Role Of Glutamine On PD-L1/PD-1-mediated Immune Escape In The Bladder Tumor Microenvironment

Object:In recent years,research on the metabolism of substances in the tumour microenvironment has become a hot topic.In particular,glutamine metabolism is closely related to the mutual regulatory function of tumour cells as well as immune cells,where it may directly influence the immune escape of tumour cells.It was found that T24 in bladder cancer cells could promote PD-L1 expression upregulation after glutamine deprivation,but the mechanism of PD-1 expression and PD-L1/PD-1-mediated tumour escape by immune cells in the immune microenvironment is not yet clear.Therefore,in this study,we aimed to study the function of tumor cells and immune cells as well as the expression changes of immune markers through glutamine deprivation in the tumor microenvironment in order to preliminarily elucidate the effect of glutamine metabolism in the tumor microenvironment on PD-L1/PD-1-mediated tumor immune escape.Methods:CD8⁺T cells were obtained by sorting from the peripheral blood of healthy adults using human CD8⁺magnetic beads.The effect of CD8⁺T cell activation versus inactivation and glutamine deprivation on the expression of killing factor（GZMB,PRF1）and cytokine IFN-γwas examined using qPCR and ELISA.The effect of glutamine on the expression levels of PD-L1,PD-1,T cell suppressor SHP2 in bladder cancer cells T24 and CD8⁺T cells was determined by qPCR and Western Blot.The proliferative effects of glutamine on bladder cancer T24 cells and activated CD8⁺T cells were measured using the CCK8 assay and live cell workstation and the expression levels of proliferation-related factors（PCNA,Ki67）were measured.The effect of glutamine on total PD-L1,PD-1,SHP2 expression was examined at the m RNA and protein levels in bladder cancer T24 and activated CD8⁺T cells co-cultures to determine the effect of glutamine on PD-L1/PD-1-mediated immune escape.The expression levels of CD8⁺T cell killing factor（GZMB,PRF1）and cytokine IFN-γin the co-culture group and the effect of glutamine on their expression were measured using ELISA.The ability of glutamine on killing bladder cancer T24 cells by CD8⁺T cells in the co-culture group was also examined using the CCK8 assay and live cell workstation.Results:1.The experimental results showed that glutamine deprivation significantly inhibited the proliferative activity of bladder cancer T24 cells and activated CD8⁺T cells,but the proliferative activity of activated CD8⁺T cells in the normal culture group was also reduced when they were deprived of continuous stimulation in vitro.2.qPCR and ELISA results showed that the activation or non-activation of CD8⁺T cells influenced their own activity as well as their killing properties.CD⁸⁺T cell activity and killing function were significantly suppressed by glutamine deprivation,while the killing activity of CD8⁺T cells was reduced by the decrease in their own activity.3.In the co-culture group,the expression of the killing factors that activate CD8⁺T cells（GZMB,PRF1）as well as the cytokine IFN-γgradually increases over time,and glutamine deprivation suppresses the expression of their cytokines.The growing T24 cells in the co-culture group continued to stimulate CD8⁺T cells,resulting in a sustained increase in killing factors and cytokines.4.In monoculture,T24 cells expressed mainly PD-L1 and low PD-1,while CD8⁺T cells expressed mainly PD-1 and low PD-L1:both T24 cells and activated CD8⁺T cells expressed the T cell suppressor SHP2.glutamine deprivation caused an upregulation of PD-L1 expression in T24 cells and a downregulation of PD-1 expression in activated CD8⁺T cells.downregulated in T24 cells and CD8⁺T cells,but had no significant effect on the expression of the T cell suppressor SHP2 in T24 cells and CD8⁺T cells.5.bladder cancer T24 cells co-cultured with activated CD8⁺T cells,glutamine deprivation after 12 h resulted in down-regulation of total PD-1,PD-L1 and SHP2expression.the change in SHP2 expression remained consistent with the total PD-1/PD-L1results.Conclusion:This study describes the effect of glutamine in the bladder tumour microenvironment on bladder cancer cell T24 and activated CD8⁺T cells and how glutamine in the microenvironment affects immune escape from bladder cancer through the PD-L1/PD-1mechanism.Glutamine deprivation in the monoculture group induced upregulation of PD-L1 expression in T24 cells and downregulation of PD-1 expression in activated CD8⁺T cells.However,total PD-L1,PD-1 expression levels were down-regulated after glutamine deprivation in the co-culture group,while SHP2 expression was down-regulated.This suggests that glutamine deprivation inhibits PD-L1/PD-1-mediated immune escape from bladder tumours.