Epigenetic Regulation Of CD8~+T Cell Memory Formation By MTORC2/AKT Signaling Axis

Tumor immunotherapy is a novel treatment strategy to fight against tumor cells by harnessing the patient's own immune system.It has been well documented that adoptive transfer of memory T cells provide better efficacy in clinics.Therefore,it is particularly important to understand the molecular mechanisms underlying the generation of memory of CD8~+T cells.The purpose of this study is to explore the effects of inhibition of the m TORC2/AKT pathway on the memory formation of CD8~+T cells,and to explore changes in metabolic regulation and epigenetics.During the course of the experiment,splenocytes of OT-1 mice were activated and proliferated in vitro,and the cells were treated with AKT inhibitor MK-2206(final concentration of 1?M)or DMSO.Detected the expression of surface activation markers and transcription factors by Flow Cytometry after 3d in vitro culture;Cell pellets were collected for ATAC-seq.After 4d in vitro culture,detected the mitochondrial related indicators and expression of cytokines by Flow Cytometry;Cell pellets were collected to extract RNA,nuclear protein and cytoplasmic protein,detected the relative expressions of genes related to memory formation,effector function,and metabolism by Fluorescence quantitative PCR,and detected the expression of proteins related to gluconeogenesis and mitochondrial synthesis by Western Blot.Flow Cytometry showed that MK-2206 treated mouse spleen cells in vitro,inhibited the expression of CD8~+T cell surface activation markers and cytokines,up-regulated the expression of CD62L and Tcf1,and enhanced Mito SOX and TMRE fluorescence signals.Fluorescent Quantitative PCR showed that MK-2206 treatment increased the relative expression of PCK1/Pygl and Pygm genes.ATAC-seq and CHIP-seq showed that MK-2206treatment led to increased accessibility of a large number of genes,and a large number of H3K4me3 rather than H3K27ac modifications appeared in both the experimental group and the control group.Taken together,by inhibiting the m TORC2/AKT pathway,it can play a role in enhancing the memory formation of CD8~+T cells,and thus inhibit the activation and effector functions of CD8~+T cells to some extent.In addition,metabolic and epigenetic test results show that inhibiting the mTORC2/AKT pathway increases mitochondrial membrane potential,ROS levels,and gluconeogenesis levels;CD8~+T cells are accompanied by dramatic changes in H3K4me3 modified chromatin accessibility.Therefore,inhibiting the m TORC2/AKT pathway improves the memory of CD8~+T cells and provides a therapeutic target for adoptive immune cell therapy.Changes in metabolism and epigenetics also better reveal the molecular mechanism of CD8~+T cell memory formation It has certain practical value in the research of tumor immunotherapy.