Molecular Mechanism Of Glutamine Metabolism Affecting T-Lymphocyte-Mediated Immune Effects Against Colorectal Cancer

Objective:Glutamine is an important source of energy for the immune system and is involved in lymphocyte proliferation,which can maintain cell viability and has an important immunomodulatory role.Tumor cells that overproliferated in the tumor microenvironment consume large amounts of glutamine,and for the competition of glutamine between T lymphocytes and tumor cells,the competitive ability of T lymphocytes is often insufficient,resulting in an insufficient ability of T lymphocytes to kill tumor cells.We consider that if we intervene in the major molecules of the signaling pathway associated with glutamine metabolism in T lymphocytes,we will be able to promote the competitiveness of T lymphocytes in the tumor microenvironment,thus enhancing their anti-tumor immune effects and preventing tumor immune escape.Methods:In this study,we targeted the T lymphocyte line JURKAT cells to investigate the mechanisms involved in glutamine metabolism in T lymphocytes and to search for new targets to enhance their anti-tumor immune effects.Firstly,we intervened JURKAT cells by PI3K,AKT,PDK1,MEK,and mTOR inhibitors respectively,and detected the expression of related molecules by WB method;next,we treated the cells with inhibitors and activators in normal and low glutamine environments to explore their effects on cell proliferation and apoptosis;further,we investigated the effects of mTOR molecules on JURKAT,PBMC,and TALL-104 cells cytokine secretion and cell activation;on this basis,we performed in vitro HCT116 colorectal cancer cell killing assay with TALL-104 and PBMC cells;finally,in animal experiments,we used mTOR activator intervention in TALL-104 cells to treat tumor-bearing nude mice and examined the rate of tumor proliferation,tumor volume,tumor weight.Results:1.PI3K,AKT,and mTOR inhibitor intervention resulted in downregulation of glutamate-pyruvate transaminase 2(GPT2),which in turn inhibited glutamine metabolism,whereas PDK1 and MEK molecule inhibition failed to downregulate GPT2 expression,with statistically significant differences(P<0.01 or P<0.001).2.The pathways and mechanisms involved in glutamine metabolism were different between JURKAT cells and HCT116 cells,and the differences were statistically significant(P<0.01 or P<0.001).3.By interfering with the signaling pathway through inhibitors,mTOR inhibitors can inhibit JURKAT cell proliferation and promote apoptosis in a low glutamine environment,and the difference is statistically significant(P<0.05,P<0.01 or P<0.01).4.By interfering with the signaling pathway through mTOR activator,mTOR activator could promote JURKAT cell proliferation and inhibit apoptosis under a low glutamine environment,and the difference was statistically significant(P<0.05).5.The protein expression of ATF4 and GPT2 was upregulated by mTOR activator intervention in the signaling pathway,and the difference was statistically significant(P<0.001).6.CD69,IL-2,and IL-4 expressions were upregulated in JURKAT and PBMC cells after mTOR activator intervention,with statistically significant differences(P<0.01 or P<0.001).7.CD8 and CD69 expression were upregulated in TALL-104 and PBMC cells after mTOR activator intervention,and the difference was statistically significant(P<0.01 or P<0.001).8.mTOR-activated JURKAT cells were more competitive for glutamine than HCT116cells,and the difference was statistically significant(P<0.05 or P<0.01).9.In in vitro experiments,mTOR activation enhanced the ability of PBMC and TALL-104to kill tumor cells,and the difference was statistically significant(P<0.05 or P<0.01).10.In the in vivo experiments,the mTOR activator intervention group showed a significant decrease in the rate of tumor proliferation,tumor volume,and tumor weight compared with the non-intervention group of nude mice,and the difference was statistically significant(P<0.05 or P<0.01).Conclusions:The above results suggest that PI3K,AKT,and mTOR inhibition then reduced the level of glutamine metabolism by inhibiting GPT2 expression;combined with past studies,we found that MEK and PDK1 molecules affected glutamine metabolism differently in HCT116 cells and JURKAT cells,i.e.,MEK and PDK1 in HCT116 cells could interfere with glutamine metabolism by affecting We also found that the activation of PI3K and AKT did not significantly affect the proliferation and apoptosis of JURKAT cells,and only when mTOR was activated did it significantly promote the proliferation of JURKAT cells in a low glutamine environment.The occurrence of apoptosis was also inhibited;next,we used mTOR activator to intervene in T lymphocytes,which promoted the anti-tumor immune activity of T lymphocytes(TALL-104,a type of CD8~+T lymphocytes)both in vivo and in vitro experiments.In conclusion,mTOR in T lymphocytes can affect glutamine metabolism while promoting T lymphocyte proliferation and activation,which in turn enhances the anti-tumor effects of T lymphocytes.Targeting T lymphocyte mTOR molecules can induce stronger antitumor immune effects,which provides new strategies and approaches for antitumor immunotherapy.