Study On The Regulatory Mechanisms Of Treg Cell Differentiation And Function By Transcription Factor EB(TFEB)

Background:Regulatory T cells（Treg cells）are essential for self-tolerance but detrimental for dampening anti-tumor immunity.The dysfunction of Treg differentiation and function can lead to a variety of autoimmune diseases.Tumor microenvironment is characterized as nutrient deprivation（glucose and amino acids）,hypoxia,acidification,and accumulation of specific metabolites（lactate and polyamines）,which suppress effector T cell survival and function but facilitate accumulation of Treg cells.Treg cells response to such environmental cues and initiate metabolically reprogrammed to promote their own survival and function.Therefore,an in-depth understanding of Treg sensing environment cues and its regulatory mechanisms is a prerequisite for development of Treg-targeting tumor immunotherapy strategies.The transcription factor EB（TFEB）belongs to Mi T/TFE family,which can transcriptionally control ysosome,mitochondria,glucose metabolism,and autophagy-related genes expression,thus modulate survival and functional fitness of cells under environmental and cellular stresses.The phosphorylation status of TFEB controlled by m TORC1 is indispensable for its transcriptional activity.m TORC1 phosphorylates the serine residues S211 of TFEB protein,promoting its binding with 14-3-3 and leading to inhibition of TFEB nuclear localization.Under starvation condition in mice,the expression of TFEB increased in many tissues,especially in liver cells,and promoted the expression of lipid metabolism and catabolism genes.Recently several studies shined a light on the regulatory effect of TFEB in immune cell function.TFEB can regulate the secretion of inflammatory cytokines of macrophages and promote the clearance of pathogens.TFEB also regulate the antigen presentation of dendritic cells and the immune senescence of B lymphocytes.However,the function of TFEB in regulating T cell differentiation and function is largely unknown.Our previous study found that starvation treatment can inhibit the activity of m TORC1 in mouse spleen CD4⁺T cells,up-regulate the expression of TFEB,and significantly increase the percentage of Treg cells in peripheral,suggesting that TFEB may regulate the differentiation and function of Treg cells.Objective:This research aims to clarify whether TFEB regulates the differentiation and function of CD4⁺T cells,especially the differentiation and function of Treg cells and related mechanisms.Methods:In vitro CD4⁺Treg cell differentiation,In vitro Treg suppression assay,RNA-Sequence,mitochondrial morphology and function detection,T cell adoptive transfer coltis,mice bone marrow chimeric model and tumor model.Results:1.TCR and IL-2 can induce the expression of TFEB in murine CD4⁺T cells in a dose-dependent manner.TCR-induced TFEB expression is dependent on the production of autocrine IL-2.IL-2 regulates TFEB expression in an m TORC1-independent pathway.2.The differentiation of Treg in Cd4^CreTcfeb^fl/flmice was significantly inhibited.The total numbers of splenocytes and lymph node cells,as well as the percentages of CD44^highCD62L^lowactivated CD4⁺and CD8⁺T cells,were higher in the spleen and p LNs isolated from Cd4^CreTcfeb^fl/flmice3.The inhibitory function of Foxp3^YFP-CreTcfeb^fl/flTreg cells was significantly impaired in vitro and in the T cell adoptive transfer coltis model,TFEB depletion in Treg cells lead to enhanced anti-tumor immunity.4.The bone marrow chimeras showed that there was significantly reduced percentages of TFEB-deficient Foxp3⁺Treg cells,compared to WT Treg cells in periphery lymphoid tissues and colonic lamina propria,the expression of effector molecules was reduced in TFEB-deficient Treg cells,which indicating TFEB endogenously regulates Treg generation and function.5.TFEB knockout did not affect autophagy and lysosomal gene expression in Treg cells,and TFEB did not regulate Treg differentiation through the autophagy-lysosomal pathway.6.TFEB deletion of Treg cells lead to impaired mitochondrial integrity and function.Overexpression of C-Myc in TFEB-deficient CD4⁺T cells can partially reverse the defect in Treg differentiation.Conclusions:We found that diverse Treg-related stimuli（Starvation）impinge on and activate TFEB,and deletion of Tcfeb inhibited Treg differentiation and function both in vitro and in vivo.Deficiency of TFEB led to reduced expression of Foxp3,as well as genes associated with Treg differentiation and function.By contrast,proinflammatory cytokines and chemokines were upregulated.Loss of TFEB resulted in impaired mitochondrial integrity and reduced levels of oxidative phosphorylation,and retrvirally C-Myc overexpression reversed the deficience of Treg differentiation and function in TFEB-deficient CD4⁺T cells,suggesting that TFEB can promote C-Myc expression to regulate cellular metabolic homeostasis and mitochondrial function in Treg cells.