Programmed death ligand 1/programmed death 1 immune checkpoint regulate radioresistance non-small-cell lung carcinoma escape from natural killer cell immunotherapy

Abstract

Objective To observe radioresistance non-small-cell lung carcinoma (NSCLC) cell escape from natural killer (NK) cell immunity in vitro and investigate the mechanism. Methods Parental NSCLC cell lines A549 and H157 were continuously radiated until radioresistance cell lines were formatted. The mRNA from parental NSCLC cell lines together with cell lines in a series of different period depending on the radiation dose and radioresistance cell lines was collected. Programmed death ligand 1 (PD-L1) level was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). The PD-L1 expression in A549 cells at 0, 4, 8, 12 Gy was 1.000±0.078, 1.440±0.112, 1.760±0.152 and 2.050±0.166, and that in H157 cells was 1.000±0.055, 1.370±0.094, 1.530±0.132 and 1.890±0.146, respectively. NK cells were isolated from peripheral blood and co-cultured with different tumor cells in different ratios, after which cytotoxicity of NK cells was tested. PD-1 levels in NK cells were also tested before and after co-culture with parental NSCLC cell lines and radioresistance cell lines. Furthermore, PD-L1 and PD-1 neutralizing antibody were added during co-culture and their effects were observed. Results PD-L1 in parental NSCLC cell lines was increased after radiation and highly expressed in radioresistance cell lines. Relative PD-L1 level in A549 cells was 1.000±0.078, and that was 4.150±0.292 in A549-Radioresistance (A549R) (t=-18.052, P=0.001). Relative PD-L1 level in H157 cells was 1.000±0.055, and that was 5.070±0.387 in H157-Radioresistance (H157R) (t=-18.034, P=0.001). The cytotoxicity of NK cells in parental NSCLC cell lines was higher than in radioresistance NSCLC cell lines in different ratios. PD-1 level in NK cells was induced after co-culture with radioresistance NSCLC cell lines not parental NSCLC cell lines. Both PD-L1 and PD-1 neutralizing antibody could increase the cytotoxicity of NK cells in radioresistance NSCLC cell lines not in parental NSCLC cell lines. Conclusion Radioresistance NSCLC cell lines escape from NK cell immunity by increasing PD-L1 expression level in tumors and induction of PD-1 exprssion level in NK cells. PD-L1/PD-1 checkpoint can be important target in reversing immune escape in radioresistance NSCLC cell lines. Key words: Radioresistance; Non-small-cell lung carcinoma; Programmed death ligand 1; Programmed death 1; Natural killer cell; Tumor immunity