Effect Of Low-glucose Microenvironment On PD-L1/PD-1 Mediated Immune Escape In Bladder Tumors

Objective:Metabolic reprogramming is one hallmarks of bladder cancer(BCa),especially reprogrammed glucose metabolism of which plays a key role in the development and progression of BCa.Low glucose is an important feature of tumor microenvironment,but its effect on PD-L1 and the mechanism of PD-L1 mediated immune escape in tumor cells has not been fully elucidated.Therefore,we developed a co-culture system of bladder tumor cells and CD8+T cells to imitating tumor low-glucose microenvironment.Based on the system,this study aims to explore the correlation between glucose metabolism and PD-1/PD-L1 mediated immune escape pathway and its possible molecular mechanism.Methods:Firstly,CD8+T cells were extracted from fresh human peripheral blood by density gradient centrifugation and magnetic bead selection,and the purity of the extracted cells was determined by flow cytometry.Based on this,a direct co-culture system of T24 bladder cancer cells and CD8+T cells was developed.We set up T24 group,T24 co-cultured with CD8+T cell group and CD8+T cell group,and they were cultured in normal 1640 medium(2000mg/L)and low glucose medium(500mg/L)respectively.The tests were carried out as follows:1.The lactate in the cell supernatant were measured by the lactic acid kit,which can reflect the glycolysis level to some extent.2.The proliferation ability of T24 in the control group and the low-glucose group was detected by the living cell workstation to determine whether low glucose could inhibit the proliferation ability of T24 cells.3.Western Blot(WB)and qRT-PCR were used to detect the expression of PD-L1 and PD-lat different time points induced by low glucose in T24 group,T24 co-cultured with CD8+T group and CD8+T group,respectively,as well as protein in EGFR/ERK/c-Jun pathway after co-cultured for 48h.4.Finally,the IFN-y and GZMB secreted by T cells in the co-culture group and CD8 group were detected by ELISA kit to explore the effect of low glucose on the killing ability of T cells.Results:1.From flesh human peripheral blood,CD8+T cells were extracted successfully and then identified them by flow cytometry.Compared with the Isotype,the proportion of CD8-positive cells in extracted cells was as high as 99%,which proved that the extracted cells have good purity.2.The images and data analysis from the living cell station showed that the proliferation of tumor cells was inhibited under low glucose condition in both the T24 and co-culture groups,meanwhile CD8+T cells also inhibited the proliferation of tumor cells.In both the T24 and co-culture groups,significant reduction in lactate was showed in low glucose group,while the lactate production in CD8+T cells group was not affected,which indicated that low glucose mainly affected the glycolysis level of tumor cells.3.PD-L1 was mainly expressed by T24 cells while PD-1 was mainly expressed by CD8+T cells.In co-culture group,WB showed that the expression of PD-L1 in the control group and the low glucose group developed an upward trend and the level of immune escape increased continuously over time.This indicated that although tumor proliferation was inhibited under co-culture condition,PD-L1/PD-1-mediated immune escape was significantly activated,and this activation was not affected in the low-glucose group at 24h.Only when glucose concentration was extremely reduced after continuous consumption at 48h,PD-L1 expression was slightly decreased compared with the normal group,which is still in the high escape activation state.In addition,the specific regulatory mechanism was analyzed by WB,and we found that after cultured for 48h,low glucose could activate the EGFR/ERK/c-Jun pathway to a certain extent and simultaneously up-regulate PD-L1 in the T24 group.However,after co-culture with CD8+T for 48h,PD-L1 in both normal and low-glucose groups increased while EGFR/ERK/c-Jun was significantly inhibited.Although PD-L1 and EGFR/ERK/c-Jun pathway in low-glucose group were lower than those in normal co-culture group,PD-L1 in low-glucose group was still much higher than that in low-glucose T24 group.The above results indicate that the tumor growth-related pathways can be significantly inhibited under the induction of T cells,but even in the low-glucose microenvironment,PD-L1/PD-1-mediated immune escape can still be highly activated.4.By detecting the killer cytokines secreted by CD8+T cell,no significant change in IFN-y level was found between the low-glucose group and the control group,while the GZMB was decreased in the low-glucose group,indicating that low glucose could inhibit the killer function of CD8+T cells to some extent.Although after co-cultured for 24h,IFN-γ and GZMB was slightly increased in the low-glucose group compared with the control group,IFN-y and GZMB were inhibited to extremely low levels at 48 hours,and no significant difference was found between the two groups.These results indicated that the killer function of CD8+T cells gradually decreased over time,which may be owing to the activation of immune escape caused by the high expression of PD-L1 after co-culture.Conclusion:In our study,the effect of low glucose on PD-L1/PD-1-mediated immune escape was explored under co-culture conditions.In co-culture group,PD-L1 is mainly expressed by T24 while PD-1 is mainly expressed by CD8+T cells CD8+T cells are the main factor of inhibiting cancer cell proliferation and enhancing immune escape,while bladder tumor cells can maintain high activation of PD-L1/PD-1-mediated immune escape under low glucose microenvironment.At the same time,the killer function of CD8+T cells greatly inhibited after co-culture,which may be due to the activation of downstream inhibitory pathways by PD-1 after recognizing PD-L1.Our study preliminarily clarified that for immune-induced tumor cells,low glucose could significantly upregulate PD-L1 while the proliferation ability of tumor cells was greatly inhibited,and then the function of immune cells was reduced,thus mediating the immune escape of tumor.