Research of T cell immunity to Epstein-Barr virus latent membrane protein 1.

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1.Epstein-Barr Virus(EBV) latent membrane protein(LMP)1,LMP2 and EBNA1 antigens are expressed in EBV-positive malignancies and candidate molecules recognized by cytotoxic T lymphocytes(CTL). To efficiently induce CTL responses to them, we applied antigen presenting cells transduced with in vitro transcribed mRNA coding each molecules to stimulate CD8+ T cells obtained from healthy donors. An HLA-A^*0206-restricted LMP1-specific CTL clone was established. The T cell clone exhibited MHC-restricted cytotoxicity to cell lines derived from EBV-positive NK lymphoma. I also established HLA-Cw^*0303-restricted EBNA1-specific CTL clone which inhibited growth of HLA-Cw^*0303-positive EBV-infected B-lymphoblastoid cell lines(LCLs). Amino acid sequences of the CTL epitopes were identified using truncated mutants of the target genes and synthetic peptides.2.An HLA-A^*2402-restricted LMP2-derived epitope whose amino acid sequence, IYVLVMLVL, had been identified by the head investigator (Blood.,2003). The epitope was presented on LCLs but not on fibroblast cells. Since IFN-γ induced the epitope presentation, I investigated the roles of subunits of so called immunoproteasome. For the sake, retrovirus vectors expressing short hairpin RNA to inhibit expression of β1i,β5i and PA28α were produced and infected to HLA^*A2402-positive LCLs. The reduced protein expression was confirmed by Western blotting. Presentation of the epitope determined by ELISPOT assay using the epitope-spepcific CTL clone, was decreased on LCLs, where either β1i,β5i or PA28α was knocked out.