Costimulatory Effect And Mechanism Of OCILRP2on Mouse T Cell Activation

BackgroundT lymphocytes mainly mediate cellular immune response in vivo, the entire response process isdivided into three stages: T cell-specific antigen recognition stage, T cell activation, proliferation, anddifferentiation stage, the effector phase of T cell responses. T cell costimulatory plays an important role inT cell activation, proliferation, differentiation, and exsistence. Interactions between receptors and ligandson the surface of effector lymphocyte are involved in costimulation. The gene of Osteoclast inhibit lectinrelated protein2was originally isolated from osteoblasts, it is highly expressed in spleen, thymus, Blymphocyte, DC and activated T cells,and the expression of OCILRP2in resting T cells is low. Reaserchshowed that C type lectin receptor molecule OCILRP2is an important activating receptor in murine T cellsactivation, the ligand of it is NKRP1f.OCILRP2is a C-type lectin-like receptor molecule.C type lectin receptor molecules play an importantrole in innate and acquired immune response by binding to its ligand. Its family member NKG2D activatesNK cells and CD8+T cells by combining with different adaptor protein for it lacks of cytoplasm signaltransduction motif.The intracellular segment of OCILRP2protein is very short, also lack of cytoplasmsignal transduction motif, it can not transmit signal.So we speculated that OCILRP2may be similar to theNKG2D and it activates downstream signaling pathways through interactions with its transmembraneadapter protein DAP12. Research showned that Lck of tyrosine phosphorylation is reduced in OCILRP2-silenced T cells, and tyrosine phosphorylation of PI3-K in activated T cells is invariant.However, the exactcostimulatory mechanism of OCILRP2on mouse T cell activation and relevant signal transduction pathwayare not clear.ObjectiveIn order to provide new clues for understanding the molecular mechanism of T cell activation, weresearch costimulatory effect and its mechanism of OCILRP2in the process of mouse T cells activation,Identify the adaptor which interacts with OCILRP2,explore the signal pathway mediated by OCILRP2in Tcell activation, enrich theory of T cell activation. MethodsCo-immunoprecipitation, GST pull down and CLSM experiments are used to identify the adaptorwhich interacts with OCILRP2.To analyze the interactive area of OCILRP2with its adaptor proteinmolecular we constructed the full-length, intracellular segment, extracellular and transmembrane domain ofrecombinant OCILRP2protein. Using CD3/CD28antibodies and PMA stimulated mouse T lymphoma EL4cells, we detedted the secretion of cytokines, cell proliferation, apoptosis, cell cycle and some otherbiological characteristics before and after stimulation. Fluorescence quantitative PCR is used to detectchanges of certain transcription factors, at the same time immunoblotting technique is used to find signaltransduction pathways associated with OCILRP2.ResultsFusion protein of GST-DAP12and GST-OCILRP2i and GST-OCILRP2t+e are successfullyconstructed.The adaptor protein molecular which interacts with OCILRP2is DAP12. GST-OCILRP2t+erecombinant protein can interact with DAP12protein, and DAP12can not interact with Ras.Confocal laser scanning results showed colocalization of OCILRP2and DAP12in the cell membraneof EL4.OCILRP2is mainly expressed in the cytoplasm in resting EL4cell line and it transported fromintracellular to the cell surface in activated EL4cells which were stimulated by CD3/CD28antibodies, andPMA treated EL4cells showed no such translocation phenomenon. The membrane protein expression ofDAP12didn’t change before and after stimulation.The secretion of IL-2level is very low in EL4cells without any stimulation and increases significantlyafter stimulated by CD3/CD28antibodies. With the increasing concentration of CD3antibody and stimulusduration, the secretion level of IL-2is also increased.So we have found the the optimal concentration ofCD3/CD28antibodies and stimulus duration to stimulate EL4cells. Different groups of EL4cells werestimulated of48h, we found that the secretion level of IL-2was decreased in the group which was addedOCILRP2antibody. Increasing concentration of CD3/CD28antibodies has a promoting effect on theproliferation of EL4cells.Proliferation of OCILRP2antibody group and silent group are slower than that ofCD3/CD28antibodies group. But the addition of exogenous IL-2antibody did not affect the proliferationof EL4cells.OCILRP2does not affect the cell cycle of EL4cells. Anti-OCILRP2antibody and si-OCILRP2plasmid were able to decrease the apoptosis of EL4cells.CD3/CD28antibodies could significantly promote mRNA synthesis of IL-2, NF-κB, NF-AT,MAPK3, MAPK8. Activation of MAPK3and MAPK8is reduced by OCILRP2-silenced.ConclusionsOCILRP2interacts with DAP12. They provide costimulatory signal for T cell activation throughMAPK pathway.