HLA-A*02/24限制的HBcAg表位特异性细胞毒T细胞克隆的建立及分析

Generation and Analysis of HLA-A*02/24 Restricted HBcAg-specific Cytotoxic T Lymphocyte Clones in a Patient with Chronic Hepatitis B.

目的建立 HLA—A*0201/2403限制的 HBcAg 表位特异性 CTL 细胞克隆。方法 HLA—A*0201/2403阳性HBV 感染者的外周血单个核细胞(PBMC)分别用限制性表位肽 HBc18～27和 HBc117～125刺激,并以有限稀释法进行克隆化,期间单独使用植物血凝素(PHA)及联合使用表位肽刺激。用免疫荧光、流式细胞术以及乳酸脱氢酶释放实验对所获克隆进行鉴定。结果从HBc18～27活化的细胞中获得29株细胞克隆,其中28株为 CD8^+T 克隆,均具有特异性细胞毒活性。从 HBc117～125激活的细胞中获得12株 CD8^+T 克隆,其中9株有明显的细胞毒活性,另3株细胞毒活性则显著低下。克隆化过程中单独使用 PHA 及联合使用表位肽,克隆形成率分别为15.62%和14.58%。结论分别用表位肽 HBc18～27和HBc117～125,能激活HLA—A*0201/2403阳性 HBV 感染者的 CD8^+T 细胞。建立 CD8^+T 克隆过程中,表位肽的使用不能提高克隆形成率。

Objective To generate HLA - A ＊ 0201 and A ＊ 2403 restricted HBcAg - specific cytotoxic T lymphocyte （CTL） clones. Methods Peripheral blood mononuclear cells （PBMCs） were derived from a HLA - A ＊ 0201/2403 - positive patient with chronic hepatitis B . PBMCs were stimulated respectively using two synthetic peptides（ HBc18-27 and HBc 117 - 125） , and epitope -specific CTL clones were generated by limiting dilution technique with PHA alone or combined with synthetic peptides. The cell clones were then characterized by IF staining,FCM and LDH release. Results After 2 weeks of in vitro stimulating PBMCs, specific CTL lines were estab- lished. 29 T clones were generated from those CTL lines using HBc18-27 stimulating. Among the 29 clones,28 clones belonged to CD8^＋ T cells and all displayed eytolytic activity. 12 CD8^＋T clones were generated from those CTL lines using HBc117-125 stimulating. Specific cytotoxie activity was observed in 9 of those clones. The other three displayed less eytolytie activity. In the process of cloning, PHA was used alone or combined with synthetic peptides,and the achievement showed a rate of 15.62% and 14.58%. Conclusion HBc18-27 and HBc117-125 are capable of activating CD8^＋ T cells in PBMCs of HLA - A ＊ 0201 /2403 patient with chronic hepatitis B. In the process of CD8^＋ T cloning, synthetic peptides could not increase the rate of successful cloning.