Effect Of IL-37 On Expression Of PD-L1 And Cytotoxic T Cell Function In Non-Small Cell Lung Cancer

Objective:Lung cancer is the most common cancer with high morbidity and mortality.The emergence of immunotherapy has brought hope to patients with lung cancer.However,patients with epidermal growth factor receptor(EGFR)mutation in non-small cell lung cancer have poor immunotherapy effects against PD-1/PD-L1.Previous studies have found that interleukin 37(IL-37)plays an important role in tumor progression.The purpose of this study was to investigate whether IL-37 affects the expression of PD-L1 in non-small cell lung cancer and the function of cytotoxic T cell,and to find the potential mechanism of immunotherapy resistance in non-small cell lung cancer,to provide directions for the exploration of lung cancer treatment.Methods:Real-time quantitative polymerase chain reaction(qRT-PCR)and Western blotting(Western blot)were used to detect the expression of PD-L1 in human lung epithelial cells BEAS-2B and human lung adenocarcinoma cells A549,PC-9,H1975.IL-37 was overexpressed by recombinant adenovirus and lentivirus respectively,the expression of IL-37 and PD-L1 was detected by qRT-PCR and Western blot,and PC-9 cells were treated with different concentrations of exogenous rhIL-37.Peripheral blood mononuclear cells(PBMC)of healthy volunteers were obtained by density gradient centrifugation and activated by CD3/CD28T cell activator and interleukin-2(IL-2).The activated T cells were co-cultured with normal PC-9 cells and PC-9 cells overexpressing IL-37.The expression of cytokines such as interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α)in the co-culture supernatant was detected by enzyme-linked immunosorbent assay(ELISA)and flow cytometry.The cytotoxicity of T cells was detected by CCK8 and flow cytometry,and further verified by PD-L1 blocking antibody.The statistical software Graph Pad Prism9 was used to analyze the experimental data.Results:The results of qRT-PCR and Western blot showed that the expression of PD-L1mRNA in A549,PC9 and H1975 cells was significantly higher than that in BEAS-2B,but there was no significant difference in PD-L1 protein expression.IL-37 was successfully overexpressed by recombinant adenovirus and lentivirus,and there was no significant difference in PD-L1 between A549 and H1975 cells overexpressing IL-37(P<0.001).The expression of mRNA and protein of PD-L1 in PC-9 cells overexpressing IL-37 was increased(P<0.001).Exogenous rhIL-37 had no significant effect on the expression of PD-L1 in PC-9 cells.Activated T cells were co-cultured with PC-9 cells and PC-9 cells overexpressing IL-37.The results of 12 cytokines detected by flow cytometry showed that the secretion of IFN-γand TNF-αwas decreased,and the result of ELISA was consistent with that of flow cytometry.The proportion of T cells after co-culture was detected by flow cytometry,and the proportion of PC-9 cells overexpressing IL-37 decreased the proportion of CD8~+T cells.At the same time,the results of CCK8 showed that the cytotoxicity of T cells was inhibited,and the difference was statistically significant(P<0.05).When PD-L1blocking antibody was added,the proportion of CD8~+T cells,cytotoxicity and IFN-γsecretion increased,the difference was statistically significant.Conclusion:This study shows that endogenous IL-37 can promote the expression of PD-L1 in PC-9 cells,and then affects the cytotoxicity and secretory function of T cells.This study reveals one of the potential mechanisms of poor efficacy of PD-L1 immunotherapy in lung adenocarcinoma with EGFR19 exon deletion,and provides a new direction for the treatment of lung adenocarcinoma.