Immune Regulatory Roles of Suppressor Of Cytokine Signaling (SOCS) Molecules

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To understand the role of SOCS family molecules in controlling cytokine signaling in T cells, we utilized a series of genetically engineered mice to alter the abundance, timing and regulation of SOCS proteins upon T cell development and differentiation. Most recently, we concluded a study on SOCS3 and its cytokine specificity to reveal that, contrary to our current view, SOCS3 does not only suppress the signaling of cytokines of the gp130 family but also cytokines of the gc family, including IL-2, IL-4 and IL-7. We previously documented that T cell stimulation by gc cytokines, such as IL-7 and IL-2, induced the expression of SOCS3, suggesting a potential role for SOCS3 in suppressing gc cytokine signaling. Indeed, genetically engineered mice that overexpress SOCS3 in T cells potently suppressed IL-2, IL-4, and IL-7 signaling in T cells, in addition to suppressing IL-6 signaling which so far had been considered the major target of SOCS3 suppression. 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 SOCS3-transgenic Foxp3+ Treg cells showed markedly decreased STAT5 phosphorylation when stimulated in vitro. Finally, the in vitro differentiation of TCR-activated naive CD4 T cells into Foxp3+ Treg cells in the presence of IL-2 and TGF-beta showed dramatically impaired generation of iTreg 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. This work has been recently published in the European Journal of Immunology. We are now following up on this study by asking potential redundancy between SOCS1 and SOCS3 in suppressing gc cytokine signaling. As described in our published study, IL-7 signaling upregulates the expression of both SOCS1 and SOCS3. It is currently unclear whether the co-expression of these two molecules is necessary for effective suppression of cytokine signaling, and whether the combined ablation of both molecules would induce a distinct effect than the deletion of only SOCS1 or SOCS3. Because SOCS3-deficiency is embryonic lethal and because SOCS1-deficiency is perinatal lethal, we are currently in the process of intercrossing SOCS1 and SOCS3 floxed mice to generate SOCS1/3 double floxed mice, and we aim to delete the SOCS1/3 expression using a T cell specific Cre transgene. We expect to gain further insight into the molecular pathways of SOCS1/3 activity, utilizing this mouse model. In addition to SOCS3, we found it important to understand the role of non-classical SOCS molecules. As such, we have been focusing on SOCS4, because we found it is highly expressed in immature thymocytes. The highly regulated expression of SOCS4 in thymocytes suggested a potential role in the development of T cells. Thus, to assess its requirement in thymopoiesis, we generated SOCS4-deficient mice utilizing a gene-trap ES cell system (SOCS4-KO mice), and we verified the absence of SOCS4 expression by real-time reverse transcription PCR and Western blot analysis. Gross phenotypic analysis of SOCS4-KO mice did not show abnormalities in their development. Regarding the T cell development thymus, we also did not observe any adverse effect of SOCS4 deficiency so that CD4 and CD8 T cell lineage commitment and maturation remained unaltered. These results suggested a potential redundancy of SOCS4 with other SOCS-family molecules, which remains to be examined. Also, whether SOCS4 is required for the cytokine-mediated generation of effector T cells need to be assessed, and we are currently analyzing the functional aspects of such SOCS4-deficient T cells. Altogether, understanding the role and requirement for SOCS4 in T cells remains a major aim of this study, and we hope to gain further mechanistic insights on whether and how SOCS4 intersect with cytokine signaling pathways in T cell development and differentiation. Finally, we are also investigating the role of another prominent member of the SOCS family, Cish, which is proposed to control both cytokine and antigen receptor signaling in T cells. 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. Moreover, Cish expression was dramatically upregulated by TCR stimulation and not by cytokine signaling, which contrasts to the induction of SOCS1 and SOCS3 that are upregulated by cytokine stimulation. These results suggested distinct roles for Cish and other SOCS family member in controlling T cell immune responses. While Cish is reported to inhibit STAT5 phosphorylation by gc cytokines, it is unclear to us why Cish expression would be induced by TCR signaling, and not by cytokine signaling. We hypothesize a previously unappreciated role for Cish in the crosstalk of TCR versus cytokine receptor signaling, and we suspect that Cish could play a role in TCR-mediated desensitization of cytokine receptor signaling. To identify and map the downstream targets of Cish, we are currently performing experiments that utilize T cells that are deficient for Cish or T cells that are forced to express Cish. Thus, mapping differences in cytokine signaling in the presence or absence of Cish will allow us the further understand the role of Cish in suppressing cytokine receptor signaling. Altogether, we expect that the comprehensive analysis of SOCS family member expression and function will provide us a clear picture of how the negative regulatory loop of cytokine signaling is controlled in T cells during their development and differentiation.