| Project/Area Number |
12670802
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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 |
Pediatrics
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| Research Institution | Aichi Cancer Center |
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Principal Investigator |
KUZUSHIMA Kiyotaka Aichi Cancer Center, Div. of Virology, Section head, 腫瘍ウイルス学部, 室長 (30311442)
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| Co-Investigator(Kenkyū-buntansha) |
TSURUMI Tatsuya Aichi Cancer Center, Div. of Virology, Chief, 腫瘍ウイルス学部, 部長 (90172072)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Epstein-Barr virus / CTL / HLA-A24 / MHC-peptide tetramer / lymphoma / cytotoxic T lymphocyte / tetrameric MHN-peptide complex / HLA-A^*2402 / LMP2 / トランスレーショナルリサーチ / LMP1 / TAT |
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| Research Abstract |
EBV is associated with several malignant diseases, including a subset of lymphomas, nasopharyngeal carcinomas, Burkitt's lymphomas and immunoblastic lymphomas seen in immunocompromised hosts. We established an efficient approach for determination of CTL epitopes in the context of HLA A^*2402 molecules through multiple screenings, consisting of a computer-assisted algorithm, an in vitro MHC stabilization assay and an enzyme-linked immuno-spot (ELISPOT) assay. HLA A24 is the most frequently encountered HLA class I allele and the genotype is almost exclusively A^*2402. We first searched for candidate,peptides with the HLA A24-binding motif by a computer-assisted algorithm among EBV proteins reported to be targeted by specific CD8^+ T cells. We selected and synthesized 42 peptides after analyzing amino acid sequences of the 5 lytic and 6 latent cycle proteins. All of them functionally stabilized HLA A^*2402 molecules which had been expressed on the peptide transporter-deficient cell line T2. Next is screening of the peptides by ELISPOT assay for their recognition by bulk EBV-specific CD8^+ T cells, established from PBMCs of EBV-immune donors positive for HLA A24.-typing. We have identified 2 possible major lytic cycle EBV-specific CTL epitopes from the amino acid sequences of BRLF1 and BMLF1 and 1 from LMP2 presented by HLA A^*2402 molecules. The newly identified peptides should prove useful for detection and/or study of EBV-specific CD8^+ T cell responses among populations positive for HLA A^*2402. We produced HLA A^*2402tetramers-incorporating the 3 peptides and 2 known epitope peptide, one derived from EBNA3A (RYSIFFDYM), and the other derived from EBNA3B (TYSAGIVQI). All the 5 tetramers successfully stained various proportions of EBV-specific CD8+ T cells. The newly identified peptides should prove useful for detection and/or study of EBV-specific CD8^+ T cell responses among populations positive for HLAA^*2402.
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