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

1. EB病毒（EBV）潜伏膜蛋白（LMP）1、LMP 2和EBNA 1抗原在EBV阳性恶性肿瘤中表达，并且是细胞毒性T淋巴细胞（CTL）识别的候选分子。为了有效地诱导对它们的CTL应答，我们应用用体外转录的编码每种分子的mRNA转导的抗原呈递细胞来刺激从健康供体获得的CD 8 + T细胞。建立HLA-A^*0206限制性LMP 1特异性CTL克隆。T细胞克隆表现出MHC限制的细胞毒性来源于EBV阳性NK淋巴瘤的细胞系。我还建立了HLA-Cw^*0303限制性EBNA 1特异性CTL克隆，其抑制HLA-Cw^*0303阳性EBV感染的B淋巴母细胞样细胞系（LCL）的生长。使用靶基因的截短突变体和合成肽鉴定CTL表位的氨基酸序列。2. HLA-A *2402限制性LMP 2衍生的表位，其氨基酸序列IYVLVMLVL已由首席研究员鉴定（Blood.，2003年）。表位在LCL上呈递，但不在成纤维细胞上呈递。由于IFN-γ诱导抗原表位呈递，我研究了所谓的免疫蛋白酶体亚基的作用。为此，制备了表达短发夹RNA以抑制β 1 i、β 5 i和PA 28 α表达的逆转录病毒载体，并感染HLA^*A2402阳性LCL。通过Western印迹证实蛋白表达降低。通过ELISPOT测定使用表位特异性CTL克隆确定的表位呈递在LCL上减少，其中β 1 i、β 5 i或PA 28 α被敲除。

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.