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

与其他SOCS家族分子相比，我们对SOCS4在免疫系统中的作用知之甚少。然而，我们发现与DP胸腺细胞相比，SOCS4在成熟T细胞中的表达下调，因此我们希望知道这种特异性表达对于维持正常的胸腺生成和T细胞稳态是否必要。为此，我们产生了SOCS4转基因小鼠，在所有T系细胞中表达小鼠SOCS4 cDNA。通过定量RT-PCR和Western blot分析，我们鉴定了3个不同的SOCS4转基因系，表达了不同水平的SOCS4转基因表达。有趣的是，我们发现胸腺细胞绝对数量的显著减少与转基因SOCS4的表达呈线性相关。这些结果表明，SOCS4的组成性表达部分抑制了T细胞的发育。对胸腺细胞亚群的详细分析进一步揭示了DN3阶段的发育阻滞，这可能是由于DN胸腺细胞增殖减少导致胸腺细胞总数减少。IL-7信号通路是DN胸腺细胞存活和增殖的重要因素，我们考虑了SOCS4干扰IL-7信号通路的可能性。然而，令人惊讶的是，通过IL-7刺激后STAT5磷酸化评估，IL-7信号在SOCS4 Tg T细胞中是正常的。接下来，为了测试STAT5以外的其他STAT信号通路是否受到影响，我们在SOCS4 Tg T细胞中评估了il -6诱导的STAT3和il -4诱导的STAT6磷酸化。奇怪的是，这些细胞因子信号都不受SOCS4过表达的影响，这表明SOCS4会干扰其他信号通路。为了验证转基因SOCS4是否会影响TCR信号传导，接下来，我们评估了短期TCR交联时Erk的磷酸化，发现SCSO4确实干扰了TCR信号传导。我们目前正在使用限制mhci的TCRS转基因来测试这一想法，我们也在研究这一观察结果背后的潜在分子机制。总的来说，我们证实了SOCS4表达的主动调节对于T细胞分化和维持是必要的，并且初步数据表明SOCS4在TCR信号传导中发挥作用，而不是细胞因子信号传导。此外，我们一直在关注另外两个SOCS家族分子，即SOCS3和Cish。我们培育了SOCS3和Cish转基因小鼠，并分析了这些小鼠的T细胞发育和T细胞功能，结果发现SOCS3影响CD8细胞发育，并与SOCS1协同抑制细胞因子信号传导，而Cish没有表现出任何突出的表型。尽管如此，我们发现SOCS1和SOCS3在T细胞中具有部分重叠但也非冗余的功能，我们目前正在一系列炎症和自身免疫性小鼠模型中研究它们在T细胞激活中的作用。在Cish的情况下，我们最初非常感兴趣，因为Cish的表达是在TCR刺激下诱导的，因为已知Cish通过与STAT5竞争来抑制IL-7信号传导。然而，奇怪的是，我们没有观察到转基因Cish对细胞因子信号传导的任何影响，即使我们通过Western blot分析证实了它在淋巴结T细胞中的正确过表达。我们目前正在考虑Cish类似于SOCS4作用于TCR信号传导而不是细胞因子信号传导的可能性，并且针对这种可能性的实验正在进行中。

In contrast to other SOCS family molecule, little is known about the role of SOCS4 in the immune system. However, we found that SOCS4 expression is donwregulated in mature T cells compared to DP thymocytes, and as such we wished to know whether such specific expression was necessary to maintain normal thymopoiesis and T cell homeostasis. To this end, we generated SOCS4 transgenic mice that expressed a mouse SOCS4 cDNA in all T lineage cells. By quantitative RT-PCR and Western blot analysis, we identified three different lines of SOCS4 transgenes which expressed graded levels of transgenic SOCS4 expression. Interestingly, we found a significant decrease in absolute thymocyte numbers in linear correlation to transgenic SOCS4 expression. These results suggested that constitutive expression of SOCS4 partially inhibited T cell development. Detailed analysis of thymocytes subpopulations further revealed a developmental block at the DN3 stage, which probably was caused by a decrease in DN thymocytes proliferation and resulting in reduction of total thymocytes numbers. IL-7 signaling is a major factor necessary for DN thymocytes survival and proliferation, and we considered the possibility that SOCS4 interferes with IL-7 signaling. Surprisingly however IL-7 signaling was normal in SOCS4 Tg T cells as assessed by STAT5 phosphorylation upon IL-7 stimulation. Next to test the possibility that other STAT signaling pathways than STAT5 were affected, we assessed IL-6-induced STAT3 and IL-4-induced STAT6 phosphorylation in SOCS4 Tg T cells. Curiously, none of these cytokine signaling were affected by SOCS4 overexpression indicating that SOCS4 interferes with other signaling pathways. To test whether transgenic SOCS4 can affect TCR signaling, next, we assessed Erk phophorylation upon short term TCR crosslinking and we found that indeed SCSO4 interfered with TCR signaling. We are currently testing this idea using MHCI-restricted TCRS transgenes and we are also investigating the potential molecular mechanism underlying this observation. Collectively, we confirmed that active modulation of SOCS4 expression is necessary for T cell differentiation and maintenance, and preliminary data from suggest a role of SOCS4 in TCR signaling instead of cytokine signaling. Additionally, we have been focusing on two other SOCS family molecules, namely SOCS3 and Cish. We have generated both SOCS3 and Cish transgenic mice and have analyzed T cell development and T cell function in these mice with the result that SOCS3 affected CD8 cell development and acted synergistically with SOCS1 to suppress cytokine signaling whereas Cish did not show any prominent phenotype. Still, we found that SOCS1 and SOCS3 have partially overlapping but also non-redundant functions in T cells, and we are currently investigating their roles during T cell activation in a series of inflammatory and autoimmune mouse models. In case of Cish, we were initially very interested because Cish expression is induced upon TCR stimulation and because Cish is known to suppress IL-7 signaling by competing with STAT5. Curiously, however, we did not observe any effect of transgenic Cish on cytokine signaling even as we confirmed its correct overexpression in lymph node T cells by Western blot analysis. We are currently considering the possibility that Cish acts analogous to SOCS4 on TCR signaling rather than on cytokine signaling, and experiments addressing this possibility are under progress.