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

由于发现SOCS 4表达在DN向DP过渡期间上调，但随后在阳性选择后下调，因此我们想评估T细胞发育期间这种阶段特异性表达是否是维持正常胸腺生成所必需的。为此，我们产生了在人CD 2微型盒控制下表达小鼠SOCS 4 cDNA的SOCS 4转基因小鼠。通过在纯化的T细胞中的定量RT-PCR，我们鉴定了具有分级水平的转基因SOCS 4表达的三种不同的SOCS 4转基因系。与转基因SOCS 4表达水平平行，我们观察到胸腺细胞绝对数量的显著减少，这表明SOCS 4的组成性过表达扰乱了T细胞发育。SOCS 4 Tg胸腺细胞的进一步分析揭示了DN 3至DN 4阶段的发育阻滞，这也通过DP胸腺细胞上CD 25表达的增加来证实。这种缺陷表明在DN胸腺细胞中前TCR信号传导或IL-7信号传导中存在潜在问题。然而，如通过IL-7刺激后的STAT 5磷酸化所评估的，IL-7信号传导在SOCS 4 Tg T细胞中是正常的。为了测试除STAT 5之外的其他STAT信号传导途径受到影响的可能性，接下来我们评估了SOCS 4 Tg T细胞中IL-6诱导的STAT 3和IL-4诱导的STAT 6磷酸化。奇怪的是，这些细胞因子信号传导途径中没有一个受到SOCS 4过表达的影响，这使得其他下游信号传导途径如PI-3 K或MAPK活化成为SOCS 4介导的细胞因子抑制的靶点的可能性是开放的。重要的是，我们不排除SOCS 4可能与TCR信号通路交叉的可能性，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SOCS 4表达的主动调节对于适当的T细胞分化是必要的，并且来自外周淋巴器官的初步数据甚至进一步表明SOCS 4在T细胞维持中的作用。SOCS 3在T细胞和胸腺细胞中的表达类似于SOCS 1的表达，我们希望分析SOCS 3过表达对T细胞功能的影响。为此，我们产生了在T谱系细胞中特异性表达小鼠SOCS 3 cDNA的SOCS 3转基因小鼠。虽然胸腺细胞总数不受影响，但SOCS 3 Tg小鼠选择性降低了CD 8 SP胸腺细胞和CD 8 T细胞的百分比和数量（减少50%）。这些数据表明SOCS 3依赖性细胞因子信号特异性参与CD 8 T细胞的谱系定型和维持。这种对CD 8 T细胞的选择性作用类似于在SOCS 1转基因小鼠中观察到的结果，其中SOCS 1过表达仅诱导CD 8 SP胸腺细胞的显著减少（大于80%）。重要的是，与SOCS 1 Tg相比，SOCS 3 Tg的作用减弱得多，但当我们产生SOCS 3 SOCS 1双转基因小鼠时，我们观察到SOCS 1和SOCS 3对阻断CD 8 T细胞发育和稳态的累加作用。因此，SOCS 1和SOCS 3在T细胞中具有部分重叠但非冗余的功能，我们的目标是在一系列炎症和自身免疫小鼠模型中研究它们在T细胞活化过程中的作用。具体地说，SOCS 3先前已显示抑制STAT 3依赖性信号传导，并且基于最近对分泌白细胞介素-17的CD 4+辅助性T细胞的发现，（Th 17细胞）和它们对STAT 3信号传导的需求，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，例如实验性自身免疫性脑脊髓炎（EAE），在Th 17细胞分化和活化的情况下。TCR刺激上调CISH在未成熟DP和成熟T细胞中的表达。重要的是，我们发现CISH表达的上调在TCR刺激的细胞中比在细胞因子信号传导的细胞中明显得多。因此，这些数据表明，CISH表达可能不仅仅是一个简单的反馈信号循环，在限制细胞因子信号。然而，虽然已知CISH抑制STAT 5磷酸化，但是在DP胸腺细胞中的阳性选择和在成熟T细胞中的活化后阻断STAT 5信号传导的意义尚不清楚。为了测试CISH抑制STAT 5的作用，我们构建了转基因载体，其在人CD 2微型盒的控制下表达FLAG标记的CISH cDNA。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SOCS 4，SOCS 3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细胞活化和稳态中调节细胞因子反应性的生物学作用。

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.