Glutamine Metabolism Regulates PD-1 Of CD8+t Cell And PD-L1 In Bladder Cancer T24/5637 Cells In Tumor Microenvironment

Objective:Immunotherapy has become one of the effective treatment plans widely used in clinical tumor treatment in addition to chemotherapy and surgery.However,tumor cells can still escape from the monitoring and clearance of the immune system through certain specific paths,which leads to the proliferation of tumor cells and the further deterioration of the patient's condition,that is the immune escape of tumor cells.Studies have shown that the activation of antigen-specific T cells and killer T cells can interfere with and down-regulate the immune response of T cells through the binding of PD-L1 from tumor cells to PD-1 receptors on the surface of lymphocytes.Therefore,finding the factors that affect the expression level of PD-1 / PD-L1 in the tumor microenvironment is essential to further explore the immune escape mechanism.Recent studies have shown that the glycolysis process can regulate the immune escape process triggered by PD-1 / PD-L1.In addition,the glutamine amino acid metabolism process may also play an important role in triggering the immune escape mechanism of bladder cancer cells.Controlling the level of glutamine metabolism may have the function of regulating the immune escape of bladder cancer cells.In this study,by observing the effects of glutamine metabolism on the PD-1 expression of CD8 + T cells and the expression of PDL1 in bladder cancer T24 / 5637 cells under single culture or co-culture conditions,we initially explored the regulation of glutamine metabolism on regulation The effect and biological mechanism of PD-1 / PD-L1 expression in bladder cancer.Methods:T24 and 5637 bladder cancer cells were purchased from the Institute of Biology,Chinese Academy of Sciences.CD8 + T cells were isolated from the peripheral blood of healthy volunteers.Glutamine metabolism levels are controlled by the glutamine analog DON(6-diazo-5-oxo-Lnorleucine).Single-culture of bladder cancer cells and co-culture of bladder cancer cells and T cells in vitro using 12-well plates.PD-1/PD-L1 gene expression in T cells and bladder cancer cells were disclosed by RT-qPCR analysis,and PD-1/PD-L1 protein expression levels in 5637 and T24 cells were detected by Western Blot.Results:First,only the glutamine metabolism level of bladder cancer cells in the single culture group was changed,and the expression of PD-L1 did not differ significantly due to the change in glutamine metabolism level.However,under the condition of adding interferon,in the single culture group of T24 / 5637 bladder cancer cells,the expression level of PD-L1 in bladder cancer cells was positively correlated with the level of glutamine metabolism level.In the co-culture system,the change of glutamine metabolism level of each group of cells(T24 / CD8 +,5637 / CD8 +)in the same direction showed the same trend of PD-1 / PD-L1 expression.Within a certain range,the higher the glutamine metabolism level,the higher the expression level of PD-1 / PD-L1.As glutamine metabolism was gradually inhibited,the expression levels of PD-L1 in T24 / 5637 cells and PD-1 in CD8 + T cells in the co-culture group gradually decreased.Conclusion:According to the research results,in the tumor microenvironment,glutamine metabolism plays an important role in the expression level of PD-1 / PD-L1 in CD8 + T cells and T24 / 5637.When bladder cancer cells are co-cultured with CD8 + cells in vitro,changing the glutamine metabolism level will regulate the expression level of PD-1 / PD-L1.It can be speculated that the control of glutamine metabolism level in the body's immune defense process may have a regulatory effect on the expression of PD-L1 and PD-1 of CD8 + T cells in bladder cancer cells.By simulating the tumor microenvironment,we draw preliminary conclusions on glutamine metabolism and PD-L1 expression.Future research should focus on the intersection of the glutamine metabolism pathway and the PD-L1 pathway or the bioinformatics analysis of related genes.