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

Division Of Basic Sciences - Nci

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Abstract

The main objective of our study is to understand the regulatory mechanisms of cytokine signaling pathways, and to assess the role of negative regulatory molecules, such as SOCS, in these processes. While the prevailing view on cytokine signaling posits that it is essential for T cell function and survival, we recently made a series of unexpected findings that revealed cytokine independent survival of T cells, and we even reported cases where cytokines are detrimental for the differentiation of T cells. In this regard, we have become interested in CD4+CD8aa+ T cells among intraepithelial lymphocytes (IELs) in the small intestine epithelium, which are derived from conventional CD4 T cells that differentiate into CD4+CD8aa+ T cells upon their recruitment in the gut. The phenotypic conversion of CD4 T cells into CD4+CD8aa+ T cells is mediated by transcriptional reprogramming that is associated with the loss of the CD4 lineage-specifying transcription factor ThPOK and the acquisition of the CD8 lineage-specifying transcription factor Runx3d. Interestingly, such reprogramming appeared to be independent of homeostatic gc cytokine signaling, as we demonstrated that neither IL-7-deficiency nor IL-15-defiency resulted in diminished differentiation of CD4+CD8aa+ T cells (Li C. et al., 2022, Cell Mol Immunol.). Instead, we found that the lack of these homeostatic gc cytokines resulted in markedly increased frequencies and numbers of CD4+CD8aa+ T cells, indicating that cytokine signaling is detrimental for their generation. The molecular mechanism of increased CD4+CD8aa+ T cell generation in the absence of IL-7 and/or IL-15, however, has been unclear. Most recently, we identified the runt-related transcriptional activator Runx3d as a key factor that limits the abundance of CD4+CD8aa+ IELs, and we showed that the lack of Runx3d results in a dramatic increase of CD4+CD8aa+ IELs. Because the expression of Runx3d is downstream of gc cytokine signaling, these results suggest that cytokine signaling could control CD4+CD8aa+ IEL differentiation through Runx3d (Li C., 2023, Cell Death Diff). Thus, we identified a new transcriptional circuitry of CD4+CD8aa+ IEL differentiation and phenotype acquisition that is negatively controlled by homeostatic gc signaling. However, it remains to be assessed what cellular signals drive and maintain this cytokine/Runx3d-mediated pathway in CD4+CD8aa+ IELs. Specifically, it is unclear whether SOCS expression could have been involved in this process, which we consider likely, because SOCS family molecules could prevent cytokine signaling in CD4 T cells to promote their differentiation. We are currently in the process of assessing SOCS expression in IELs to assess these issues. Another ongoing study on the role of SOCS molecules is to interrogate the role of SOCS4 which remains a poorly characterized SOCS family member that is abundantly expressed in immature thymocytes but less so on mature T cells. Such developmentally regulated expression of SOCS4 suggested a potential role for SOCS4 in thymic T cell generation. To examine its role and requirement in thymopoiesis, we previously generated SOCS4-deficient mice utilizing a gene-trap ES cell system and verified the absence of SOCS4 mRNA expression by real-time reverse transcription PCR. Disappointingly, assessing the T cell development in the thymus of SOCS4-deficient mice did not reveal any adverse effect of SOCS4-deficiency. We failed to observe significant changes in thymocyte numbers, positive selection, and lineage differentiation in the absence of SOCS4. Whether the lack of any discernible effects in T cell development would be due to potential redundancy with other SOCS-family molecules is currently not clear to us. However, we have recently established new mouse models to address this question. Among others, we introduced the SOCS4-KO allele into HY TCR transgenic mice to test the role of SOCS4 in TCR signaling and cytokine signaling using monospecific TCR expressing thymocytes. A potential connection of SOCS4 with the TCR signaling pathway came from our recent observation that SOCS4-deficient T cells showed higher responsiveness to TCR signaling, so that we found it necessary to test the effect of SOCS4 on TCR signaling. Here, we found that gc family cytokine signaling in SOCS4-deficient thymocytes and T cells remained unaltered but the magnitude of TCR signaling evidently increased. Why SOCS4 would interfere with TCR- instead of cytokine receptor signaling is currently unclear to us. It also needs to be assessed whether the lack of SOCS4 interferes with TCR-induced activation and differentiation of T cells, and we aim to assess assessing the functional aspects of SOCS4-deficient T cells in our future studies. In parallel to our studies on SOCS4, we have been studying the mechanistic aspect of SOCS1 controlled regulation of gc signaling. SOCS1 expression is dynamically regulated upon T cell development, and immature thymocyte express the largest amounts of SOCS1 which is downregulated upon positive selection. We have previously demonstrated that SOCS1 in immature CD4, CD8 double-positive thymocytes is critical to prevent cytokine signaling in pre-selection thymocytes. However, it was unclear what drives SOCS1 expression in a stage-specific manner in developing thymocytes. We have been addressing this question using 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 have been using these mice to monitor SOCS1 transcription under different stimulatory conditions both in vivo and in vitro. Additionally, our recent studies suggested a partially redundant role of SOCS3 in the suppression of gc cytokine signaling in thymocytes, so that we are currently in the process of generating SOCS1-floxed, SOCS3-floxed CD4-Cre mice to examine the redundancy among major SOCS family molecules in T cells. We are aiming to employ these mouse models to gain better understanding on the regulatory mechanisms of cytokine receptor signaling under homeostatic and inflammatory conditions.