南方医科大学学报2017，Vol.37Issue(5)：581-587,7.DOI:10.3969/j.issn.1673-4254.2017.05.03
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
Construction of specific artificial antigen-presenting cells for in vitro activation of CD19 chimeric antigen receptor T cells
摘要
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.关键词
人工抗原提呈细胞/CD19/嵌合抗原受体修饰T细胞/增殖/杀伤Key words
artificial antigen presenting cells/CD19/chimeric antigen receptor T cells/proliferation/cytotoxicity引用本文复制引用
彭耀军,吴其艳,刘鸿宇,赵健,危华锋..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[J].南方医科大学学报,2017,37(5):581-587,7.基金项目
国家自然科学基金(81372528) （81372528）
国家高技术研究发展计划“863”计划(2014AA020704) Supported by National Natural Science Foundation of China (81372528). （2014AA020704）
