| Project/Area Number |
13670901
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Dermatology
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| Research Institution | Keio University |
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Principal Investigator |
SAKURAI Toshiharu Keio University, School of Medicine Instructor, 医学部, 助手 (20101933)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Yuriko Keio University, School of Medicine Instructor, 医学部, 助手 (40255435)
FUJITA Tomonobu Keio University, School of Medicine Instructor, 医学部, 助手 (20199334)
KAWAKAMI Yutaka Keio University, School of Medicine Professor, 医学部, 教授 (50161287)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2001: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Keywords | HLA tetramer / melanoma-antigen peptide / HLA-A2.1V / HLA-A2.6 / TIL / CMV-antigen peptide / HLA-A^*0201 / HLA-A^*2402 / CTL / HLA-A2.1 / HLA-A2.6 |
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| Research Abstract |
The purpose of this project is the studies on the cloning and immune-response monitoring of the melanoma antigen-specific cytotoxic T lymphocytes using soluble peptide-HLA tetramers produced.
1. The tumor-infiltrating T-lymphocyte (TIL) 620 line (recognized gp-100-209) was double-stained with anti-CD8 mAb-PC5 and HLA-A2.1/gp100-209 PE-tetramer. The tetramer-binding CD8+ T cells (14 %) were sorting from TIL 620 line by fluorescence-activated cell sorting (FACS) and expanded in vitro. The cultured tetramer-binding CD8+ T cells were stained 98.4 % with HLA-A2.1/gp 100-209 PE-tetramer and released IFN-γ more than 7 fold of that from original TIL 620 line. It become is able to cloning the antigen-specific active T lymphocytes using HLA tetramers.
2. HLA-A2.6 and A2.7 heavy chains were constructed from HLA-A2.1 by mutagenesis kit, synthesized and purified using a prokaryotic expression system. HLA-A2.6/gp100-280 tetramer was produced, and stained 4-5 % of TIL 660 (recognized A2.l/gp-100-280 epitope) and 7.97 % of the induced T cell line from PBMC of HLA-A2.6+ healthy donor by gp100-280 peptide. These results suggested that the HLA-A2.1 restricted melanoma-antigen peptide might be cross-recognized by other A2-supertype molecules.
3. As the model of monitoring tumor-specific T lymphocytes using HLA tetramers, fluorescent HLA-CMV peptide tetramer were used to monitor the recovery of CMV-specific T lymphocytes in recipients of allogeneic stem cell transplants, (1) CMV pp65-495 specific T cells were induced, using CMV pp65-495 peptide, in 7 of 8 PMBC from A2.1+ healthy donor, and stained with HLA-2.1/ CMV pp65-495 PE-tetramer. Then CMV pp65-495 is immunodominant peptide. (2) The use of HLA-peptide tetramer to quantify CMV specific T cells is valuable for monitoring and studying T-cell responses after allogeneic stem cell transplantation.
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