Mechanism Exploration Of LAT-involved TCR Signaling In Regulation Peripheral T Cells Activation And Differentiation

Linker for activation of T cell (LAT), the transmembrane proteinwhich plays a key role in the early stage of TCR signaling transductionafter the ligation of TCR and MHC-peptide complex, is one of the mostimportant substrates of the Syk family protein tyrosine kinase likeZAP-70. Being activated by ZAP-70, phosphorylated intracellulartyrosine residues of LAT provide docking sites for molecules like Grb2,PLC-γ1and Gads. When recruited and activated by LAT, PLC-γ1canhydrolyze PIP2into IP3and DAG thus initiating the mobilization ofcalcium influx and activation of Ras-MAPK pathway, while Gadsinvolves in the cell skeleton reconstitution and integrin activation afterbinding to SLP-76. LAT can also activate Ras-MAPK pathway bybinding and recruiting Grb2-SOS to the cell membrane. All thosemolecules recruited directly or indirectly to LAT form the complexnamed as “LAT signalsome”, which converts the extracellular signalrecognized by TCR into the intracellular signaling pathways, which plays important roles on the development, maturation, proliferation, activationand differentiation of T cells.It has been proved that LAT plays critical roles on T celldevelopment and T cell activation by using lat gene knocking down miceor lat-/-T cells. However, results from LATY136Fmice and mice with latconditional knock out in the peripheral revealed that there exhibits theimpairment of TCR down-stream signaling and abnormal T cellproliferation and differentiation, which indicates that LAT moleculemight function differently in peripheral mature T cells. Based on theseprevious findings, we suggest new mechanisms that LAT regulates T cellin mature T cells in periphery.In our study, LAT specific siRNA was transfected into T cell viaAmaxa transfection system in vitro. With this in vitro knocking downstrategy, LAT-/-primary T cells and T cell lines are obtained with highefficacy and specificity for further study. We first investigated theproliferation and cytokine secretion of LAT-/-T cell upon TCR/CD28stimulation. Our results indicated that T cell displayed no differences inproliferation but higher IL-2secretion capacity compared with the controlgroup. Both the mRNA level and cytokine secretion of IFN-decreaseswhereas IL-4and GATA3mRNA levels increased, suggesting theTh2-like polarization of T cells upon TCR/CD28stimulation with latdeficiency. In order to figure out the molecular mechanisms involved in thealteration of cytokine secretion in T cells with LAT knock down, wefurther analyzed the phosphorylation of key molecules in TCR signalingpathways. The results showed that when LAT deficient primary T cellwas stimulated by TCR/CD28, the phosphorylation of Lck which locatesupstream of LAT stayed intact whereas the phosphorylation ofdownstream protein PLC-γ1dramatically decreased leading to thediminished calcium influx. Meanwhile, the activation of Erk1/2was alsosignificantly down-regulated. More interstingly, we found that during theactivation of LAT deficient T cell, the lipid molecule PIP3accumulatedin the cytosol. As PIP3is an important messenger in the activation ofPI3K/AKT pathway, we also observed the over-activation of PI3K/AKTand prolonged phosphorylation of NF-B.Based on all the findings mentioned above, we imply that inperipheral T cells LAT might function as a regulator of co-stimulatorypathways like CD28pathway, in which PIP2is the key hub molecule insignal switching between TCR pathway and co-stimulator pathway. In Tcell with normal expression of LAT, signaling from TCR/CD28canhydrolyze PIP2into IP3and DAG conducting the normal activationsignals while under the circumstance of LAT deficiency PIP2convertsinto PIP3resulting in the enhancement of PI3K/AKT activation mediatedby CD28involvement, thus modifies the T cell functionality via bypass activation. Our findings, for the first time, proved the new regulatorymodel of LAT molecule in peripheral mature T cells and provided directevidence to how LAT-involved TCR signaling regulates co-stimulatorypathways.