AIM:Peptide vaccines have been conceived as promising therapies for tumor-inflicted patients due to their easy production and capability of inducing specific immune response required for defending the tumor.During our previous research,4 HLA-A2-restricted peptides had been identified as immunogenic in vivo.In this study, we aimed to testify the in vivo immunogenicity of the 4 peptides.METHODS: BALB/c mice were vaccinated with HLA-A2 restricted peptides emulsified in incomplete Freund's adjuvant(IFA)subcutaneously in combination with the epitope at the adjacent location.After the 3rd peptide vaccination for 10 d,the peptide-specific immune response was evaluated by ELISPOT and ELISA.The ability to induce T cell response was investigated by using cytotoxicity assay in vivo and the presence of pep-tide-specific CD8+T cells capable of recognizing the MHC-peptide was detected by flow cytometry.RESULTS: Among the 4 candidate HLA-A2 restricted peptides,the immune response elicited by P2004-1Y9V was superior to that of the other 3 peptides.The CTLs induced by P2004 and P2004-1Y9V lysed CAPAN-2 cells.P2004-1Y9V peptide-specific CTLs showed higher cytotoxicity against pancreatic tumor cell lines of CAPAN-2 than the native peptide-specific CTLs.Intracellu-lar cytokine staining assay indicated the presence of P 2004-1Y9V specific CD8 +T cells in the P2004-1Y9V vaccinated mice.CONCLUSION:P2004-1Y9V is the most immunogenic peptide in vivo, and can be explored as potential tumor peptide vaccine in the future clinical study.%目的:检测来源于黏蛋白4(MUC4)的人白细胞抗原(HLA)-A2限制性表位肽免疫BALB/c小鼠后产生细胞免疫应答的水平,筛选出体内具有免疫原性的HLA-A2限制性表位肽.方法:将来源于MUC4的候选HLA-A2限制性表位肽、通用性Th表位和弗氏不完全佐剂联合应用皮下免疫BALB/c小鼠,用酶联免疫斑点实验(ELISPOT)和酶联免疫吸附实验(ELISA)检测小鼠脾细胞表位肽特异性细胞免疫应答的水平,体内细胞毒实验活性检测候选肽诱导细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL)的能力,并用流式细胞术检测表位肽特异性的CD8 +T细胞所占比例.结果:在检测的4个表位肽中,P2004-1Y9V诱导BALB/c小鼠产生的细胞免疫应答最强,细胞毒实验结果显示,P2004和P2004-1Y9V对CAPAN-2细胞均有一定的杀伤作用,且P2004-1Y9V特异性CTLs对CAPAN-2细胞杀伤率高于原肽(P<0.05).胞内因子染色实验进一步表明P2004-1Y9V免疫后可产生特异性的CD8 +T细胞.结论:在来源于MUC4的HLA-A2限制性表位肽中,P2004-1Y9V具有较强的体内免疫原性,可以作为潜在的肿瘤多肽疫苗应用于临床研究.
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