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

我们一直专注于三个SOCS家族成员的作用，即SOCS4， CISH和SOCS3。我们的目标是这些分子，因为它们在胸腺细胞或活化的T细胞中高度表达，但它们在T细胞功能中的作用仍然不为人所知。为了评估它们的生物学意义，我们制作了新的试剂，使我们能够研究它们在体内的生物学功能。在此之前，我们培育了一个T细胞特异性的SOCS4转基因小鼠，我们现在用它来评估SOCS4在T细胞发育和T细胞激活中的作用。我们将HY TCR转基因引入SOCS4转基因小鼠，然后使这些小鼠进一步缺乏rag2，从而使唯一表达的TCR来源于TCR转基因。值得注意的是，固定TCR特异性加剧了SCSO4过表达的影响，并揭示了SOCS4在胸腺细胞阳性选择中的作用。具体来说，我们发现HY SOCS4转基因雌性小鼠的T细胞成熟和稳态受损，SOCS4影响胸腺细胞的TCR水平和TCR反应性。因此，尽管SOCS4最初被确定为细胞因子信号的潜在抑制因子，但我们现在报道了SOCS4在TCR信号传导中的新作用。重要的是，我们进行了一系列的生化研究，并能够确定SOCS4与TCR信号复合物的直接相互作用。我们目前正在进一步进行这一观察。有趣的是，转基因SOCS4没有影响细胞因子信号传导，因为我们没有观察到胸腺细胞和成熟T细胞中IL-7下游信号传导有任何差异。我们发现SOCS4与TCR信号通路相交，这表明SOCS分子的作用比抑制细胞因子信号传导要广泛得多。在研究SOCS4的同时，我们还进行了SOCS3作用的实验，因为SOCS3在胸腺细胞中高度表达，而且SOCS3的表达是由细胞因子信号传导诱导的。为此，我们培育了SOCS3转基因小鼠，研究SOCS3在T细胞发育和反应性中的作用。虽然总胸腺细胞数量不受影响，但我们发现SOCS3 Tg小鼠选择性地减少了CD8SP胸腺细胞和外周CD8 T细胞的百分比和数量（减少50%）。有趣的是，SOCS3过表达抑制IL-6信号通路，但不抑制il -7诱导的STAT5磷酸化，这表明SOCS3具有细胞因子特异性作用。此外，在评估SOCS3、SOCS1双转基因小鼠时，我们发现SOCS1和SOCS3在T细胞中具有部分重叠但非冗余的功能。因此，我们的目标是通过一系列炎症和自身免疫小鼠模型来研究它们的作用。具体来说，SOCS3先前已被证明可以抑制stat3依赖的信号传导。基于最近对白细胞介素17分泌CD4+ T细胞（Th17细胞）及其对STAT3信号的需求的研究，我们旨在利用SOCS3转基因小鼠，在Th17细胞分化和激活的背景下，利用自身免疫性疾病模型，如实验性自身免疫性脑脊髓炎（EAE），测试SOCS3在T细胞功能中的作用。SOCS3是否也在TCR信号传导中发挥作用，如SOCS4，是一个悬而未决的问题，我们的目标是在未来的研究中解决。最后，我们对了解Cish的作用也很感兴趣，因为我们发现Cish在TCR刺激的细胞中被诱导表达。这些数据表明Cish作为TCR和细胞因子信号传导的潜在反馈机制。与此一致的是，已知Cish抑制STAT5磷酸化，而STAT5磷酸化反过来对胸腺细胞发育和T细胞活化至关重要。为了了解体内对Cish的需求，我们利用基因陷阱技术培育了Cish缺陷小鼠，并在人CD2迷你盒的控制下，通过表达flag标记的Cish cDNA，培育了Cish转基因小鼠。我们一直在分析这些小鼠的T细胞功能，但没有发现主要影响。然而，最近的报道表明，Cish可以通过抑制STAT4、STAT5和STAT6的激活来负向调节Th2和Th9亚群的分化。我们计划用我们的试剂来解决这些问题。

We have been focusing on the role of three SOCS family members, i.e. SOCS4, CISH and SOCS3. We targeted these molecules because they are highly expressed in thymocytes or in activated T cells but their role in T cell function has remained not well known. To assess their biological significance, we have generated new reagents that allowed us to study their biological functions in vivo. Previously, we generated a T cell-specific SOCS4 transgenic mouse, which we have now used to assess the role of SOCS4 in T cell development and T cell activation. We introduced a HY TCR transgene into SOCS4 transgenic mice and then made these mice additionally deficient for RAG-2 so that the only TCR that is expressed is derived from the TCR transgene. Remarkably, fixing the TCR specificity exacerbated the effect of SCSO4 overexpression and revealed a role for SOCS4 in thymocyte positive selection. Specifically, we found that T cell maturation and homeostasis were impaired in HY SOCS4 transgenic female mice, and that SOCS4 affected TCR levels and TCR reactivity in thymocytes. Thus, even as SOCS4 was originally identified as a potential suppressor of cytokine signaling, we now report a new role for SOCS4 in TCR signaling. Importantly, we performed a series of biochemical studies and were able to identify direct