The Mechanism Of MTORC1 Pathway Regulating T Cell Immunobiological Process Of Tilapia

T cells are the main component of the adaptive immune system and play an essential role in resisting the infection of viruses,bacteria and parasites and tumorigenesis.Mature T cells are divided into CD8⁺cytotoxic T cells and CD4⁺helper T cells.Once encountering antigen,CD8⁺T cells activate,proliferate and differentiate to perform immune function.CD8⁺T cells differentiate into effector or memory CD8⁺T cells,which mediate target cell killing,infection clearance and provide long-term immune protection;CD4⁺T cells are heterogeneous cell groups,which can differentiate into a variety of helper T cells such as Th1,Th2 and Th17.By secreting different cytokines,CD4⁺T cells can recruit and activate other immune cells and enhance their immune activity,so as to coordinate a variety of immune responses,protect the body from pathogenic bacteria,and play a key role in adaptive immune response.However,our theory of T-cell immunity is limited to mammalian models,and it is little known about the process and regulation mechanism of T-cell-mediated adaptive immunity in lower vertebrates.Mammalian/mechanistic target of rapamycin（m TOR）pathway is an important signal pathway,which promotes cell survival,growth,proliferation and differentiation.Taking Nile tilapia as the model,this study investigated the regulatory mechanism of m TORC1 pathway on the biological process of fish T cell immunity,which is of great significance to explore the origin and evolution of adaptive immunity and reveal the nature of immune defense in living beings.Tilapia encodes complete m TORC1 pathway elements.Bioinformatics analysis found that the tertiary structure of each element is highly conserved compared with mammals,suggesting that fish m TORC1 pathway may perform similar functions to mammals.In the adaptive immune stage of tilapia infected with Streptococcus agalactis,there was obvious inflammatory infiltration in the liver,accompanied by the increase of total number of spleen lymphocytes and the proportion of T cells in peripheral blood,indicating that T cells participated in the adaptive immune response of tilapia.Meanwhile,the m RNA,total protein and phosphorylation levels of m TORC1 pathway components in spleen lymphocytes were significantly up-regulated,indicating that m TORC1 pathway may regulate tilapia lymphocyte mediated adaptive immune response.The phosphorylation level of various components of m TORC1 pathway increased significantly after activation of lymphocytes in vitro;After blocking m TORC1 pathway with rapamycin,it was found that the phosphorylation levels of S6and 4EBP1 decreased significantly,and T cells activated induced the expression of IFN-γwas also significantly inhibited,indicating that m TORC1 pathway regulates the activation of tilapia T cells through phosphorylation.In the primary immune response against S.agalactiae infection,the proportion of T cells in peripheral blood decreased significantly after blocked the m TORC1 pathway,indicating that m TORC1 pathway regulated the proliferation of tilapia T cells,which was further confirmed by Brd U incorporation experiment in vivo and CFSE labeling analysis in vitro.In addition,the inhibition of m TORC1 pathway reduces the expression of pro-inflammatory cytokines,cytotoxic-related genes and pro-apoptotic genes in lymphocytes,impairs the ability of inflammatory infiltration and infection clearance,and ultimately aggravated the death of tilapia,indicating that m TORC1 pathway regulates the effector function of tilapia T cells.During the activation of tilapia T cells,the m RNA levels of glycolysis,glutamine and lipid synthesis related genes were significantly up-regulated,indicating that the activation of fish T cells was accompanied by multiple metabolic reprogramming.After blocking m TORC1 pathway,the uptake of glucose by lymphocytes was significantly inhibited;Meanwhile,the m RNA levels of genes related to glycolysis,glutamine and lipid synthesis were down-regulated,and the activities of key rate limiting enzymes of glycolysis and glutamine were weakened,indicating that m TORC1 pathway regulates tilapia T-cell immunity through metabolic reprogramming.Mechanistically,the inhibition of m TORC1 pathway down-regulated the levels of the key transcription factors c-myc and HIF-1αand SREBP of glycolysis,glutamine and lipid synthesis,indicating that m TORC1 pathway regulates the metabolic reprogramming of tilapia by regulating transcription factors.Therefore,the above results show that m TORC1pathway promotes the activation,proliferation and adaptive immune response of tilapia T cells by coupling immune signals and metabolic reprogramming.Using anti-CD3,CD4-1 and IFN-γ,we found that tilapia had already evolved IFN-γ-producing CD4⁺T cells,which revealed that the differentiation of Th cells originated from the bony fish of lower vertebrates 450 million years ago.During the activation of tilapia T cells,the m RNA levels of T-bet,STAT1 and STAT4,the key transcription factors of Th1 cell differentiation,were significantly up-regulated,accompanied by the phosphorylation of STAT1.Overexpression of T-bet or STAT1 in 293T cells can promote the expression of IFN-γ;Meanwhile,T-bet significantly enhanced activity of tilapia IFN-γpromoter;The activity of STAT1 was specifically inhibited,and the transcription level of T-bet was significantly down-regulated,indicating that tilapia regulates the transcription of IFN-γthrough STAT1-T-bet axis.In addition,cytokines IL-2,IL-12 and IL-27 induced the high expression of T-bet,STAT1,STAT4 and IL-12Rβ2,which jointly determines the fate of Na（?）ve CD4⁺T cells differentiate into Th1cells in tilapia.At the same time,IFN-γbinds to its receptors,and promotes the expression of T-bet,STAT1 and STAT4,as well as regulating the phosphorylation of STAT1,which forms the positive feedback loop through IFN-γ-STAT1-T-bet-IFN-γto enhance the commitment of tilapia Th1 cells.During the adaptive immune stage of Edwardsiella piscicida infection,the percentage and absolute number of IFN-γ-producing CD4⁺T cells increased significantly.IFN-γadministration professionally activated tilapia macrophages,and enhanced their phagocytosis,which promoted elimination of the infection and improved the survival rate of tilapia,indicating that early vertebrate has used differentiated Th1 cells secreting cytokine IFN-γto resist intracellular bacterial infections.After inhibiting m TORC1 pathway,the ability of CD4⁺T cells to produce IFN-γwere obviously impaired,indicating that m TORC1 pathway regulates the differentiation of teleost Th1 cells.Mechanistically,blockaed of m TORC1 signaling impaired the expression of the key transcription factors T-bet,STAT1,STAT4 and T cell surface receptor IL-12Rβ2,and inhibited the activation and proliferation of CD4⁺T cells,indicating that tilapia utilized m TORC1 signaling to ensure Th1 cell development,by modulating cellular activation and proliferation and the expression of the key transcription factors.In summary,the study took Nile tilapia as a model,we investigated the mechanisms of m TORC1 pathway regulating T cell activation,proliferation,differentiation and anti-infective immunity.The results not only enrich the regulatory mechanism of T cell immunity in early vertebrates,but also provide a new perspective and evidence for understanding the origin and evolution of adaptive immunity.