PD-1抑制剂Nivolumab对CD19嵌合抗原受体T细胞体外增殖和杀伤活性的影响

Effect of PD-1 inhibitor Nivolumab on the proliferation and cytotoxicity of anti-CD19 chimeric antigen receptor T cells

目的评估程序性细胞死亡受体1（PD-1）抑制剂Nivolumab对CD19-CAR—T细胞体外增殖和杀伤活性的影响。方法收集5例外周血PD-1高表达恶性淋巴瘤患者的外周血T细胞制备CD19-CAR—T细胞，在培养第8天加入终浓度分别为72、36、18pg／ml的Nivolumab，同时设患者T细胞联合72μg／mlNivolumab及正常人CD19-CAR—T细胞为对照，采用CCK．8法、LDH细胞毒性检测、ELlSA法比较各组的增殖活性、杀伤活性及炎症因子水平。结果①PD—1高表达患者CD19-CAR-T细胞转染率与正常人接近[（32．80±7．22）％对（35．10±5．84）％，t=-0．554，P=0．593]。②72gg／ml Nivolumab联合CD19．CAR—T细胞不影响其增殖，但联合应用24、48h对Pfeiffer细胞的杀伤率均优于单用患者CD19-CAR—T细胞及患者来源T细胞＋72gg／ml的Nivolumab（P值均〈0．001），其中48h时各组的杀伤率分别为（71．61±9．50）％、（6．77±1．26）％、（15，33±4．11）％。72、36μg／ml Nivolumab联合来源CD19-CAR—T细胞对Pfejffer细胞的杀伤率差异无统计学意义（P值分别为0．281、0．267），二者均高于患者来源CD19-CAR—T细胞＋18μg／ml Nivolumab组（P值均〈0．001）。③不同剂量Nivolumab联合患者来源CD19-CAR-T细胞，不影响炎症因子IFN-γ、TNF-α水平（P值均〉0．05）。结论终浓度为36g／ml的Nivolumab与CD19-CAR—T细胞联合应用，可在减轻药物毒副作用同时增强CD19-CAR-T细胞的杀伤活性。

Objective To Evaluation the effect of PD- 1 inhibitor Nivolumab on the proliferation and cytotoxicity of anti-CD19 chimeric antigen receptor T cells （CD19-CAR-T） in vitro. Methods Five patients with high PD- 1 expression in peripheral blood and five healthy volunteers were selected. These peripheral blood mononuclear cells were used as the source of T cells to prepare CD19-CAR-T cells. Different doses （72, 36, 18 pg/ml） of Nivolumab was added on day 8 to the culture medium. Patient T cells incubated with 72 pg/ml Nivolumab and CD19-CAR-T cells of healthy volunteers were used as controls. CCK-8, lactate dehydrogenase （LDH） cytotoxicity assay and ELASA were used to detect the proliferation capacity, the specific cytotoxicity and the inflammatory factor secretion. Results ①T cells from patients with high expression of PD-1 as the source of CD19-CAR-T cells did not affect transfection rate compared with that of healthy volunteers I（32.80±7.22）% vs （35.10±5.84）%, t = -0.554, P = 0.593 ]. ②Incubation of CD19-CAR-T cells with 72μg/ml Nivolumab did not affect CDIg-CAR-T cell proliferation, but its cytotoxicity was significantly higher than that of CD19-CAR-T cells alone or patients＇ T cells ＋72 μg/ml Nivolumab （all P 〈 0.001）, there was no significant difference in the killing activity between the 72 μg/ml and 36 μg/ml Nivolumab treated CD19-CAR-T cells on Pfeiffer cells （P = 0.281, 0.267, respectively）, and they were all higher than those of 18 μg/ml Nivolumab treated CD19-CAR-T cells （all P〈 0.001）. ③ Different doses of PD- 1 inhibitor Nivolumab combined with CD19-CAR-T cells does not affect the secretion of IFN-γ and IFN-α （all P 〉 0.05）. Conclusion Combination of 36 μg/ml PD-1 inhibitor and CD 19-CAR-T cells could reduce the drug toxicity and enhance the cytotoxicity.