Our efforts to understand the role of SOCS family molecules in T cell development and differentiation have been focused on four members, namely SOCS1, SOCS3, SOCS4 and Cish. SOCS1 is of importance because it is highly expressed in immature thymocytes and was found to be necessary to suppress cytokine signaling in pre-selection thymocytes. We recently demonstrated that the transcription factor ThPOK, which specifies CD4 lineage differentiation, also induces expression of SOCS1. Consequently, CD4, CD8 lineage choice in the thymus is controlled by a ThPOK-dependent pathway of SOCS molecule expression. We generated a series of ThPOK transgenic mice with graded expression of transgenic ThPOK, and we assessed CD4 versus CD8 lineage choice and T cell development in the thymus of these mice, either in the presence or absence of SOCS1. As an extension of this previous study, we are currently assessing the effect of ThPOK on mature CD4 T cells to address a continued role of ThPOK in cytokine receptor signaling. While SOCS1 potently suppresses all JAK, STAT-mediated cytokine signaling, SOCS3 is another inhibitory molecule with a KIR domain, so that redundant roles for SOCS3 and SOCS1 had been proposed. To formally examine this possibility, we are currently generating SOCS1, SOCS3-conditional KO mice that have deleted both SOCS1 and SOCS3 in T cells. Once generated, we wish to compare their T cell phenotype and function to those of SOCS1 or SOCS3 single deficient mice. Moreover, to assess whether SOCS1 and SOCS3 can exert synergistic effects on inhibiting cytokine signaling, we generated and characterized SOCS3 transgenic mice where the transgene is driven by a human CD2 mini-cassette. Overall T cell development in SOCS3-transgenic mice was comparable to wild type mice, except for a selective loss (about 50 percent reduction) of CD8SP thymocytes and peripheral CD8 T cell numbers. These data agree with a cytokine requirement for CD8 lineage commitment and homeostasis, and they demonstrate that SOCS3 also plays a role in CD4, CD8 lineage choice. To directly determine the effect of SOCS3 on cytokine receptor signaling, we examined IL-4, IL-6, IL-7 and IFN-g signaling in SOCS3-transgenic T cells by assessing downstream STAT6, STAT3, STAT5 and STAT1 activation, respectively. Interestingly, we found a broad inhibitory effect of SOCS3 on all tested cytokines similar to SOCS1. These results suggested that SOCS3 interferes with cytokine receptor signaling, utilizing a mechanism that possibly involved direct inhibition of JAK activation. Unlike SOCS1 and SOCS3 which are highly expressed in T cells, we recently found that Cish is expressed only at low levels in thymocytes and in resting T cells. Notably, Cish expression also differed in its response to TCR signaling because it was upregulated by TCR stimulation, and not by cytokine signaling, which contrasts to the regulation of SOCS1 and SOCS3 expression. These results suggest distinct roles for Cish and SOCS1, SOCS3, that need to be interrogated. In T cells, Cish was previously 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. Thus, to further assess the role for Cish, we generated Cish transgenic mice that express a FLAG-tagged Cish cDNA under the control of the human CD2 mini-cassette. Notably, we did not find any major changes in thymocyte development or T cell homeostasis, compared to WT mice. Moreover, we also did not find any effects of Cish overexpression on cytokine receptor signaling. To verify these observations in vivo, we are currently further addressing these issues using Cish-deficient mice and by adoptive transfer of Cish-transgenic or Cish-deficient T cells into lymphopenic mice to examine their homeostatic proliferation and survival. Finally, based on our previous observation that SOCS4 is highly expressed in immature thymocytes, we wished to understand its requirement in T cell development by generating SOCS4-deficient mice. We utilized a gene-trap ES cell system to produce SOCS4 deficient mice, and verified the absence of SOCS4 expression by real-time reverse transcription PCR. The phenotypic and functional characterization of these mice are currently under progress. To further address the role of SOCS4 in T cells, we also generated a T cell-specific SOCS4 transgenic mouse. Here we found that SOCS4 overexpression suppresses T cell development and T cell activation. 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 apoptotic activity was markedly increased. Understanding the downstream effects of SOCS4 overexpression and, or SOCS4 deficiency are the major aims of this study, where we hope to gain further mechanistic insights how SOCS4 interferes with T cell development and differentiation.
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