EB病毒LMP2A特异性CTL的体外诱导及分析

用EBV LMP2A重组痘苗病毒 (rVV LMP2A )转染人树突状细胞 (DC ) ,转染后的DC分别在第 1、 7、 14天刺激相同MHC背景的T细胞 ,在IL 2作用下诱导LMP2A特异性CTL。用LDH释放法检测CTL杀伤活性 ;流式细胞术 (FACS )检测CTL诱导分化过程中CD3+ 、CD4 + 、CD8+ 、CD5 6 + 等细胞的分群变化 ;RT PCR检测细胞分化过程中FasLmRNA表达 ;生物活性法检测功能性细胞因子IFN γ的分泌。结果显示本法诱导的CTL对靶细胞有特异性杀伤活性 ,第 2次和第 3次DC刺激后杀伤活性有所上升 ;在CTL诱导分化的第 7、 14、 2 1天细胞分群以CD4 + 、CD8+ 细胞为主 ;RT PCR证实所诱导的细胞内有FasLmRNA的表达 ;随细胞培养天数的增加IFN γ分泌增加 ,在第 14天达到较高水平。研究表明重组痘苗病毒载体rVV LMP2A转染的DC刺激T细胞可诱导出EBV LMP2A特异性CTL。

Immunological Analysis of EBV LMP2A-Specific CTL Induced by DC Transfected with rVV-LMP2A in vitro

With the help of IL-2,EBV-specific CTL were induced by coculturing EBV-LMP2A recombinant vaccinia virus (rVV-LMP2A) transfected DC with the same donor-derived T cells. Specific cytotoxicity of the CTL was measured by LDH Release Kit. The dynamic alteration of expression of the surface antigen,such as CD3?CD4?CD8 and CD56 were detected by FACS. The level of IFN-γ was measured by biological activity method and the expression of FasL mRNA was detected by RT-PCR.The results showed that the CTL induced in vitro showed specific cytotoxicity to the target cells transfected with LMP2A and the cytotoxicity increased after the second or the third stimulation of DC. The populations of induced cell were composed mainly of CD4 + and CD8 +T cells. Expression of FasL in the induced cell was confirmed by RT-PCR. Secretion of IFN-γ raised as CTL cultured time prolonged but reached a rather high level on day 14. It concludes that strong and functional EBV-specific CTL can be generated by autologous mature DC transfected with rVV-LMP2A. This study may be useful to the applications of CTL-based immunotherapeutic approaches to EBV-associated maliganancies.