interaction of SOCS4 with the TCR signaling complex. We are currently in the process of further pursuing this observation. Interestingly, transgenic SOCS4 did not affect cytokine signaling as we failed to observe any difference in IL-7 downstream signaling in thymocytes and mature T cells. Our finding that SOCS4 intersects with the TCR signaling pathway suggests a much broader role for SOCS molecules than in suppression of cytokine signaling. In parallel to our studies on SOCS4, we also proceeded with experiments addressing the role of SOCS3, because SOCS3 is highly expressed in thymocytes and because SOCS3 expression is induced upon cytokine signaling. To this end, we generated SOCS3 transgenic mice to study SOCS3 effect in T cell development and reactivity. While total thymocyte numbers were not affected, we found that SOCS3 Tg mice had selectively reduced percentage and numbers ( 50% reduction) of CD8SP thymocytes and peripheral CD8 T cells. Interestingly, SOCS3 overexpression suppressed IL-6 signaling but not IL-7-indcued STAT5 phosphorylation suggesting a cytokine-specific effect of SOCS3. Furthermore, when assessing SOCS3, SOCS1 double transgenic mice, we found that SOCS1 and SOCS3 have partially overlapping but also non-redundant functions in T cells. Consequently, we aim to study their roles using a series of inflammatory and autoimmune mouse models. Specifically, SOCS3 has been previously shown to suppress STAT3-dependent signaling. Based on recent findings on interleukin-17-secreting CD4+ T cells (Th17 cells) and their requirement for STAT3 signaling, we aim to utilize SOCS3 transgenic mice to test the role of SOCS3 in T cell function using autoimmune disease models such as experimental autoimmune encephalomyelitis (EAE) in context of Th17 cell differentiation and activation. Whether SOCS3 also plays a role in TCR signaling, such as SOCS4, is a pending question that we aim to address in future studies. Finally, we were also interested in understanding the role of Cish, because we found that CISH expression was induced in TCR stimulated cells. These data suggest a role for Cish as a potential feedback mechanism for TCR and cytokine signaling. In agreement, Cish is known to inhibit STAT5 phosphorylation which in turn is critical for thymocyte development and T cell activation. To understand the in vivo requirement for Cish, we generated Cish-deficient mice using gene trap technology and also generated Cish transgenic mice by expressing a FLAG-tagged Cish cDNA under the control of the human CD2 mini-cassette. We have been analyzing the T cell function of these mice, but did not find a major effect. However, recent reports suggested that Cish could negatively regulate differentiation of Th2 and Th9 subsets by inhibiting activation of STAT4, STAT5 and STAT6. We plan to address these points using our reagents.