Molecular Mechanism Of TGF-β1 Negative Regulation Of T Cell Immunity In Tilapia

T cells are a key weapon in adaptive immunity,playing an integral role in tumor surveillance and elimination of infection,and these cells can also employ complex mechanisms to avoid an overactive immune response,thereby ensuring that the organism is protected from inflammatory damage.Transforming growth factor-β1(TGF-β1)is an important anti-inflammatory cytokine,which negatively regulates T cell immunity through a variety of ways to maintain immune homeostasis.Fish are the vertebrates that most evolved T cell adaptive immunity.Many studies have elucidated the regulatory mechanism of T cell effector function in teleost,but the negative regulation of T cell immunity in early vertebrates is still not fully understood.In this study,we using a Nile tilapia(Oreochromis niloticus)model,investigated the negative regulatory role of TGF-β1 in adaptive T cell immunity and analyzed the regulatory mechanism.Nile tilapia encodes an evolutionarily conserved form of TGF-β1,a cytokine that is widely expressed in lymphoid tissues or organs.TGF-β1 m RNA and protein expression in lymphocytes were significantly induced during both adaptive immunity by Edwardsiella piscicida infection and T cell activation,suggesting that TGF-β1 is involved in T cell-mediated adaptive immunity in tilapia.In vitro stimulation of lymphocytes with TGF-β1 not only down-regulated the expression of pro-inflammatory cytokines such as TNF-αand IL-2,but also inhibited the transcription of T cell activation markers CD122,IL-2,IFN-γand the phosphorylation of AKT,Erk,S6,indicating that TGF-β1 limited the activation of T cells from tilapia.Administration of TGF-β1 during E.piscicida infection limits the activation,proliferation,survival,and cytotoxic functions of T cells and impairs the body’s ability to clear pathogenic bacteria.In addition,TGF-β1 inhibited phytohaemagglutinin(PHA)-induced transcription of the key transcription factor T-bet and GATA3 of Th1 and Th2 cells,suggesting that TGF-β1 may limit Th1 and Th2 cell differentiation.These results indicate that TGF-β1negatively regulates T cell-mediated adaptive immunity in Nile tilapia.TGF-β1 could bind to TGF-βR1 and TGF-βR2 in vitro,and bind to Nile tilapia CD3~+T cells,especially CD3~+CD4~+T cells,indicating that TGF-β1 could transmit signals through TGF-βR complex on T cells.Treatment of lymphocytes with TGF-β1upregulated transcription of key elements of the TGF-βR/Smad axis and phosphorylation of Smad2 and Smad3,which was blocked by specific TGF-βR inhibitors.In addition,T cell activation also significantly induced the transcription of key components of the TGF-βR/Smad axis and the phosphorylation of Smad2 and Smad3,indicating that the TGF-βR/Smad axis is a pathway for TGF-β1 signaling and this axis is involved in the T cell immunity of Nile tilapia.T cells once activated,Smad2 and Smad3 are rapidly transported to the nucleus,and Smad3 interacts with transcription partners NFAT1,T-bet and Foxp3 to form a transcriptional regulatory complex.Moreover,Smad3 overexpression decreased the promoter activities of pro-inflammatory cytokines IL-2 and IFN-γand enhanced the inhibitory factors CTLA4 and Foxp3,suggestting that TGF-β1 could negatively regulate T cell immunity by using Smad3 to interact with transcription factors or directly regulate the expression of effector genes.TGF-β1 stimulation significantly induced Nile tilapia Foxp3 transcriptional expression.Foxp3 as a homodimer bind to T-bet or directly regulate the prduction of target genes,including reducing IL-2 and IFN-γand enhancing TGF-β1,IL-10 and Foxp3 promoter activities.Therefore,TGF-β1 can induce the expression of Foxp3 to negatively regulate T cell immunity.These results suggest that TGF-β1 can directly or indirectly regulate the expression of target genes by triggering Smad3 to enter the nucleus and induce the expression of Foxp3,thereby negatively regulate T cell-mediated adaptive immunity.In summary,TGF-β1 negatively regulates T cell adaptive immunity by restricting the transcription of effector genes by activating the TGF-βR/Smad signaling pathway,and further uses the transcription factor Foxp3 to achieve a cascade regulation of target gene expression.This study systematically explored the detailed mechanism of TGF-β1 negative regulation of T cell immunity in Nile tilapia,and provided new scientific evidence for the mechanism of T cell immune tolerance and the evolution of adaptive immune system in teleost.