摘要目的:探讨程序性死亡受体配体1(PD-L1)对CLL-1 CAR-T细胞抗急性髓系白血病(AML)作用的影响。方法:通过构建PD-L1表达载体、制备慢病毒、转导、单克隆筛选技术获得稳定表达PD-L1的THP-1单克隆细胞株(THP1-PDL1)，然后以前期制备的CLL-1 CAR-T细胞为效应细胞，以THP-1、THP1-PDL1单克隆细胞株作为靶细胞，分别通过LDH检测、CBA法、CFSE法评价PD-L1过表达对CLL-1 CAR-T细胞裂解功能、炎性因子释放、细胞增殖等功能的影响。结果:①成功制备了PD-L1慢病毒，并筛选获得了稳定表达PD-L1的THP1-PDL1单克隆细胞株，流式细胞术及PCR验证成功。②PD-L1过表达抑制了CLL-1 CAR-T细胞裂解THP-1细胞的能力；效靶比为10∶1时，CLL-1 CAR-T细胞对THP1-PDL1细胞的杀伤效率明显低于对THP-1细胞的杀伤效率[(15.70±9.90)%对(51.95±2.52)%， P<0.05]。③PD-L1过表达减弱了CLL-1 CAR-T细胞释放细胞因子的能力[与THP1-PDL1细胞共培养时对与THP-1细胞共培养时：IFN-γ(115.66±3.13)pg/ml对(1708.16±26.76)pg/ml， P<0.05；IL-6(17.37±0.72)pg/ml对(124.92±4.26)pg/ml， P<0.05；IL-10(5.69±0.13)pg/ml对(124.12±3.02)pg/ml， P<0.05]；同时抑制了CLL-1 CAR-T细胞的增殖能力。 结论:成功构建了表达PD-L1的THP1-PDL1单克隆细胞株，同时证实了PD-L1过表达对CLL-1 CAR-T细胞抗AML的不利的影响，为通过PD-1/PD-L1通路调控CLL-1 CAR-T细胞功能提供了一定的理论基础。

abstractsObjective:To investigate the effects of programmed death receptor ligand 1(PD-L1)on CLL-1 CAR-T against acute myeloid leukemia(AML).Methods:In this experiment, the PD-L1 expression vector was constructed, and then the lentivirus vector was packaged by three-plasmid packaging system. THP-1 monoclonal cell lines stably expressing PD-L1 were set up. The CLL-1 CAR-T was developed by our team, as the effector cell for co-culture with the THP-1 or THP1-PDL1 cell lines, respectively. Then, the LDH was tested using the kit, the supernatant cytokine was detected by CBA, and the CLL-1 CAR-T cell proliferation was demonstrated by flow cytometry(FCM)with CSFE labeled.Results:①The PD-L1 lentivirus vector was successfully constructed, and monoclonal cell lines of THP-1 with stable PD-L1 was set up and verified by FCM and PCR. ②The overexpression of PD-L1 inhibited CLL-1 CAR-T's ability to lyse THP-1 cells(E∶F ratio 10∶1); the killing efficiency of CLL-1 CAR-T on THP1-PDL1 cells was lower than that of THP-1 cells[(15.70±9.90)% vs(51.95 ± 2.52)%, P<0.05]. ③The overexpression of PD-L1 decrease the release of cytokine[THP1-PDL1 group vs THP-1 group: IFN-γ(115.66±3.13)pg/ml vs(1708.16 ± 26.76)pg/ml, P<0.05; IL-6(17.37±0.72)pg/ml vs(124.92±4.26)pg/ml, P<0.05; IL-10(5.69±0.13)pg/ml vs(124.12±3.02)pg/ml, P<0.05]. Additionally, the proliferation of CLL-1 CAR-T was also inhibited. Conclusion:Monoclonal cell lines of THP-1 with stable PD-L1 expression were successfully constructed, and the adverse effect of PD-L1 overexpression on CLL-1 CAR-T anti-AML was confirmed, which provided a theoretical basis for the regulation of CLL-1CAR-T through the PD-1/PD-L1 pathway.