特异性人工抗原提呈细胞体外激活CD19嵌合抗原受体T细胞的构建

摘要

目的 构建CD19特异性人工抗原提呈细胞(aAPC)用于体外激活扩增CD19嵌合抗原受体(CAR)修饰T细胞(CD19-CAR-T),并考察其杀伤效应.方法 通过慢病毒介导的方法制备以NIH3T3为细胞骨架、表达共刺激分子CD86和/或CD137L的CD19特异性aAPC (NIH3T3-CD 19/86、NIH3T3-CD19/86/137L).采用照射的CD19特异性aAPC与CD 19-CAR-T细胞按一定比例混合培养激活扩增CD19-CAR-T细胞,台盼蓝染色法检测并绘制CD 19-CAR-T细胞生长曲线;流式细胞术检测CD19-CAR-T细胞CAR表达变化及分化表型;生物发光细胞毒性法检测扩增的CD19-CAR-T细胞体外靶特异杀伤效应.结果 流式检测结果显示NIH3T3-CD19/86和NIH3T3-CD19/86/137L细胞表面分别高表达CD19、CD86和/或CD137L分子;NIH3T3-CD19/86和NIH3T3-CD19/86/137L细胞都能够高效扩增CD 19-CAR-T细胞,NIH3T3-CD19/86/137L细胞具有更好的扩增效应,其与CD19-CAR-T细胞混合培养14d后,扩增的T细胞数量明显高于NIH3T3-CD19/86细胞组(P＜0.05);同时,与刺激前相比NIH3T3-CD 19/86和NIH3T3-CD19/86/137L细胞刺激扩增的T细胞中CD19-CAR-T细胞比例显著增加(P＜0.05);扩增的CD 19-CAR-T细胞具有靶特异杀伤效应,能够特异性杀伤CD19阳性靶细胞;流式检测显示NIH3T3-CD19/86/137L扩增的CD19-CAR-T细胞含有约20％作用中心记忆性T细胞.结论 成功制备CD19特异性aAPC,其可在体外特异性扩增功能性CD 19-CAR-T细胞,为初步建立制备高质量临床级CD19-CART细胞的方法提供了技术支撑.%Objective To construct CD19-specific artificial antigen-presenting cells (aAPCs) for in vitro activation and expansion of CD19 chimeric antigen receptor (CAR)-modified T cells (CD19-CAR-T) and investigate their cytotoxic effect.Methods CD19-specific aAPCs (NIH3T3-CD19/86,NIH3T3-CD19/86/137L) expressing costimulatory molecules CD86 and/or CD137L were prepared on the basis of NIH3T3 backbone cells by lentivirus-mediated gene transfer.Irradiated CD19-specific aAPCs were co-cultured with CD19-CAR-T cells to activate and amplify CD19-CAR-T cells.The growth curve of CD19-CAR-T cells was determined by trypan blue exclusion assay,and CD19CAR expression and phenotype on CD19-CAR-T cells were detected by flow cytometry.The in vitro cytotoxicity of CD19-CAR-T cells against the target cells was evaluated by bioluminescence-based cytotoxicity assay.Results Flow cytometry showed that NIH3T3-CD19/86 and NIH3T3-CD19/86/137L expressed high levels of CD19,CD86 and/or CD137L.Both NIH3T3-CD19/86 and NIH3T3-CD19/86/137L cells could amplify CD19-CAR-T cells efficiently,but NIH3T3-CD19/86/137L cells had better amplification effect.After 14 days of co-culture with NIH3T3-CD19/86/137L cells,the number of CD19-CAR-T cells was significantly greater than that of NIH3T3-CD19/86 cells (P＜0.05),and the proportion of CD19-CAR-T cells in the total T cells increased significantly (P＜0.05).CD19-CAR-T cells amplified by CD19-specific aAPCs produced target-specific cytotoxicity and were able to specifically kill CD19-positive target cells.About 20％ central memory T cells were present in the final products expanded by NIH3T3-CD19/86/137L.Conclusion We successfully prepared CD19-specific aAPCs that can specifically amplify functional CD19-CART cells in vitro,which facilitates the acquisition of clinical-scale high-quality CD19-CART cells.