研究者詳細 - 河上　裕

論文 - 河上　裕

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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年09月

ISSN 0008-5472

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.

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年06月

ISSN 0270-9139

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 < .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.

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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年06月

ISSN 0925-5710

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.

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年05月

ISSN 0008-5472

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>T (K2729N)and 8732C>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>A nonsense mutation was changed into a missense mutation by a secondary alteration, 8731T>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.

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年05月

ISSN 0146-0404

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.

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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年04月

ISSN 0165-5728

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.

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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年03月

ISSN 1042-8194

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.

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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年03月

研究論文（学術雑誌）, 共著

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年01月

ISSN 0006-4971

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.

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Cellular immunotherapy of cancer

Kawakami Yutaka

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

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年12月

ISSN 0022-1759

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.

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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年12月

研究論文（学術雑誌）, 共著

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年10月

ISSN 0004-3591

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.

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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年09月

ISSN 0003-4967

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.

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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年09月

研究論文（学術雑誌）, 共著

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年09月

ISSN 0914-2223

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.

Correspondence re

Fodor János, Kawakami Yutaka, Kiniwa Yukiko

Cancer Research 62 （ 16 ） 4836 - 4837 2002年08月

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年08月

ISSN 0004-3591

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.

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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年08月

研究論文（学術雑誌）, 共著

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年07月

研究論文（学術雑誌）, 共著

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