekly restimulation with irradiated tumor cells in conjunction with IL-4 allowed enhanced growth of TIL. With low-dose IL-2 (10 U/ml) and IL-4, expanded TIL had little cytolytic activity against Daudi or allogeneic tumors. Further, IL-4 augmented the total lytic activity against autologous tumors in most cases. With high-dose IL-2 (1,000 U/ml), IL-4 addition decreased nonspecific killing activity against Daudi or allogeneic melanomas in many cases, and reciprocally augmented cytolytic activity against the autologous melanoma in many cases. This suggests the possible use of IL-4 in cancer therapy, especially in adoptive cellular immunotherapy using TIL or in conjunction with IL-2 administration.</p>

Ultrastructural study of megakaryocyte maturation

Ogawa T., Miyake K., Kawakami Y., Kizaki M.

Journal of Clinical Electron Microscopy 17 ( 5-6 ) 938 - 939 1984

ISSN  0021-4981