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

由于SOCS4的表达在DN到DP的转变过程中上调，但在阳性选择过程中下调，我们想要评估在T细胞发育过程中是否需要这种阶段特异性表达来维持正常的胸腺生成。为此，我们在人CD2 mini-cassette的控制下，产生了表达小鼠SOCS4 cDNA的SOCS4转基因小鼠。通过在纯化的T细胞中进行定量RT-PCR，我们鉴定出了三种不同的SOCS4转基因系，这些系的SOCS4转基因表达水平是分级的。与转基因SOCS4表达水平平行，我们观察到胸腺细胞绝对数量显著减少，这表明SOCS4的组成性过表达干扰了T细胞的发育。进一步分析SOCS4 Tg胸腺细胞发现DN3至DN4期发育阻滞，DP胸腺细胞上CD25表达增加也证实了这一点。这种缺陷提示DN胸腺细胞的tcr前信号或IL-7信号可能存在问题。然而，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。为了测试STAT5以外的其他STAT信号通路是否受到影响，接下来我们评估了il -6诱导的STAT3和il -4诱导的SOCS4 Tg T细胞中STAT6的磷酸化。奇怪的是，这些细胞因子信号通路都没有受到SOCS4过表达的影响，这使得其他下游信号通路（如PI-3K或MAPK激活）可能成为SOCS4介导的细胞因子抑制的目标。重要的是，我们不排除SOCS4可能与TCR信号通路相交的可能性，我们目前正在进行实验来解决这些问题。总的来说，我们证实了SOCS4表达的主动调节对于T细胞的正常分化是必要的，来自外周淋巴器官的初步数据甚至进一步表明SOCS4在T细胞维持中的作用。SOCS3在T细胞和胸腺细胞中的表达与SOCS1类似，我们希望分析SOCS3过表达对T细胞功能的影响。为此，我们产生了SOCS3转基因小鼠，在T系细胞中特异性表达小鼠SOCS3 cDNA。虽然总胸腺细胞数量不受影响，但SOCS3 Tg小鼠选择性地减少了CD8SP胸腺细胞和CD8 T细胞的百分比和数量（减少50%）。这些数据表明，依赖socs3的细胞因子信号特异性地参与CD8 T细胞的谱系承诺和维持。这种对CD8 T细胞的选择性作用类似于在SOCS1转基因小鼠中观察到的结果，SOCS1过表达仅导致CD8SP胸腺细胞显著减少（大于80%）。重要的是，与SOCS1 Tg相比，SOCS3 Tg的作用明显减弱，但当我们产生SOCS3 - SOCS1双转基因小鼠时，我们观察到SOCS1和SOCS3在阻断CD8 T细胞发育和稳态方面的加性效应。因此，SOCS1和SOCS3在T细胞中具有部分重叠但也非冗余的功能，我们的目标是在一系列炎症和自身免疫性小鼠模型中研究它们在T细胞激活过程中的作用。具体而言，SOCS3先前已被证明可以抑制STAT3依赖性信号传导，基于最近对白细胞介素17分泌CD4+辅助性T细胞（Th17细胞）及其对STAT3信号传导的需求的研究结果，我们的目标是利用SOCS3转基因小鼠，在Th17细胞分化和激活的背景下，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））来测试SOCS3在T细胞功能中的作用。TCR刺激上调未成熟DP和成熟T细胞中CISH的表达。重要的是，我们发现CISH表达的上调在TCR刺激细胞中比在细胞因子信号细胞中更为明显。因此，这些数据表明CISH表达在限制细胞因子信号传导方面可能不仅仅是一个简单的反馈信号循环。然而，虽然已知CISH可以抑制STAT5磷酸化，但阻断STAT5信号在DP胸腺细胞阳性选择和成熟T细胞激活时的意义尚不清楚。为了测试CISH对STAT5的抑制作用，我们构建了在人CD2迷你盒控制下表达flag标记的CISH cDNA的转基因载体。这些CISH转基因小鼠现在已经产生，我们正在分析这些小鼠的T细胞功能。总的来说，我们正在研究三个SOCS家族成员，即SOCS4， SOCS3和CISH的功能，并使用一组转基因小鼠和一系列体外研究，我们正在确定SOCS家族分子在T细胞激活和稳态中调节细胞因子反应的生物学作用。

Because SOCS4 expression was found to be upregulated during DN to DP transition but then downregulated upon positive selection, we wanted to assess whether such stage-specific expression during T cell development was necessary to maintain normal thymopoiesis. To this end, we generated SOCS4 transgenic mice that expressed a mouse SOCS4 cDNA under the control of the human CD2 mini-cassette. By quantitative RT-PCR in purified T cells, we identified three different lines of SOCS4 transgenes with graded levels of transgenic SOCS4 expression. Parallel to transgenic SOCS4 expression levels, we observed a significant decrease in absolute thymocyte numbers which suggested that constitutive overexpression of SOCS4 disturbed T cell development. Further analysis of SOCS4 Tg thymocytes revealed a developmental block in the DN3 to DN4 stage that was also confirmed by increased CD25 expression on DP thymocytes. Such a defect suggested either a potential problem in pre-TCR signaling or in IL-7 signaling in DN thymocytes. IL-7 signaling, however, was normal in SOCS4 Tg T cells as assessed by STAT5 phosphorylation upon IL-7 stimulation. To test the possibility that other STAT signaling pathways than STAT5 were affected, next we assessed IL-6-induced STAT3 and IL-4-induced STAT6 phosphorylation in SOCS4 Tg T cells. Curiously, none of these cytokine signaling pathways were affected by SOCS4 overexpression leaving the