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

Division Of Basic Sciences - Nci

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Abstract

In our studies to understand the role of SOCS molecules in T cells, we made progress in assessing the effect of SOCS3 which has been considered a major downstream suppressor of the IL-6 signaling pathway. Interestingly, not only stimulation by IL-6 but also stimulation by gc cytokines, such as IL-7 and IL-15, also induced the expression of SOCS3 in T cells. Therefore, we suspected a potential role for SOCS3 in suppressing gc cytokine signaling also. We have generated and examined transgenic mice that overexpress SOCS3 in T cells, and we found that SOCS3 significantly suppressed the downstream signaling of the gc cytokines IL-2, IL-4 and IL-7. This study was recently published [Luckey MA et al., 2020, Eur. J. Immunol.], and here we documented a previously underappreciated role of SOCS3 in controlling IL-2 signaling. Because IL-2 is a critical mediator of Foxp3+ Treg cell development, we first assessed the generation of Foxp3+ Treg cells in SOCS3 transgenic mice. Here, 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 showed markedly decreased STAT5 phosphorylation when stimulated with IL-2 in vitro. Finally, the 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 Foxp3+ Treg cells. Collectively, our results revealed and documented that SOCS3 is a potent negative regulator of gc cytokine signaling, in addition to its suppressive effect on IL-6 and other gp130 family cytokines. Therefore, these results further suggest that the induction of SOCS3 and its role in inhibiting cytokine signaling should be considered in a broader context that also includes gc cytokines. Because SOCS3 contains a KIR domain that can directly inhibit JAK1 and JAK3 kinases, we are currently investigating the detailed mechanism how SOCS3 would interfere with gc cytokine signaling and whether it utilizes the same mechanism to suppress IL-6 signaling. We are also addressing the role of SOCS3 in the suppression of other gc cytokines in the context of CD8 memory T cell generation, and iNKT cell development and function. SOCS4 remains a poorly characterized member of the SOCS family, which is abundantly expressed in immature thymocytes but downregulated on mature T cells. Such developmentally regulated expression of SOCS4 suggested a potential role in the process of T cell maturation. To examine its role and requirement in thymopoiesis, we have recently generated SOCS4-deficient mice utilizing a gene-trap ES cell system. We were able to verify the absence of SOCS4 mRNA expression by real-time reverse transcription PCR. Gross phenotypic analysis of SOCS4-deficient mice did not show abnormalities in their development, and SOCS4-deficient mice were born at Mendelian ratio. Regarding the T cell development in the thymus, we also did not observe any adverse effect of SOCS4-deficiency. Whether the lack of any discernible effects is due to potential redundancy with other SOCS-family molecules is currently not clear to us. To address this possibility, we isolated naive CD8 T cells from SOCS4-deficient and wildtype mice and performed bulk RNAseq analysis. Notably, we did not find any significant differences in the expression of other SOCS family members, suggesting that it is unlikely that there is a compensatory effect of upregulating the expression of other SOCS molecules in SOCS4-deficient T cells. Nonetheless, we observed a significant increase in TCR responsiveness in SOCS4-deficient T cells, indicating that SOCS4 might intersect with the TCR signaling pathway. Curiously, cytokine signaling in SOCS4-deficient thymocytes and T cells was unaltered. Whether the lack of SOCS4 would interfere with the activation and differentiation of T cells is yet unclear to us, and we are currently assessing the functional aspects of SOCS4-deficient T cells. To examine if the forced expression of SOCS4 would affect TCR or cytokine signaling in T cells, we also generated T cell-specific SOCS4-transgenic mice. Here, we found that SOCS4 overexpression suppressed the development and differentiation of T cells. Specifically, we found that constitutive overexpression of SOCS4 impaired peripheral T cell survival and homeostasis so that naive T cell numbers were significantly reduced. Along these lines, T cell apoptosis was markedly increased in SOCS4 transgenic mice. The detailed mechanisms how SOCS4 would interfere with TCR or cytokine signaling so that it would lead to impaired T cell development and homeostasis is not clear to us, and addressing this issue remains a major aim of this study. In addition to SOCS4 and SOCS3, we also continued our research on the mechanistic aspect of SOCS1 regulation. While we have previously shown that the nuclear factor ThPOK upregulates the transcription of SOCS1 (and SOCS3)[Luckey MA et al., 2014, Nat. Immunol.], ThPOK is only expressed in CD4 T cells and absent on CD8 T cells and immature thymocytes. Therefore, it has not been clear how SOCS1 is induced in CD4, CD8 double positive thymocytes or in cytokine stimulated CD8 T cells. To this end, we obtained SOCS1 reporter mice where a human CD4 cDNA is inserted into the SOCS1 gene after a STOP cassette. Deletion of the STOP cassette using thymocyte specific Cre transgenes permitted the expression of the human CD4 reporter, and we were able to utilize these mice to monitor SOCS1 transcription under different conditions both in vivo and in vitro. Currently, we are expanding our studies using these SOCS1 reporter mice into different disease models to gain better understanding of the regulatory mechanisms of SOCS1 under homeostatic and inflammatory conditions. Unlike SOCS1, SOCS3, and SOCS4 which are highly expressed in both thymocytes and T cells, we found that the SOCS family member Cish is expressed only at low levels in resting T cells. Moreover, Cish expression was upregulated by TCR stimulation and not by cytokine signaling which contrasts to SOCS1 and SOCS3 whose expression is induced by cytokine signaling but not by TCR signaling. 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, the biological significance of Cish induction by TCR signaling, and not by cytokine signaling, was unclear to us. To address this question, we generated Cish-transgenic mice that express a FLAG-tagged Cish cDNA under the control of the human CD2 promoter/enhancer so that Cish is overexpressed in all T lineage cells. Curiously, 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 not affect T cell function under steady-state condition. We also did not find any effects of Cish overexpression on cytokine receptor expression or signaling so that gc signaling remained comparable between Cish-transgenic and wildtype T cells. To identify the 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 how cytokine signaling is controlled in T cells during their development and differentiation.