To understand the roles of SOCS family molecules in controlling cytokine signaling in T cells, we utilized a multi-pronged approach to alter the expression of SOCS molecules and assess their effects during T cell development and differentiation. We generated a series of genetically engineered mice that either lack or overexpress individual SOCS family molecules. Using these tools, among others, we made significant advances in assessing the role of SOCS3, which has been considered a major downstream suppressor of the IL-6 signaling pathway. Interestingly, stimulation by gc cytokines such as IL-7 and IL-15 also induced expression of SOCS3, so that we suspected a potential role for SOCS3 in suppressing gc cytokine signaling. Indeed, genetically engineered mice that overexpress SOCS3 in T cells suppressed IL-6 signaling as expected, but we found that SOCS3 also substantially inhibited downstream signaling of the gc cytokines IL-2, IL-4, and IL-7. Because IL-2 is a critical mediator of Foxp3+ Treg cell generation, we further assessed the generation of Foxp3+ Treg cells in SOCS3 transgenic mice. As expected, we observed a substantial defect in Foxp3+ Treg cell development in the thymus of SOCS3 transgenic mice. Moreover, IL-2 signaling in Foxp3+ Treg cells of SOCS3-transgenic mice markedly decreased STAT5 phosphorylation when stimulated in vitro. Finally, in vitro differentiation of naive CD4 T cells into Foxp3+ Treg cells by TCR activation in the presence of IL-2 and TGF-beta showed dramatically impaired generation of Treg cells. Collectively, these results revealed a previously unappreciated role of SOCS3 in controlling gc cytokine signaling and consequently in suppressing cellular processes that depend on gc cytokines such as IL-2. In addition to SOCS3, we have been focusing on SOCS4, because we found it is highly expressed in immature thymocytes, suggesting a potential role in the maturation process of T cells. To assess its requirement in thymopoiesis, we generated SOCS4-deficient mice utilizing a gene-trap ES cell system, and we verified the absence of SOCS4 expression by real-time reverse transcription PCR. Gross phenotypic analysis of these mice did not show abnormalities in their development. Detailed cellular and functional characterization of these mice are currently under progress. Regarding T cell development, however, we did not observe any adverse effect of SOCS4 deficiency in producing T cells in the thymus, indicating potential redundancy with other SOCS-family molecules. We are currently assessing the functional aspects of SOCS4-deficient T cells. To examine if enforced SOCS4 expression would affect T cells, we also generated T cell-specific SOCS4-transgenic mice. Here, we found that SOCS4 overexpression suppresses the development and differentiation of T cells. Specifically, we found that constitutive expression of SOCS4 impaired peripheral T cell survival and homeostasis so that naive T cell numbers were significantly reduced, and apoptosis was markedly increased. Understanding the downstream effects of SOCS4 in T cells remains the major aim of this study, and we hope to gain further mechanistic insights on how SOCS4 interferes with T cell development and differentiation. Unlike SOCS1, SOCS3, and SOCS4 which are highly expressed in both thymocytes and T cells, we found that Cish is expressed only at low levels in resting T cells. Notably, we found that Cish expression upregulated by TCR stimulation and not by cytokine signaling, which contrasts to the regulation of SOCS1 and SOCS3 expression. These results suggested distinct roles for Cish and other SOCS family member in controlling T cell immune responses. Previously, Cish had been reported to inhibit STAT5 phosphorylation by gc cytokines. However, why Cish expression is induced by TCR signaling, and not by cytokine signaling, was unclear to us. We have now generated Cish-transgenic mice that express a FLAG-tagged Cish cDNA under the control of the human CD2 promoter/enhancer. We did not find any major changes in thymocyte development or T cell homeostasis in the presence of increased Cish expression, indicating that Cish does affect T cell function under steady-state condition. Moreover, we also did not find any effects of Cish overexpression on cytokine receptor expression or signaling. To identify the exact downstream targets of Cish, we are currently performing experiments that utilize Cish-deficient or Cish-overexpressing T cells, and we are mapping differences in their activation and differentiation compared to wildtype T cells. Altogether, we expect that the comprehensive analysis of SOCS family member expression and function will provide us a clear picture of how cytokine signaling is controlled in T cells during their development and differentiation.
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