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

发布时间：2018-01-27 06:51

本文关键词： 人工抗原提呈细胞 CD 嵌合抗原受体修饰T细胞增殖杀伤　出处：《南方医科大学学报》2017年05期 　论文类型：期刊论文

【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a APC(NIH3T3-CD19/86、NIH3T3-CD19/86/137L)。采用照射的CD19特异性a APC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(P0.05);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(P0.05);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a APC,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 cell (APC) to activate and amplify CD19 chimeric antigen receptor (CAR) to modify T cell (CD19-CAR-T) in vitro. Methods the cytoskeleton of NIH3T3 was prepared by lentivirus-mediated method. CD19 specific a APC(NIH3T3-CD19/86 expressing costimulatory molecule CD86 and / or CD137L. NIH3T3-CD19 / 86 / 137L). CD19-CAR-T cells were activated by mixed culture of irradiated CD19 specific a APC and CD19-CAR-T cells in a certain proportion. Trypan blue staining was used to detect and draw the growth curve of CD19-CAR-T cells. The changes of CAR expression and differentiation phenotype of CD19-CAR-T cells were detected by flow cytometry. In vitro target specific cytotoxicity of amplified CD19-CAR-T cells was detected by bioluminescence cytotoxicity assay. Results NIH3T3-CD19/86 and NIH3T3-CD were detected by flow cytometry. The surface of 19 / 86 / 137L cells respectively expressed CD19. CD86 and / or CD137L molecules; Both NIH3T3-CD19/86 and NIH3T3-CD19/86/137L cells can amplify CD19-CAR-T cells efficiently. NIH3T3-CD19/86/137L cells had better amplification effect and were mixed with CD19-CAR-T cells for 14 days. The number of expanded T cells was significantly higher than that of NIH3T3-CD19/86 cells. At the same time. The percentage of CD19-CAR-T cells in T cells stimulated by NIH3T3-CD19/86 and NIH3T3-CD19/86/137L cells increased significantly compared with those before stimulation. P0.05; The amplified CD19-CAR-T cells have target specific killing effect and can specifically kill CD19 positive target cells. Flow cytometry showed that CD19-CAR-T cells amplified by NIH3T3-CD19/86/137L contained about 20% central memory T cells. Conclusion the specificity of CD19 was successfully prepared. A. APC. It can specifically amplify functional CD19-CAR-T cells in vitro and provide technical support for the preliminary preparation of high quality clinical grade CD19-CART cells.
【作者单位】：解放军总医院肿瘤中心实验室;南开大学医学院;第二军医大学肿瘤研究所;
【基金】：国家自然科学基金(81372528) 国家高技术研究发展计划“863”计划(2014AA020704)~~
【分类号】：R730.51
【正文快照】： 海200433Construction of specific artificial antigen-presenting cells for in vitro activation of CD19 chi-meric antigen receptor T cellsPENG Yaojun1,WU Qiyan1,LIU Hongyu2,ZHAO Jian1,3,WEI Huafeng11Cancer Center Key Laboratory,General Hospital of PLA,Beiji

【相似文献】