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 μg/ml) of Nivolumab was added on day 8 to the culture medium. Patient T cells incubated with 72 μg/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 [(32.80±7.22)% vs (35.10±5.84)%, t=-0.554, P=0.593]. ②Incubation of CD19-CAR-T cells with 72 μg/ml Nivolumab did not affect CD19-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). ③Different 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 CD19-CAR-T cells could reduce the drug toxicity and enhance the cytotoxicity.
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、18 μg/ml的Nivolumab，同时设患者T细胞联合72 μg/ml Nivolumab及正常人CD19-CAR-T细胞为对照，采用CCK-8法、LDH细胞毒性检测、ELISA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]。②72 μg/ml Nivolumab联合CD19-CAR-T细胞不影响其增殖，但联合应用24、48 h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+72 μg/ml的Nivolumab（P值均<0.001），其中48 h时各组的杀伤率分别为（71.61±9.50）%、（6.77±1.26）%、（15.33±4.11）%。72、36 μg/ml Nivolumab联合来源CD19-CAR-T细胞对Pfeiffer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+18 μg/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36 μg/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。.
Keywords: Chimeric antigen receptor; Lymphoma; PD-1 inhibitor.