Research On The Mechanism That SEA Counteracts The Inhibitory Effection Of Imatinib On TCR Signaling Pathway

Objective:To investigate the effects of superantigen staphylococcal enterotoxins A (SEA) onTCR-signaling by detecting expression of the relevant kinases at mRNA and protein level;To ascertain whether SEA can counteract the immunosuppressive influence on T cellscaused by imatinib and go further to explore the molecular mechanism.Methods:1. T cell lines including both Jurkat and Molt4were stimulated with SEA at a seriesof concentrations for24h respectively, thereafter the early kinases in TCR signalingpathway such as Lck, Fyn and ZAP70were detected by qRT-PCR and Western blotting tostudy their expressions at both mRNA and protein level.2. The Jurkat cells and the primary human T lymphocytes from healthy donors wereselected as research objects. According to whether pretreated with SEA（20ng/mL）for24h or not, cells were divided into two groups. Thereafter, cells were treated withimatinib （40nmol/L）for1h and control groups without imatinib treatment were setted.The four experimental groups were as follows: control group, SEA solely treated group,imatinib solely treated group and imatinib treated following SEA treatment group. Cellsof each group were stimulated with anti-CD3/CD28antibody for24h, then IL-2expression was detected by ELISA; Cells of each group were stimulated withanti-CD3/CD28antibody for15seconds, then the phosphorylations of Lck, ZAP70, LATand PLCγⅠwere examined by Western blotting.Results：1. Jurkat and Molt4cells were stimulated with SEA(10ng/mL) for24h, the results ofboth cell lines were identical at mRNA level. the expression of Lck mRNA wasup-regulated; ZAP70was down-regulated; Fyn was unchanged.2. After stimulation with SEA for24h, the expression of Lck and ZAP70protein wasup-regulated and this effects were dose-dependent.3. Kinases in TCR signaling pathway including Lck, ZAP70, LAT and PLCγ wereactivated by anti-CD3/CD28antibody in both Jurkat cells and primary human Tlymphocytes; Imatinib inhibites the phosphorylations of the kinases above and decreases IL-2production, while SEA attenuates the imatinib-mediated down-regulation of TCRsignaling and IL-2production by inhibiting tyrosine phosphorylation of these kinases inTCR signaling pathway.Conclusion:Superantigen SEA up-regulates the expressions of Lck and ZAP70at mRNA andprotein level; Superantigen SEA antagonize the suppressive effect of imatinib on T-cellactivation by upregulating phosphorylation level of the TCR pathway kinase Lck, ZAP70,LAT and PLCγI. Lck expression level regulates the threshold of activation in T cells. Lckoverexpression decreases the threshold of TCR activation while Lck at low expressionincreases it.