PD-L1表达对NK细胞杀伤急性髓系白血病细胞株活性的影响及其机制研究

Effect of PD-L1 Expression on Activity of NK Killing AML Cell Lines and Its Mechanisms

目的:探讨不同急性髓系白血病(acute myelogenous leukemia, AML)细胞株表面程序性死亡因子配体1(programmed death receptor-ligand 1, PD-L1)表达水平对NK细胞杀伤效应的影响及其可能的机制。方法 :抽取健康人外周血,应用miniMACS免疫磁珠阴选法分出NK细胞;流式细胞术检测AML细胞株表面PD-L1表达水平;LDH释放法和单抗体阻断实验分析NK细胞对AML细胞株的杀伤效应;ELISA法检测效靶细胞共培养上清中IFN-γ和IL-2含量。结果:CD3-CD56+NK细胞含量由分选前的(12.44±3.48)%提高到(71.29±5.65)%;流式细胞分析结果表明,KG-1a细胞表面低表达PD-L1(18.35±4.12)%,而THP1细胞表面高表达PD-L1(76.42±26.54)%,同时发现NK细胞对KG-1a具有高效杀伤效应,而对THP1杀伤效果相对较差;联合应用PD-L1单抗可不同程度加强NK细胞对5种AML细胞的杀伤作用,同时促进细胞因子IFN-γ和IL-2分泌。结论:NK细胞对AML细胞株的杀伤活性与靶细胞表面PD-L1表达量有关,PD-L1高表达可抑制NK的效靶杀伤活性,而阻断PD1/PD-L1结合则有益于靶细胞对NK细胞的杀伤作用的敏感性,其机制可能与促进IFN-γ和IL-2释放有关。

Objective:To expolore the effect of programmed death receptor ligand 1(PD-L1)expression level on killing effect of different cell lines of acute myeloid leukemia(AML)and its possible mechanism.Methods:Peripheral blood from healthy individuals was collected routinely;NK cells were isolated using immunomagnetic beads;PD-L1 expression level was detected by flow cytometry;the killing effect of NK cells on acute myelogenous leukemia cell lines was evaluated with LDH release method and monoclonal antibody blocking experiment;the expression levels of IFN-γand IL-2 in the supernatants from the co-cultured effector/targer cells were measured by ELISA.Results:The ratio of CD3-CD56+NK cells increased from(12.44±3.48)%to(71.29±5.65)%.The flow cytometry showed that KG-1a cells lowly expressed PD-L1(8.35±4.12)%,but the THP cells a highly expressed PD-L1(76.42±26.54)%.Meanwhile,the NK cells displayed a more efficient killing effect on KG-1a cells than that of THP1 cells(P<0.05).Moreover,PDL1 monoclonal antibody could reinforce NK cell killing effect and,promote the secretion of IFN-γand IL-2 in 5 acute myelogenous leukemia cell lines to varying degree.Conclusion:The killing effect of NK cells on acute myelogenous leukemia cell line is inversely proportional to PD-L1 expression;blocking PD1/PD-L1 binding can significantly enhance the killing efficiency of effector-target cells,which way be related with promoting the release of IFN-γand IL-2.