Details of a Researcher - Kawakami, Yutaka

Papers - Kawakami, Yutaka

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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

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.

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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

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.

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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

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.

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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

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.

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

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.

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

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.

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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

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.

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

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 (<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.

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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

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.

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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

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.

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