CD19 CAR-T细胞培养过程中PD-1蛋白、mRNA水平及细胞杀伤活性变化  

PD-1 expression,mRNA level and cytotoxicity changes in CD19CAR-T cells

作　　者：蒲业迪[1] 王嘉[1] 邓琦[1] 朱海波[1] 江嫣雨 孟娟霞[1] 李玉明[1] Pu Yedi;Wang Jia;Deng Qi;Zhu Haibo;Jiang Yanyu;Meng Juanxia;Li Yuming(Department of Hematology,Tianjin First Central Hospital,Tianjin 300192,China)

机构地区：[1]天津市第一中心医院血液科,300192

出　　处：《中华血液学杂志》2019年第9期759-763,共5页Chinese Journal of Hematology

摘　　要：目的 探讨CD19 CAR-T细胞培养过程中其PD-1蛋白、mRNA水平及细胞杀伤活性变化.方法 收集6例外周血PD-1高表达恶性淋巴瘤患者、6例健康志愿者的外周血T细胞,作为CAR-T培养的T细胞来源.流式细胞术检测PD-1蛋白表达、PCR法检测PD-1 mRNA水平,CCK-8法检测细胞增殖,LDH法检测细胞杀伤活性.结果 ①PD-1高表达患者T细胞来源CD19 CAR-T细胞,与志愿者T细胞来源者相比,转染率无差异(P>0.05);②PD-1高表达T细胞来源CAR-T细胞与PD-1抑制剂联合与否,以及健康志愿者CAR-T之间,细胞增殖差异无统计学意义(P>0.05);③PD-1高表达T细胞与CAR-T细胞对淋巴瘤细胞株杀伤活性,低于二者联合PD-1抑制剂及志愿者CAR-T细胞(P<0.001),而PD-1高表达T细胞来源CAR-T细胞联合PD-1抑制剂与健康志愿者CAR-T细胞间差异无统计学意义(P>0.05);④各组细胞培养过程中PD-1表达均下降,差异无统计学意义(P>0.05),但各组细胞培养过程中,PD-1 mRNA的变化差异无统计学意义(P>0.05);⑤PD-1高表达T细胞来源CAR-T收获后,与PD-1抑制剂共培养与否,其PD-1表达差异无统计学意义(P>0.05),但CAR-T与淋巴瘤细胞株接触后,其PD-1表达随培养时间延长而增高,加入PD-1抑制剂可拮抗该作用;各组间PD-1 mRNA的变化差异无统计学意义(P>0.05).结论 PD-1高表达T细胞来源CAR-T细胞与肿瘤细胞接触后,其PD-1表达随培养时间延长而增高;而包括PD-1抑制剂在内,不能改变其PD-1 mRNA的表达.Objective To observe the changes of PD-1 expression,mRNA level and cytotoxic activity of CD19 CAR-T cells during the culture process of CAR-T cells.Methods The peripheral blood T cells of 6 lymphoma patients with high expression of PD-1 and 6 healthy volunteers were the source of CAR-T cells.The expression of PD-1 was analyzed by flow cytometry.The mRNA level of PD-1 was analyzed by PCR.The cell proliferation was analyzed by CCK-8 assay.The cytotoxicity was analyzed by LDH assay.Results①The transfection efficiency of high PD-1 expression T cells and healthy volunteer T cells were as the same(P>0.05).②The cell proliferation capacity of CD19 CAR-T cells from high PD-1 expression T cells or healthy volunteer T cells,with or without PD-1 inhibitor were as the same(P>0.05).③The cytotoxicity to lymphoma cells of high PD-1 expression T cells and CAR-T cells were lower than that of these two T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer T cells(P<0.001).There was no difference of the cytotoxicity between the CAR-T cells from high PD-1 expression T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer(P>0.05).④There was no difference of the expression of PD-1 in all CAR-T cell groups during the culture process(P>0.05).There was no difference of mRNA level of PD-1 in all groups during the culture process(P>0.05).⑤The PD-1 expression of CAR-T cells increased by the time of culture after contacting with lymphoma cells(P<0.001).The PD-1 inhibitors could antagonize this effect.There was no difference of mRNA level of PD-1 in all groups after contacting with lymphoma cells(P>0.05).Conclusion The PD-1 expression of CAR-T cells from high PD-1 expression T cells increased by the time of culture after contacting with lymphoma cells.However,the mRNA level of PD-1 of all groups did not change,even if PD-1 inhibitor was applied.

关 键 词：PD-1 mRNA 转染 T细胞 嵌合抗原受体T细胞 

分 类 号：R73[医药卫生—肿瘤]