一个新的MAGE—1表位为肝癌细胞表面HLA—B7分子递呈

目的：肿瘤抗原的存在是机体识别肿瘤并激活免疫系统的物质基础。机体抗肿瘤免疫以细胞免疫为主，故寻找被T细胞识别的肝癌细胞抗原肽，为肝癌免疫治疗奠定基础。方法：对于肝癌细胞系HHCC（HLA-A2，A29，B7，B51），应用细胞膜酸洗技术使抗原肽从细胞膜表面脱落，经过凝胶层析，反相高效液相色谱层板得到不同组份多肽，高效液相色谱=质谱仪联用技术获得抗原肽的一级结构。通过氨基酸锚定位点分析，预测其HLA配体类型；互联网上氨基酸同源性分析确定其同源性用技术获得抗原肽的一级结构，通过氨基酸锚定位点分析，预测其HLA配体类型，互联网上氨基酸同源性分析确定其同源性序列；人工合成抗原肽，HLA同型树突状细胞递呈合成抗原肽刺激特异性CTL反应，^51Cr杀伤实验检测CTL对肿瘤细胞系的杀伤作用。结果：经过反相高效液相色谱层析后可以获得10多个组份，其中一个组份经过液质联用鉴定和氨基酸同源性分别确定其序列为EPVTKAEML，是黑色素瘤抗原-1，MAGE-1（121-129）表位。由HLA-B7分子递呈，体外诱导实验表明其可以诱导出较强的CTL反应。结论：液质联用技术是寻找抗原肽的有效方法，MAGE-1抗原肽EPVTKAEML地于HLA-B7阳性及阳性的肝癌患者具有潜在的免疫治疗作用。

A new MAGE-1 epitope naturally processed and presented by HLA-B7 on human hepatocellular carcinomas

Objective:The current approach to immunotherapy is mainly reliant on the role of T lymphocyte and tumor antigen recognized by T lymphocyte.To broaden the clinical applicability of peptide-based immunotherapy in human hepatocellular carcinomas,there is a need to isolate and identify tumor antigen peptides bound to HLA class I molecules on the human hepatocellular carcinomas.Methods:The prime structure characterization of HLA binding peptide by a human hepatocellular carcinoma cell line with a HLA-A2,A29,B7,B51 was performed.Extract of MHC-associated peptides by mild acid wash of viable hepatocellular carcinomas cells,and collected by gel filtration,then,fractionated by reversed phase high pressured liquid chromatography (RP-HPLC).HPLC-fractionated samples were sequenced and identified by HPLC-MS-MS (tandom mass spectrometry).Protein database in the internet were used as additional tools for structure analysis and for determination of the protein source of the eluted peptides,potential peptide binding HLA alleles had been identified using a computer program by comparing anchor amino acid residues.Allologous dendritic cells (DCs) having the same HLA alleles pulsed the synthetic peptide,PBMCs were stimulated by peptide-pulsed autologous DCs.The specific cytotoxicity against the hepatocellular carcinoma cell line were determined by 51 Cr release assay.Results:RP-HPLC showed that there were teens different fractions of peptides derived from HHCC human hepatocellular carcinoma cell line,HPLC-ESI-MS-MS showed the amino acid sequence of the fractions.One of the most promising candidates for T cell epitope is nonamers peptide EPVTKAMEL,derived from MAGE1 121-129 ,had the HLA-B7 peptide binding motify.PBMC cocultured with the peptide-pulsed DCs could induce the relevant peptide specific CTLs,moreover,they showed specific cytotoxicity against HHCC cell line.Conclusion:Sensitive sequencing by HPLC-MS may provide a powerful method of identifying tumor specific antigenic peptides,nonamers peptide EPVTKAEML,can be used for hepatocellular carcinoma vaccine strategies with confidence that it is identical to the naturally processed peptide epitopes presented at the surface of hepatoma cells in association with HLA-B7 molecules.