We have devised a multi-pronged approach to understand the roles and requirements of SOCS family molecules in T cells. We have generated a series of genetically engineered mice that either lack or overexpress SOCS4 family molecules, and we have been analyzing how T cell functions are affected by altered expression of SOCS. Among others, we have been focusing on SOCS4, because we found it highly expressed in immature thymocytes, suggesting a potential role in the maturation process of T cells. To this end, we generated SOCS4-deficient mice utilizing a gene-trap ES cell system to produce SOCS4 deficient mice, and we verified the absence of SOCS4 expression by real-time reverse transcription PCR. Gross analysis of these mice did not show abnormalities in their development. Detailed phenotypic and functional characterization of these mice are currently under progress. Regarding T cell development, we did not observe any adverse effect of SOCS4 deficiency in producing T cells in the thymus. However, functional assays of SOCS4-deficient T cells have not been finished and awaits further results. To examine if enforced SOCS4 expression would affect 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 apoptotis 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 how SOCS4 interferes with T cell development and differentiation. In parallel to SOCS4, we also assessed expression of SOCS3 during T cell development and homeostasis. In addition to SOCS1, SOCS3 is the only other SOCS family molecule with a KIR domain. Thus, redundant roles for SOCS3 and SOCS1 had been proposed. To formally examine this possibility, we are currently in the process of generating SOCS1/SOCS3-conditional KO mice that have deleted both SOCS1 and SOCS3 in T cells. Once generated, we plan 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 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% 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-gamma 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 cytokines that was tested, even as surface expression of their proprietary receptors was not altered. These results suggested that SOCS3, similar to SOCS1, interferes with cytokine receptor signaling, and that it potentially utilizes a mechanism that involves direct inhibition of JAK activation. Unlike SOCS1, SOCS3 and SOCS4 which are highly expressed in thymocytes and T cells, we found that Cish is expressed only at low levels in resting T cells. Notably, Cish expression also differed in its response to TCR signaling because its expression was 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 repsonses. In T cells, Cish was previously reported to inhibit STAT5phosphorylation 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. 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 utilizes Cish-deficient or overexpressing T cells, and we are mapping differences in their activation and differentiation compared to wildtype T cells.
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