Among the SOCS family members, SOCS1, SOCS3 and Cish are molecules already known to be involved in modulating cytokine response in T cells. In this study, we have additionally identified SOCS4 as a new SOCS family member to play a role in T cell immunity, and thus we are currently focusing on the expression, regulation and function of SOCS1, SOCS3, Cish and also SOCS4. SOCS1 is highly expressed in immature DP thymocytes to suppress cytokine signaling in pre-selection thymocytes. Suppression of cytokine signaling is a critical mechanism to prevent survival of thymocytes which have failed to get positively selected. On the other hand, cytokine signaling is necessary to provide survival of mature thymocytes and also to direct CD8 lineage choice during CD4/CD8 lineage commitment. In fact, intrathymic cytokine signaling induces expression of the CD8 lineage specifying factor Runx3, and desensitization of cytokine receptor signaling is critical to suppress Runx3 expression and to promote CD4 lineage differentiation. As a potential mechanism to suppress cytokine signaling during CD4 lineage choice, we assessed expression of SOCS family molecule expression in CD4 single positive (SP) thymocytes. We found that both CD8SP and CD4SP thymocytes expressed SOCS1, SOCS3 and Cish, but that CD4 lineage cells expressed significantly higher levels of SOCS1 and Cish than CD8SP thymocytes. SOCS expression is induced by cytokine signaling, so that SOCS molecule expression during CD8 lineage differentiation is a consequence of intrathymic cytokine signaling. However, it was not clear why CD4SP cells expressed high levels of SOCS molecules, and even at higher levels than CD8SP thymocytes. As such, we wished to know if transcription factors that are specifically expressed in CD4 lineage cells would drive SOCS expression in CD4 cells. ThPOK is a zinc-finger nuclear factor that is specifically expressed in CD4 lineage cells and which is induced by strong/persistent TCR signaling during CD4 lineage differentiation. To examine the scenario that ThPOK would induce SOCS expression to inhibit cytokine signaling and direct CD4 lineage choice, we generated a series of ThPOK-transgenic mice with increasing levels of transgenic ThPOK expression. We found that increased ThPOK expression resulted in increased SOCS1, SOCS3 and Cish expression in CD4 T cells. We further identified that ThPOK directly induced SOCS1 transcription in luciferase reporter assays and in ThPOK-transgenic SOCS1 reporter mice. Importantly, ThPOK function was dependent on SOCS1 expression because CD4 lineage redirection by ThPOK was impaired in SOCS1-deficient ThPOK transgenic mice. Reciprocally, transgenic expression of SOCS1 was sufficient to generate CD4 T cells in the absence of ThPOK expression, indicating that the major function of ThPOK is to induce expression of SOCS1. In sum, these data reveal a novel role for SOCS in T cells, revealing that SOCS1 and potentially other SOCS molecules are directly involved in CD4/CD8 lineage decision in the thymus. Because of its potential redundancy with SOCS1, we also proceeded with experiments addressing the role of SOCS3 in T cells. SOCS3 is highly expressed in thymocytes, but only induced to high levels in mature T cells by cytokine stimulation. To understand the downstream effect of SOCS3 under steady state conditions, we generated SOCS3 transgenic (SOCS3 Tg) mice under the control of the human CD2 mini-cassette. While overall T cell development in these mice was comparable to wildtype mice, interestingly, we found that SOCS3 Tg mice had a selective loss ( 50% reduction) of CD8SP thymocytes and peripheral CD8 T cells. SOCS3 overexpression potently suppressed IL-6-induced STAT3 activation but only modestly suppressed IL-7-indcued STAT5 phosphorylation. These results suggest a cytokine-specific effect of SOCS3 which we are currently investigating with a broader panel of different cytokines. Based on recent findings on IL-17-secreting CD4+ T cells (Th17 cells) and their requirement for STAT3 signaling, we aim to utilize SOCS3 Tg mice to test the role of SOCS3 in T cell function in vivo. Additionally, we were also interested in understanding the role of Cish, because we found that TCR stimulation induced expression of Cish. In T cells, Cish inhibits STAT5 phosphorylation by gc cytokines, but why Cish expression is induced by TCR signaling rather than by cytokine signaling is unclear. Potentially, Cish could play a role as a mediator in the interplay between TCR and cytokine signaling, which in turn is critical for thymocyte development and T cell activation. To understand the in vivo requirement for Cish, we generated Cish-deficient mice using gene trap technology and further generated Cish transgenic mice by expressing a FLAG-tagged Cish cDNA under the control of the human CD2 mini-cassette. Initial analysis of T cell functions in these mice did not reveal a major difference to wildtype mice. However, there are recent reports that suggest Cish could negatively regulate differentiation of Th2 and Th9 subsets by inhibiting activation of STAT4, STAT5 and STAT6. We plan to address these points using these newly generated reagents. Finally, we also found that SOCS4 is highly expressed in thymocytes and wished to understand its role in T cells. To address this question, we generated a T cell-specific SOCS4 transgenic mouse, which we employed to assess the role of SOCS4 in T cell development and T cell activation. We further introduced a HY-specific TCR transgene into SOCS4 transgenic mice and then made these mice additionally deficient for RAG-2 so that the only TCR that is expressed is derived from the TCR transgene. Remarkably, fixing the TCR specificity exacerbated the effect of SOCS4 overexpression and revealed SOCS4 overexpression suppressed thymocyte positive selection. Specifically, we found that T cell maturation and homeostasis were impaired in HY SOCS4 transgenic female mice, and that SOCS4 affected TCR levels and TCR reactivity in thymocytes. Moreover, we performed a series of biochemical studies and were able to identify direct interaction of SOCS4 with the TCR signaling complex. We are currently in the process of further analyzing its mechanism. Thus, our finding that SOCS4 intersects with the TCR signaling pathway suggests a much broader role for SOCS molecules than in suppression of cytokine signaling.
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