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.

abstractsObjective 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. Results ①The 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.