肝素酶CTL表位负载的树突状细胞疫苗体外抗肿瘤免疫效应研究

目的研究肝素酶(Hpa)多肽疫苗体外能否诱发肝素酶特异性CTL反应,为肝素酶多肽疫苗的临床应用提供理论依据。方法5条HLA-A2限制性肝素酶抗原表位Hpa(525-533)(PAFSYSFFV)、Hpa(353-361)(PLLSDTFAA)、Hpa(277-285)(KMLKSFLKA)、Hpa(400-408)(PLPDYWLSL)、Hpa(405-413)(WLSLLFKKL)]分别负载正常人外周血来源(PBMC)的树突状细胞(DC),诱导肝素酶特异性细胞毒T淋巴细胞(CTL),采用4 h标准51Cr释放实验检测上述CTL对不同肿瘤细胞的免疫杀伤效应,采用ELISPOT方法检测肝素酶表位肽负载的DC疫苗刺激的效应细胞IFN-γ的释放。结果5条肝素酶多肽表位中,只有Hpa(525-533)、Hpa(277-285)和Hpa(405-413)体外可以诱导肝素酶表位特异性CTL反应,对Hpa阳性且HLA-A2阳性的KATO-Ⅲ胃癌细胞、U2OS骨肉瘤细胞、SW480结肠癌细胞具有明显的杀伤效应,对HLA-A2阴性的HepG2肝癌细胞和Hpa阴性的MCF-7乳腺癌细胞不具有杀伤效应,但对转染了HLA-A2的HepG2细胞和转染了肝素酶质粒的MCF-7细胞恢复杀伤效应,对自体淋巴细胞不具有杀伤效应,进一步研究表明,Hpa(525-533)、Hpa(277-285)和Hpa(405-413)表位肽可以促进CTL细胞IFN-γ的释放。结论人肝素酶特异性抗原表位Hpa(525-533)、Hpa(277-285)、Hpa(405-413)不仅能激发机体特异性的抗肿瘤效应,而且还能诱导一个非特异性抗肿瘤的良性循环,这种肝素酶多肽疫苗具有广谱、高效、特异、安全的优点,为肝素酶表位多肽疫苗的临床应用提供了理论依据。

Heparanase epitope peptide-pulsed DCs elicit immune responses against various tumors in vitro

Objective The present paper is designed to study whether the heparanase eptitopes elicit heparanase-specific CTL against various tumors in vitro and provide theoretical evidence for their clinical application.Methods Dendritic cells(DCs) were pulsed with five HLA-A2-restricted epitopes of heparanase respectively to induce heparanase-specific cytotoxic T lymphocytes(CTLs).Specific lysis to various tumor cell lines was examined by a 4 h standard 51 Cr release assay.The IFN-γ releasing of effector cells was tested by ELISPOT.Results Of the five epitopes of heparanase,only Hpa(525-533),Hpa(277-285) and Hpa(405-413) could elicit potent specific lysis to KATO-Ⅲ,U2OS and SW480 cells which were heparanase and HLA-A2 positive.Effector induced by the above epitopes could not only lyse MCF-7 cells,which was heparanase negative,But also lyse as HepG2 cells,which was HLA-A2 negaive.But the specific lysis reoccurred after MCF-7 cells transduced with a plasmid contained full-length cDNA of heparanase or HepG2 cells transduced with a plasmid contained full-length cDNA of HLA-A2.Futher study demonstrated these heparanase-specific effectors induced by the above epitopes could not lyse autologous lymphocytes.In addition,we also found that Hpa(525-533),Hpa(277-285) and Hpa(405-413) heparanase epitopes could increase IFN-γ releasing of effectors.Conclusions The above studies show for the first time that the heparanase epitopes Hpa(525-533),Hpa(277-285) and Hpa(405-413) are capable of eliciting a specific anti-tumor immune response by heparanase-specific CTLs,as well as a non-specific anti-tumor immune response by enhancing cytokines releasing.These findings show that heparanase epitope vaccines have various advantages such as broad-spectrum,high-effect,high specificity and safety,which will provide theoretical evidence for their clinical application.