possibility open of other downstream signaling pathways such as PI-3K or MAPK activation being the target of SOCS4-mediated cytokine suppression. Importantly, we are not excluding the possibility that SOCS4 might intersect with the TCR signaling pathway and we are currently performing experiments to address these issues. Collectively, we confirmed that active modulation of SOCS4 expression is necessary for proper T cell differentiation, and preliminary data from peripheral lymphoid organs even further suggest a role of SOCS4 in T cell maintenance. SOCS3 expression in T cells and thymocytes is analogue to SOCS1 expression, and we wished to analyze the effect of SOCS3 overexpression on T cell function. To this end, we generated SOCS3 transgenic mice that expressed a mouse SOCS3 cDNA specifically in T lineage cells. While total thymocyte numbers were not affected, SOCS3 Tg mice had selectively reduced percentage and numbers ( 50% reduction) of CD8SP thymocytes and CD8 T cells. These data suggested that SOCS3-dependent cytokine signals participate specifically in lineage commitment and maintenance of CD8 T cells. Such selective effect on CD8 T cells is analogue to the results observed in SOCS1 transgenic mice where SOCS1 overexpression induced a dramatic decrease ( greater than 80%) in CD8SP thymocytes only. Importantly, the effect of SOCS3 Tg was much blunted compared to SOCS1 Tg but when we generated SOCS3 SOCS1 double transgenic mice, we observed an additive effect of SOCS1 and SOCS3 on blocking CD8 T cell development and homeostasis. Thus, SOCS1 and SOCS3 have partially overlapping but also non-redundant functions in T cells, and we aim to investigate their roles during T cell activation in a series of inflammatory and autoimmune mouse models. Specifically, SOCS3 has been previously shown to suppress STAT3-dependent signaling, and based on recent findings on interleukin-17-secreting CD4+ helper 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. TCR stimulation upregulates CISH expression in both immature DP and mature T cells. Importantly, we found that upregulation of CISH expression was much more pronounced in TCR stimulated cells than in cytokine signaled cells. Thus, these data suggest that CISH expression might serve more than just a simple feedback signaling loop in limiting cytokine signaling. However, while CISH is known to inhibit STAT5 phosphorylation, the significance of blocking STAT5 signaling upon positive selection in DP thymocytes and upon activation in mature T cells is not known. To test the role of STAT5 inhibition by CISH, we have constructed transgenic vectors that express a FLAG-tagged CISH cDNA under the control of the human CD2 mini-cassette. These CISH transgenic mice have been now generated, and we are in the process of analyzing the T cell function of these mice. Collectively, we are investigating the functions of three SOCS family members, i.e. SOCS4, SOCS3 and CISH, and using a panel of transgenic mice and a series of in vitro studies, we are in the process of determining the biological role of regulating cytokine responsiveness by SOCS family molecules in T cell activation and homeostasis.