南方医科大学学报

南方医科大学学报 ›› 2017, Vol. 37 ›› Issue (05): 581-.

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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

彭耀军，吴其艳，刘鸿宇，赵健，危华锋   

  • 出版日期:2017-05-20 发布日期:2017-05-20

Construction of specific artificial antigen-presenting cells for in vitro activation of CD19 chimeric antigen receptor T cells

  • Online:2017-05-20 Published:2017-05-20

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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-CD19/86、NIH3T3-CD19/86/137L）。采用照射的CD19 特异性aAPC与CD19-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CD19-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CD19-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14 d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-CD19/86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CD19-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 CD19-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。

Abstract: 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.