肿瘤特异性TCRαβ基因转染诱导记忆性T细胞分化及其免疫功能的研究

目的研究特异性TCR基因转染T细胞被肿瘤抗原激活后,记忆性T细胞的分化情况,并明确其表型特征和免疫功能。方法密度梯度离心法分离PBMC,重组TCR腺病毒感染T细胞,流式细胞术检测外源TCR表达效率。AFP表位肽刺激T细胞,流式细胞术检测TCR基因转染T细胞经抗原刺激后,记忆性T细胞标志分子表达。MTT法检测T细胞对不同肿瘤细胞株的杀伤活性。Annexin V-PI双染法检测靶细胞凋亡比例。ELISA法检测T细胞作用于靶细胞后IFN-γ与IL-2分泌水平。结果重组腺病毒载体感染3 d后,外源TCR表达比例接近30%。特异性TCR基因转染可有效促进T细胞识别肿瘤抗原后活化,CD45RO+细胞比例逐渐上升至接近50%。CD45RO+细胞以CD62L–CD44+表型为主。此后CD62L+细胞比例逐渐上升。最终出现分群明显的CD62L+CD44+TCM表型细胞。特异性TCR基因转染能够促进T细胞杀伤AFP+靶细胞,诱导靶细胞凋亡,并促进IFN-γ分泌。抗原预先刺激能够进一步增强TCR基因转染T细胞抗肿瘤免疫效应。结论 TCR基因转染能够有效促进T细胞识别肿瘤抗原后活化。经肿瘤抗原预先刺激的TCR基因转染T细胞将启动记忆性T细胞分化,在再次遭遇表达相同抗原的肿瘤细胞时发挥更为强烈的免疫效应。

The differentiation and anti-tumor immunity of memory T cells after specific TCR gene transfection and tumor antigen induction

Objective To investigate the differentiation of memory T cells after the activation of TCR gene transfected T cells by tumor antigen stimulation in vitro, and to determine the phenotype and anti-tumor immunity of the induced memory T cells. Methods PBMC were isolated by density gradient centrifugation. T cells were infected by recombinant TCR adenovirus and the expression efficiency of transgene in T cells was detected by FACS. T cells were stimulated by 9-amino long HLA-A2＋ epitope from AFP218-226. Expression of molecular markers of memory T cell on TCR gene transfected T cells after activation was detected by FACS. Specific lysis of T cells was evaluated by MTT assay. The ratio of apoptotic cells in the target cells was detected by Annexin V/PI double-labeled FACS. The secretion of cytokine IFN-γ and IL-2 of T ceils was quantified by ELISA assay. Results The ratio of exogenous TCR positive cells was about 30% of T cells 3 days after infection. Tumor-specific TCR gene modification redirected the antigen specificity of T cells effectively. TCR gene transfected T cells were activated effectively by stimulation of AFP. The ratio of CD45RO＋ cells was increased after activation and reached about 50% of T cells 14 days after stimulation. Most of CD45RO＋ cells were CD62L-CD44-. The ratio of CD62L positive ceils was then increased. Cell clusters with CD62L＋CD44＋ TCM phenotype appeared in effector T cells eventually. Tumor specific TCR gene modification enhanced the abilities of T cells to lyse HLA-A2＋ AFP＋ target cells, to induce HepG-2 apoptosis, and to secret IFN-γ. Stimulation ofTCR gene transfected T cells by tumor antigen effectively enhanced the anti-tumor activity of T cells. Conclusion Tumor specific TCR gene modification redirected antigen specificity of T cells effectively. Antigen specific T cells stimulated by tumor antigen initiate the differentiation of memory T characters of TcM and mediate enhanced response upon re-exposure to tumor antigen.