Studies On Mechanism Of Murine T Lymphocytes Activation By MHC ?-independent Staphylococcus Enterotoxin C2 Mutant

Staphylococcal enterotoxin C2(SEC2),a MHCII-dependent T cell superantigen,causes rapid clonal expansion of lymphocytes and secretion of T cell growth factors,leading to a severe inflammatory response within tissues.Although previous studies have shown that ST-4,a SEC2 mutant with enhanced recognition of V? regions of T-cell receptors(TCRV?),can activate an increased number of T cells and produce more cytokines than SEC2.However,the signaling mechanisms of SEC2/ST-4-mediated immune activation have not been addressed.First,we used specific inhibitors to investigate the functions of PI3K/mTOR and NF-?B signaling pathways in SEC2/ST-4-induced T cell activation.In this study,we found that SEC2/ST-4 could effectively induce splenocytes activation in BALB/c mouse.Meanwhile,we observed significantly upregulated expression of p70S6K,cyclin E,cyclin D3,and NF-KB/p65,but downregulated expression of p27kip during SEC2/ST-4-driven T cells activation.ST-4 intensifies PI3K/mTOR and NF-?B signaling transduction than SEC2.Moreover,we found that IL-2 secretion was positively associated with p65 expression in a time-and dose-dependent manner.The PI3K inhibitor LY294002,mTOR inhibitor rapamycin,and NF-?B inhibitor Bayl1-7085 could suppress SEC2/ST-4-induced proliferation,CD69/CD25 expression,cell-cycle progression,and IL-2 production in BALB/c mouse splenocytes.SEC2/ST-4-induced changes in cell cycle and PI3K/mTOR signaling were significantly relieved by either LY294002 or rapamycin,and the induction of NF-KB/p65 induced was significantly downregulated by Bay11-7085.These seemed that the involvement of PI3K/mTOR and NF-?B signaling pathways in SEC2/ST-4-induced T cell activation.PKC? links the TCR signal to activate the NF-?B for IL-2 secretion through a unique pathway.This study used the specific inhibitor,siRNA,and the blocking antibody to determine the detailed signaling mechanism of NF-KB/p65 and IL-2 induced by SEC2/ST-4 in T cells.We demonstrated that IL-2 secretion in response to either SEC2 or ST-4 stimulation was accompanied by upregulation of PKC?,IKK?/?,I?B?,and NF-?B in mouse splenocytes.PKC? inhibitor AEB071 significantly suppressed SEC2/ST-4-induced T cell proliferation,CD69 and CD25 expression,and IL-2 secretion.Meanwhile,SEC2/ST-4-induced changes in PKC?/NF-?B signaling were significantly relieved by AEB071 in a dose-dependent manner.Using lymphocyte specific tyrosine kinase(Lck)siRNA,we found that Lck controlled SEC2/ST-4-induced phosphorylation of PKC?.Furthermore,we also demonstrated that the IL-2R/STAT5 pathway is essential for SEC2/ST-4-induced T-cell activation by blocking IL-2.These seemed that the involvement of PKC?/NF-?B and IL-2R/STAT5 signaling pathways in SEC2/ST-4-induced T cell activation.The dependence of SEs on MHC ? molecules may be mediated by the interaction between SEs and TCR,suggesting that the affinity between SEs and TCR may affect the dependence of MHC ? molecules.In order to determine whether ST-4 could stimulate T cells activation independent the MHC ?.We chose the CD4+T cells(without MHCII molecular)by immunomagnetic beads separation.Then,we found that compared with SEC2,ST-4 could induce BALB/c mouse CD4+T cells proliferation in the V?8.2 and 8.3 manners in the absence of MHC ? molecules.Furthermore,we demonstrated that only ST-4 could activate CD4+T cells in the absence of MHC ? molecules through the PKC?/NF-?B signaling pathway.The PKC? inhibitor AEB071 significantly suppressed ST-4-induced CD4+T cells proliferation,CD69 and CD25 expression,and IL-2 secretion.Further,ST-4-induced changes in PKC?/NF-?B signaling were significantly relieved by AEB071 in a dose-dependent manner.Using Lck siRNA and blocking antibody,we found that IL-2R/STAT5 pathway was essential for ST-4-induced CD4+T cell activation in the absence of MHC ?.This process was also controlled by Lck.Taken together,our findings demonstrate the involvement of PI3K/mTOR,PKC?/NF-?B,and IL-2R/STAT5 signaling pathways in SEC2/ST-4-induced T cell activation.ST-4 intensifies PI3K/mTOR,PKC?/NF-?B,and IL-2R/STAT5 signaling transduction,ultimately leading to enhance T cell activation.ST-4 intensifies PKC?/NF-?B and IL-2R/STAT5 signaling pathways,ultimately leading to induce CD4+T cell activation in the absence of MHC ?.This study will provide theoretical references for modification and application of SEC2.