Because SOCS4 expression was found to be upregulated during DN to DP transition but then downregulated upon positive selection, we wanted to assess whether such stage-specific expression during T cell development was necessary to maintain normal thymopoiesis. To this end, we generated SOCS4 transgenic mice that expressed a mouse SOCS4 cDNA under the control of the human CD2 mini-cassette. By quantitative RT-PCR in purified T cells, we identified three different lines of SOCS4 transgenes with graded levels of transgenic SOCS4 expression. Parallel to transgenic SOCS4 expression levels, we observed a significant decrease in absolute thymocyte numbers which suggested that constitutive overexpression of SOCS4 disturbed T cell development. Further analysis of SOCS4 Tg thymocytes revealed a developmental block in the DN3 to DN4 stage that was also confirmed by increased CD25 expression on DP thymocytes. Such a defect suggested either a potential problem in pre-TCR signaling or in IL-7 signaling in DN thymocytes. IL-7 signaling, however, was normal in SOCS4 Tg T cells as assessed by STAT5 phosphorylation upon IL-7 stimulation. To test the possibility that other STAT signaling pathways than STAT5 were affected, next we assessed IL-6-induced STAT3 and IL-4-induced STAT6 phosphorylation in SOCS4 Tg T cells. Curiously, none of these cytokine signaling pathways were affected by SOCS4 overexpression leaving the possibility open of other downstream signaling pathways such as PI-3K or MAPK activation being the target of SOCS4-mediated cytokine suppression. Importantly, we are not excluding the possibility that SOCS4 might intersect with the TCR signaling pathway and we are currently performing experiments to address these issues. Collectively, we confirmed that active modulation of SOCS4 expression is necessary for proper T cell differentiation, and preliminary data from peripheral lymphoid organs even further suggest a role of SOCS4 in T cell maintenance. SOCS3 expression in T cells and thymocytes is analogue to SOCS1 expression, and we wished to analyze the effect of SOCS3 overexpression on T cell function. To this end, we generated SOCS3 transgenic mice that expressed a mouse SOCS3 cDNA specifically in T lineage cells. While total thymocyte numbers were not affected, SOCS3 Tg mice had selectively reduced percentage and numbers ( 50% reduction) of CD8SP thymocytes and CD8 T cells. These data suggested that SOCS3-dependent cytokine signals participate specifically in lineage commitment and maintenance of CD8 T cells. Such selective effect on CD8 T cells is analogue to the results observed in SOCS1 transgenic mice where SOCS1 overexpression induced a dramatic decrease ( greater than 80%) in CD8SP thymocytes only. Importantly, the effect of SOCS3 Tg was much blunted compared to SOCS1 Tg but when we generated SOCS3 SOCS1 double transgenic mice, we observed an additive effect of SOCS1 and SOCS3 on blocking CD8 T cell development and homeostasis. Thus, SOCS1 and SOCS3 have partially overlapping but also non-redundant functions in T cells, and we aim to investigate their roles during T cell activation in a series of inflammatory and autoimmune mouse models. Specifically, SOCS3 has been previously shown to suppress STAT3-dependent signaling, and based on recent findings on interleukin-17-secreting CD4+ helper T cells (Th17 cells) and their requirement for STAT3 signaling, we aim to utilize SOCS3 transgenic mice to test the role of SOCS3 in T cell function using autoimmune disease models such as experimental autoimmune encephalomyelitis (EAE) in context of Th17 cell differentiation and activation. TCR stimulation upregulates CISH expression in both immature DP and mature T cells. Importantly, we found that upregulation of CISH expression was much more pronounced in TCR stimulated cells than in cytokine signaled cells. Thus, these data suggest that CISH expression might serve more than just a simple feedback signaling loop in limiting cytokine signaling. However, while CISH is known to inhibit STAT5 phosphorylation, the significance of blocking STAT5 signaling upon positive selection in DP thymocytes and upon activation in mature T cells is not known. To test the role of STAT5 inhibition by CISH, we have constructed transgenic vectors that express a FLAG-tagged CISH cDNA under the control of the human CD2 mini-cassette. These CISH transgenic mice have been now generated, and we are in the process of analyzing the T cell function of these mice. Collectively, we are investigating the functions of three SOCS family members, i.e. SOCS4, SOCS3 and CISH, and using a panel of transgenic mice and a series of in vitro studies, we are in the process of determining the biological role of regulating cytokine responsiveness by SOCS family molecules in T cell activation and homeostasis.
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