In contrast to other SOCS family molecule, little is known about the role of SOCS4 in the immune system. However, we found that SOCS4 expression is donwregulated in mature T cells compared to DP thymocytes, and as such we wished to know whether such specific expression was necessary to maintain normal thymopoiesis and T cell homeostasis. To this end, we generated SOCS4 transgenic mice that expressed a mouse SOCS4 cDNA in all T lineage cells. By quantitative RT-PCR and Western blot analysis, we identified three different lines of SOCS4 transgenes which expressed graded levels of transgenic SOCS4 expression. Interestingly, we found a significant decrease in absolute thymocyte numbers in linear correlation to transgenic SOCS4 expression. These results suggested that constitutive expression of SOCS4 partially inhibited T cell development. Detailed analysis of thymocytes subpopulations further revealed a developmental block at the DN3 stage, which probably was caused by a decrease in DN thymocytes proliferation and resulting in reduction of total thymocytes numbers. IL-7 signaling is a major factor necessary for DN thymocytes survival and proliferation, and we considered the possibility that SOCS4 interferes with IL-7 signaling. Surprisingly however IL-7 signaling was normal in SOCS4 Tg T cells as assessed by STAT5 phosphorylation upon IL-7 stimulation. Next to test the possibility that other STAT signaling pathways than STAT5 were affected, we assessed IL-6-induced STAT3 and IL-4-induced STAT6 phosphorylation in SOCS4 Tg T cells. Curiously, none of these cytokine signaling were affected by SOCS4 overexpression indicating that SOCS4 interferes with other signaling pathways. To test whether transgenic SOCS4 can affect TCR signaling, next, we assessed Erk phophorylation upon short term TCR crosslinking and we found that indeed SCSO4 interfered with TCR signaling. We are currently testing this idea using MHCI-restricted TCRS transgenes and we are also investigating the potential molecular mechanism underlying this observation. Collectively, we confirmed that active modulation of SOCS4 expression is necessary for T cell differentiation and maintenance, and preliminary data from suggest a role of SOCS4 in TCR signaling instead of cytokine signaling. Additionally, we have been focusing on two other SOCS family molecules, namely SOCS3 and Cish. We have generated both SOCS3 and Cish transgenic mice and have analyzed T cell development and T cell function in these mice with the result that SOCS3 affected CD8 cell development and acted synergistically with SOCS1 to suppress cytokine signaling whereas Cish did not show any prominent phenotype. Still, we found that SOCS1 and SOCS3 have partially overlapping but also non-redundant functions in T cells, and we are currently investigating their roles during T cell activation in a series of inflammatory and autoimmune mouse models. In case of Cish, we were initially very interested because Cish expression is induced upon TCR stimulation and because Cish is known to suppress IL-7 signaling by competing with STAT5. Curiously, however, we did not observe any effect of transgenic Cish on cytokine signaling even as we confirmed its correct overexpression in lymph node T cells by Western blot analysis. We are currently considering the possibility that Cish acts analogous to SOCS4 on TCR signaling rather than on cytokine signaling, and experiments addressing this possibility are under progress.
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