We initiated this study by establishing a new set of real time-polymerase chain reaction (PCR) assays that specifically and quantitatively report messenger RNA (mRNA) expression of each members of the SOCS family. Then, using various organs from C57BL/6 mice, we performed a comprehensive tissue distribution analysis of SOCS molecules expression. In agreement with prior reports, we confirmed that SOCS1 was highly expressed in the thymus and specifically in developing thymocytes indicating its critical role in T cell development and differentiation. Surprisingly, mRNA expression of the other two immune cell-related SOCS family members, namely SOCS3 and CISH, were found to be expressed only in low levels in thymus and spleens whereas they were found in high levels in lung and kidney, respectively. The biological significance of such high level SOCS3 and CISH expression in these tissues is currently not clear to us. However, these data suggest that, in contrast to SOCS1, SOCS3 and CISH have distinct developmental expression patterns during T cell development, and that their expression is normally maintained at a basal level only to be induced upon cytokine signaling. In fact, when we stimulated purified CD4+ or CD8+ T cells with cytokines such as IL-7 or IL-4, we observed a strong induction of both SOCS3 and CISH expression which presumably limits further signaling by these cytokines. As for other members of the SOCS family, we observed different patterns of expression depending on the tissues which collectively suggested that each individual SOCS member might have a specific function for different cells. For example, we observed a highly specific expression of SOCS7 in the brain whereas high levels of SOCS2 expression were detected in kidney, live and lung but in no immune tissues. Most interestingly, however, we found SOCS4 mRNA to be enriched in the thymus and particularly to be highly expressed in thymocytes which closely resembled the expression pattern of SOCS1. To further investigate whether SOCS4 expression is developmentally regulated as SOCS1, we electronically sorted and purified thymocyte subpopulations and we found that SOCS4 mRNA is highly upregulated in immature CD4+CD8+ double positive (DP) thymocytes but then down regulated in mature CD4+ single positive (SP) and CD8+ SP thymocytes as well as in mature T cells. Western blot analysis further showed that SOCS4 protein expression correlated to SOCS4 mRNA expression in that SOCS4 protein expression was high in immature DP thymocytes but then downregulated in mature thymocytes and peripheral T cells. It is well documented that positive selecting T cell receptor (TCR) signal downregulates SOCS1 expression during T cell development. To assess whether SOCS4 expression is also under the control of TCR signals, next we isolated mature T cells and immature DP thymocytes and TCR stimulated them in vitro to determine its effect on SOCS4 mRNA expression. As in case of SOCS1, both plate-bound anti-TCR antibody stimulation as well as pharmacological mimics of TCR signals both strongly downregulated SOCS4 mRNA expression. These data suggest that that SOCS4 could be potentially a redundant molecule of SOCS1 that protects immature thymocytes from getting prematurely cytokine signaled. We are currently in the process of generating a T-lineage specific SOCS4 transgenic mouse and a SOCS4 conditional knock-out (KO) mouse to assess the effects of SOCS4 overexpression and conditional deletion on immune homeostasis and activation. Ultimately, we wished to generate SOCS1-deficient, SOCS4-deficient double KO mice, which will enable us to address potential redundant roles of SOCS1 and SOCS4 and also to determine the specific role of SOCS4 in limiting cytokine signaling during T cell differentiation and activation. In addition to a redundant role of SOCS1 with SOCS4, however, we are also interested in a potential cross-talk and functional redundancy of SOCS1 with other SOCS molecules. In particular, SOCS3 is of great interest to us as we found SOCS3 expression to be unaffected during T cell development but strongly modulated by cytokine signaling. Since we found SOCS3 mRNA to be constitutively expressed to a significant level in both thymocytes and resting T cells, we further considered the possibility that maintaining SOCS3 expression would be critical for dampening cytokine signaling during immune homeostasis. To this end, we generated mice where Socs3 was specifically deleted in immature thymocytes and in mature T cells. To do so, we crossed SOCS3 floxed mice with mice transgenic for a CD8-double positive enhancer drive Cre transgene, and we are currently in the process of analyzing these mice. The initial data suggest that thymocyte development and T cell homeostasis are relatively normal in these mice albeit with a slight increase in CD8 T cell numbers. Functional analysis of these T cells and their further phenotypic analyses are currently in process. Finally, to examine the effect of SOCS3 overexpression in T cells, we have generated SOCS3 transgenic mice that specifically express SOCS3 in all T lineage cells. Preliminary data suggest that cytokine signaling is severely impaired in these cells and that both T cell development and homeostasis were affected caused by diminished cytokine signaling. Importantly, SOCS3 has been previously implicated in suppressing STAT3-mediated immune responses, and in light of the recent findings on interleukin-17-secreting CD4+ helper T cells (Th17 cells) and their requirement for STAT3 signaling, we consider it important to utilize these SOCS3 transgenic mice to test autoimmune disease models such as experimental autoimmune encephalomyelitis (EAE) in context of Th17 cell differentiation and activation. In sum, the major findings and accomplishments can be summarized as follows: 1. Comprehensive analysis and tissue distribution of SOCS family molecule expression 2. Identification of SOCS4 as a potential and novel immune regulatory SOCS molecule 3. Generation and characterization of conditional knock-out mice with T lineage specific deletion of SOCS3 4. Generation and characterization of transgenic mice overexpressing SOCS3 under the control of a human CD2 mini-cassette. 5. Analysis of regulatory mechanisms that control SOCS molecule expression in T cells
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