The Role Of PD-L1 Molecules In Immune Escape Of Lung Cancer Mediated By Tumor Associated Macrophage

Over the past several years, the immunotherapy in the treatment of tumors is growing by leaps and bounds. The immune system has the function of immune surveillance to remove tumor cells, playing a critical role in the occurrence, development and metastasis of tumors. The immunotherapy has close relation with the tumor microenvironment. The tumor microenvironment is composed of tumor cells, stromal cells, micrangium,dendritic cells,macrophages and so on. As the site for the modulation of immune responses, many factors are associated with tumor immunity in the tumor microenvironment, including the negative regulation of cytokines, suppressive immunoregulatory Treg, inhibitory response between ligand and receptor and negative regulation of T cells. Programmed death ligand 1(PD-L1) has attracted a mounting attention in recent years. The activation of PD-1/PD-L1 signaling pathway can mediate immune escape of tumor and promote the growth of tumor, by inhibiting the proliferation and activation of CD4+T and CD8+T cells. This study focuses on the analysis of the expression of PD-L1 in the immune cells and tumor cells, exploring the regulatory mechanism and biological significance, adding new understanding on the mechanisms of immune escape of lung cancer.Part I The expression of PD-L1 in the tumor-associated macrophages[Objective] To research the expression and clinical significance of PD-L1 in the tumor-associated macrophages.[Methods] Collectingthe clinical data of 76 patients with pleural effusion from respiratory department of the First Affiliated Hospital of Soochow University between October 2012 to October 2013. Among them, 52 patients were lung cancer, including 32 adenocarcinoma, 20 squamous cell carcinoma, 24 patients being tuberculous pleurisy. The pleural effusion mononuclear cells(PEMC) were separated by ficoll hypaque gradient. Using Flow cytometry to analyse the expression of PD-L1 and B7-H4 on the The role of PD-L1 molecules in immune escape of lung cancer mediated by tumor associated macrophage. Abstract surface of tumor-associated macrophages.[Results] Compared with tuberculous pleural effusion,malignant pleural effusion had a higher expression of PD-L1 on the surface of tumor-associated macrophages(30.16±16.64%vs 14.09±11.89,P<0.01). This phenomenon could also be detected in the expression of B7-H4(16.97±10.32%vs 7.17±5.52%,P<0.01) and the co-expression of PD-L1 and B7-H4(11.05±9.51% vs 3.99±5.03%,P<0.01). While in the squamous cell carcinoma and adenocarcinoma, the result had no significant difference. The expression of PD-L1 and B7-H4 and the co-expression of PD-L1 and B7-H4 were(28.73±15.03% vs 32.44±19.13,P=0.440),(17.83±10.73% vs 16.58±12.35%,P=0.701) and(10.06±7.43% vs 12.63±12.17 %, P=0.348).[Conclusion] Compared with tuberculous pleural effusion, malignant pleural effusion caused by lung cancer had higher expression of PD-L1 and B7-H4 and the co-expression of PD-L1 and B7-H4. This result have good diagnostic value for malignant pleural effusion.Part IIThe related factorson the regulation of PD-L1 in lung cancer cells[Objective] To investigate the regulation effects of TAMs on the expression of PD-L1 in lung cancer cells and its biological significance.[Methods](1) Using the pleural effusionand supernatant of tumor-associated macrophage(TAMs supernatant) cultured lung cancer cells(A549 cells). The expression of PD-L1 were detected withflow cytometer. The invasion capacity was measured in vitro using transwell migration assays.(2)The pleural effusion mononuclear cells(PEMC) were separated by ficoll hypaque gradient. Using flow cytometry to analyse the expression of IL-6、IL-10、TNF-α、IFN-γin the tumor-associated macrophages.(3)The IL-6、IL-10、TNF-α、IFN-γstimulated A549 cells and dectected the expression of PD-L1 by flow cytometry.(4) Analyzing the expression levels of p-ERK, p-AKT p-Stat3 with western blot after stimulated with IFN-γ.(5) Using MAPK/ERK signaling pathway blocker PD98059, P13K/AKT signaling pathway blocker LY294002 and JAK/STAT3 signaling pathway inhibitor AG490 to culture A549 cells. Detected the expression of PD-L1 by flow cytometry after stimulated with IFN-γ.(6)The PD-1 fusion protein combined with PD-L1 on A549 cells stimulated by IFN-γ, we detected the cell proliferation of A549 cells using CCk-8 assay, cell apoptosis using a flow cytometer and the invasion capacity using transwell assays.(7) After confirming the knockdown of the si RNAs targeting PD-L1, we also detected the cell proliferation of A549 cells, cell apoptosis and the invasion capacity.[Results](1) Compared with unstimulated A549 cells, the expression of PD-L1 after stimulated with pleural effusion and TAMs supernatant was increased. The same result also showed on invasion capacity.(2)The expression levels of IL-6 、 IL-10 、 TNF-αand IFN-γwere 30.1%, 2.76%, 40.2% and 12.5% respectively.(3)The expression of PD-L1 increased after stimulated with IFN-γ. There were no change in IL-6、IL-10、TNF-α.(4) The levels of p-AKT and p-Stat3 stimulated with IFN-γ were changed dramatically. While the levels of p-ERK remained. Compared with unstimulated A549 cells, the levels of p-AKT and p-Stat3 stimulated with IFN-γ were higher. While the levels of p-ERK remain the same.(5) It were found that treatment with LY294002(P13K/AKT signaling pathway inhibitor) and AG490(JAK/STAT3 signaling pathway inhibitor) could inhibit the expression of PD-L1 on the surface of A549 cells. While PD98059(MAPK/ERK signaling pathway inhibitor) couldn’t decreased the expression of PD-L1.(6)After the PD-1 fusion protein combined with the A549 cells stimulated by IFN-γ, the cell proliferation increased, invasion capacity enhanced and cell apoptosis decreased with the differentconcentration of PD-1 fusion protein.(7) After the interference of PD-L1 expression, cell proliferation decreased, invasion capacity weakened and cell apoptosis increased.[Conclusion] The tumor associated macrophages in the pleural effusion could upregulate the expression of PD-L1 on the A549 cells. P13K/AKT signaling pathway and JAK/STAT3 signaling pathway both participate this process. The induced expression of PD-L1 could increase the proliferation and decrease the apoptosis of the lung cancer cells. The intervention of the expression of PD-L1 in lung cancer cells could inhibit the immune escape of lung cancer cells to some extent.