NY-ESO-1和MAGE-A3抗原肽诱导肝癌患者的免疫应答研究

目的利用合成的HLA-A02限制性NY—ESO-1 p157—165或MAGE—A3p271—279抗原肽，体外通过抗原递呈细胞诱导HCC患者外周血T淋巴细胞，从而成功诱导出特异性CD8^＋T淋巴细胞免疫应答。方法通过SSP方法筛选20例HLA—A＊02的HCC患者的HLA亚型；通过逆转录多聚酶链反应和测序分析NY—ESO-1和MAGE—A3在肝癌组织中的表达以及多肽表位密集区的核苷酸变异状况；体外用细胞因子培养HCC患者外周血来源的树突状细胞（DC）；人工合成HLA—A＊02限制性NY—ESO-1p157—165或MAGE—A3 p271—279多肽，直接或用去除CD8^＋T淋巴细胞的PBMC或DC递呈，体外同T淋巴细胞孵育16d后，利用Elispot、细胞内细胞因子染色和四聚体实验检测T细胞免疫应答。结果20例HLA—A＊02患者中，NY-ESO-1mRNA阳性患者为11例，MAGE—A3mRNA阳性患者12例。中国HCC患者表达的NY—ESO-1和MAGE—A3序列高度保守。经多肽直接或用去除CD8^＋T淋巴细胞的PBMC或DC递呈，体外活化外周血T淋巴细胞，4例（36．4％）NY—ESO-1阳性HCC患者以及4例（33．3％）MAGE．A3患者产生针对多肽特异性的CD8’T细胞免疫应答。不同HLA—A＊02亚型HCC患者可产生针对NY—ESO-1p157—165或MAGE—A3p271—279抗原肽的免疫应答。结论NY—ESO-1p157-165或MAGE—A3p271—279抗原肽可以诱导出针对抗原肽的特异性CD8^＋T淋巴细胞免疫应答。提示HLA—A＊02限制性的NY-ESO-1 p157-165或MAGE—A3p271—279抗原肽可以作为有潜力的肝癌免疫治疗疫苗。

Immune responses to NY-ESO-1 and MAGE-A3 peptides in hepatocellular carcinoma patients

Objective To induce specific immune responses in vitro against the peptides of NY-ESO-1 p157-165 and MAGE-A3 p271-279 of hepatocellular carcinoma (HCC) patients. Methods HLA-A*02 subtypes were typed by SSP typing kit in 20 HCC patients. The expressions of NY-ESO-1 and MAGE-A3 were detected by RT-PCR and the products were sequenced for determining the mutation in genes. Dendritic cells (DCs) were obtained from peripheral bloods mononuclear cells (PBMCs) of HCC patients under cytokines coculture. The NY-ESO-1 p157-165 or MAGE-A3 p271-279 peptides presented by PBMCs with CD8~+T deleted or DCs were cultured with T lymphocytes for 16 days. The immune responses of CD8~+ T lymphocytes were evaluated by Elispot assay, Cytospot assay, and Tetramer staining. Results Eleven and twelve out of 20 HCC patients were NY-ESO-1 mRNA and MAGE-A3 mRNA positive, respectively. None of mutation was found in these genes. Four out of 11 NY-ESO-1 mRNA~+ patients (36.4%) and four out of 12 MAGE-A3 mRNA~+ patients (33.3%) were detected immune respon- ses of CD8~+ T lymphocytes. The immune responses were observed in patients with all existing HLA-A*02 subtypes. Conclusion The immune responses to NY-ESO-1 p157-165 and MAGE-A3 p271-279 epitopes can be induced in CD8~+ T lymphocytes of HCC patients in vitro, which suggest NY-ESO-1 and MAGE-A3 are potential vaccine candidates for immune therapies of HCC.