Expression Of PD-L1 In Lung Cancer Cell Lines And Its Role In Tumor Immunity

Objective: The expression screening of different pathological types of lung cancer cell line PD-L1 on the surface,to provide a basis for immunotherapy of PD-L1 as the predicted targets.Through the screening results of different expression levels of PD-L1,on the one hand,the upper and lower expression level,to explore the characteristics of lung cancer cell biology;on the other hand,activation and sorting CD8+T cells with different expression levels lung cancer lines were co cultured to test the detection of apoptosis of lung cancer cells in vitro.Simulating the in vivo immune microenvironment,testing cell apoptosis of different expression levels was to explore PD-L1,PD-L1 expression level in the immune function of the PD-1/PD-L1 pathway in the treatment.Simulation of PD-L1 antibodies by siRNA interference of PD-L1 in the blocking effect of PD-1/PD-L1 pathway in immune treatment provides a theoretical basis for immunotherapy targeting PD-1/PD-L1 pathway.Methods: Flow cytometry was used to detect different pathological types of lung cancer cell lines(HCC827,H1299,A549(adenocarcinoma),H226,H520,H460(squamous cell carcinoma)(large cell carcinoma),H446(small cell carcinoma))by surface PD-L1 expression of immune-fluorescence labelling.Differential expression analysis of the lung cancer cell surface PDL1 by fluorescence value,and then choose high,low expression cell lines.Adding cytokines IL-4,IL-6,IL-13,TGF-?-1,IFN-? gamma induced lung cancer cell lines were to select the choose of cytokine upping the levels of PD-L1 by Western blot technique.SiRNA interference expression technique was used to screen siRNA fragments which can effectively down regulate the expression of PD-L1 by Western blot.Transwell assay was used to detect the cell migration ability of the lung cancer cell lines induced by up regulation and down regulation.PBMC was isolated from peripheral blood of healthy volunteers by lymphocyte isolation solution and cultivated by T cells activation kits in vitro to activate and maintain the activity of T cells in vitro.The sorted CD8+T cells,lung cancer cell lines with different expression levels were co cultured,and analyze the correlation between th apoptosis rate and the expression levels of PD-L1 by detecting the apoptosis by flow cytometry.Results: Through flow cytometry,the expression level of PD-L1 in lung cancer cell lines was HCC827,H460(high expression),H1299,H226,H446,H520(expressed),A549(low expression).After the addition of cytokines IL-4? IL-6?IL-13?TGF-?-1?IFN-?,TGF-?-1and IFN-? have up-regulated the level of PD-L1 expression in lung cancer cells.After the synthesis of 3 siRNA fragments,the high efficiency interference fragment si-PD-L1 could significantly down regulate the expression of PD-L1 in lung cancer cells with high expression of PD-L1.Selection of adenocarcinoma cell lines H1299,A549 with the migration ability of Transwell,Transwell experimental results showed that TGF-?-1and IFN-? can enhance the ability of cell migration significantly compared to the control group with the increased expression of lung cancer cell line PD-L1.Si-PD-L1 interference expression in lung cancer cell line PD-L1,the expression level of PD-L1 was significantly reduced,the ability of cell migration was significantly decreased;cell line added TGF-?-1and IFN-? can be enhanced cell migration with PD-L1 higher expression then cell migration will be significantly weakened after siRNA interference.PBMC of peripheral blood was separated by lymphocyte separation.PD-1 expression was up-regulated by flow cytometry after activation of T cells activation kits in vitro.The CD8+T cells from the magnetic beads with lung cancer cell line of different expression levels(HCC827,H460,H446(high expression),H226(expressed),A549(low expression))after co culture,flow cytometry to detect cell apoptosis,apoptosis and PD-L1 expression level was negatively correlated.The apoptosis rate of lung cancer cell line by siRNA interference reducing PD-L1 co cultured with CD8+T cells increased significantly.Conclusion: Different pathological types of lung cancer cell lines had different levels of PD-L1 expression.The expression of PD-L1 can promote the migration of lung cancer cells and may be involved in lung cancer metastasis.The effect level of the immunotherapy was correlated with PDL1 expression.Lung cancer cell lines of different PD-L1 expression levels cultured in CD8+T cells had different apoptosis rates.Downregulation of PD-L1 expression and blocking PD-1/PD-L1 pathway can enhance the cytotoxicity of CD8+T cells to lung cancer cells.