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

我们一直专注于SOCS家族的三个成员，即SOCS 4，CISH和SOCS 3的作用。我们靶向这些分子，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，我们已经产生了新的试剂，使我们能够在体内研究它们的生物学功能。以前，我们产生了T细胞特异性SOCS 4转基因小鼠，我们现在已经用来评估SOCS 4在T细胞发育和T细胞活化中的作用。我们将HY TCR转基因引入SOCS 4转基因小鼠中，然后使这些小鼠另外缺乏RAG-2，使得表达的唯一TCR源自TCR转基因。值得注意的是，固定TCR特异性加剧了SCSO 4过表达的影响，并揭示了SOCS 4在胸腺细胞阳性选择中的作用。具体来说，我们发现HY SOCS 4转基因雌性小鼠的T细胞成熟和稳态受损，并且SOCS 4影响胸腺细胞中TCR水平和TCR反应性。因此，即使SOCS 4最初被鉴定为细胞因子信号传导的潜在抑制剂，我们现在报告SOCS 4在TCR信号传导中的新作用。重要的是，我们进行了一系列生物化学研究，并能够鉴定SOCS 4与TCR信号复合物的直接相互作用。我们目前正在进一步落实这一意见。有趣的是，转基因SOCS 4不影响细胞因子信号传导，因为我们未能观察到胸腺细胞和成熟T细胞中IL-7下游信号传导的任何差异。我们发现SOCS 4与TCR信号通路交叉，这表明SOCS分子的作用比抑制细胞因子信号传导更广泛。与我们对SOCS 4的研究平行，我们还进行了解决SOCS 3作用的实验，因为SOCS 3在胸腺细胞中高度表达，并且因为SOCS 3表达在细胞因子信号传导后被诱导。为此，我们建立了SOCS 3转基因小鼠，以研究SOCS 3在T细胞发育和反应性中的作用。虽然胸腺细胞总数不受影响，但我们发现SOCS 3 Tg小鼠选择性降低了CD 8 SP胸腺细胞和外周CD 8 T细胞的百分比和数量（减少50%）。有趣的是，SOCS 3过表达抑制IL-6信号传导，但不抑制IL-7诱导的STAT 5磷酸化，这表明SOCS 3的精氨酸特异性作用。此外，当评估SOCS 3、SOCS 1双转基因小鼠时，我们发现SOCS 1和SOCS 3在T细胞中具有部分重叠但非冗余的功能。因此，我们的目标是使用一系列炎症和自身免疫小鼠模型来研究它们的作用。具体而言，SOCS 3先前已显示抑制STAT 3依赖性信号传导。基于最近对分泌白细胞介素-17的CD 4 + T细胞（Th 17细胞）及其对STAT 3信号传导的需求的研究结果，我们的目标是利用SOCS 3转基因小鼠来测试SOCS 3在T细胞功能中的作用，使用自身免疫性疾病模型，如实验性自身免疫性脑脊髓炎（EAE），在Th 17细胞分化和活化的背景下。SOCS 3是否也在TCR信号传导中发挥作用，如SOCS 4，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，我们也有兴趣了解Cish的作用，因为我们发现CISH表达在TCR刺激的细胞中被诱导。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的作用。与此一致，已知Cish抑制STAT 5磷酸化，而STAT 5磷酸化又对胸腺细胞发育和T细胞活化至关重要。为了理解体内对Cish的需求，我们使用基因捕获技术产生Cish缺陷小鼠，并且还通过在人CD 2微型盒的控制下表达FLAG标记的Cish cDNA产生Cish转基因小鼠。我们一直在分析这些小鼠的T细胞功能，但没有发现重大影响。然而，最近的研究表明，Cish可以通过抑制STAT 4、STAT 5和STAT 6的活化来负性调节Th 2和Th 9亚群的分化。我们计划使用我们的试剂解决这些问题。

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.