Development of viral antigen specific immunotherapy for Epstein-Barr virus related gastric cancer.

(1)潜伏膜蛋白2(LMP2)表位表位负载树突状细胞(DCs)诱导细胞毒性T细胞(CTL)：合成LMP2多肽TYGPVFMCL(HLA-A24限制性)，以负载LMP2表位的DC体外刺激健康供者PBMC，建立CTL细胞系。(2)自体EB病毒转化B细胞株(LCL)的CTL诱导，将B95-8细胞的浓缩上清液加入健康供者的PBMC中，建立LCL。用自体LCL体外刺激健康供者的PBMC。这种LCL诱导的CTL识别EBV潜伏期抗原的全部谱系。(3)聚合酶链式反应(PCR)检测EBV-DNA。为了检测我所胃切除患者中EBV相关性胃癌，我们进行了聚合酶链式反应和Southern杂交。在此基础上，我们尝试建立了LMP2阳性的胃癌细胞系，为以CTL为基础的免疫治疗提供了有用的靶点。

(1) Cytotoxic T cell(CTL) induction with latent membrane protein 2(LMP2) epitope peptide -loaded dendritic cells(DCs).We synthesized the LMP2 peptide, TYGPVFMCL(HLA-A24 restricted), and established CTL line by stimulating PBMCs from HLA-A24 positive healthy donors in vitro with LMP2 peptide-loaded DCs. This CTL could lyse the target cells pulsed with this peptide, but couldn't lyse LMP2-CDNA transformed target cells.(2) CTL induction with autologous EBV transformed B cell line(LCL).In order to establish LCL, concentrated supernatant from B95-8 cultures, a human type1 EBV-transformed marmoset B-cell line, was added to a healthy donor's PBMCs. PBMCs from healthy donors were stimulated in vitro with autologous LCL. This LCL-induced CTL recognized the full repertoire of EBV latency antigens. We selected LMP2-specific CTL line after 5lCr release assay.(3) Detection of EBV-DNA by polymers chain reaction (PCR).In order to detect EBV related gastric cancers from patients underwent gastorectomy in our institute, we performed PCR and southern hybridization. After that, we tried to establish LMP2 positive cell lines of gastric cancer which could be useful targets for CTL-based immunotherapy.