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

为了了解SOCS家族分子在控制T细胞中细胞因子信号传导中的作用，我们利用多管齐下的方法来改变SOCS分子的表达并评估它们在T细胞发育和分化期间的作用。我们产生了一系列的基因工程小鼠，要么缺乏或过度表达个别SOCS家族分子。使用这些工具，我们在评估SOCS 3的作用方面取得了重大进展，SOCS 3被认为是IL-6信号通路的主要下游抑制因子。有趣的是，通过gc细胞因子如IL-7和IL-15的刺激也诱导SOCS 3的表达，因此我们怀疑SOCS 3在抑制gc细胞因子信号传导中的潜在作用。事实上，如预期的那样，在T细胞中过表达SOCS 3的基因工程小鼠抑制了IL-6信号传导，但我们发现SOCS 3也基本上抑制了gc细胞因子IL-2、IL-4和IL-7的下游信号传导。因为IL-2是Foxp 3 + Treg细胞产生的关键介质，我们进一步评估了SOCS 3转基因小鼠中Foxp 3 + Treg细胞的产生。正如预期的那样，我们观察到SOCS 3转基因小鼠胸腺中Foxp 3 + Treg细胞发育的实质性缺陷。此外，在体外刺激时，SOCS 3转基因小鼠的Foxp 3 + Treg细胞中的IL-2信号传导显著降低STAT 5磷酸化。最后，在IL-2和TGF-β存在下通过TCR活化将初始CD 4 T细胞体外分化为Foxp 3 + Treg细胞显示Treg细胞的产生显著受损。总的来说，这些结果揭示了SOCS 3在控制gc细胞因子信号传导中以及因此在抑制依赖于gc细胞因子如IL-2的细胞过程中的先前未被认识到的作用。除了SOCS 3之外，我们一直关注SOCS 4，因为我们发现它在未成熟的胸腺细胞中高度表达，这表明它在T细胞的成熟过程中具有潜在的作用。为了评估其在胸腺生成中的需求，我们利用基因捕获ES细胞系统产生SOCS 4缺陷小鼠，并且我们通过实时逆转录PCR验证了SOCS 4表达的缺乏。这些小鼠的总体表型分析未显示其发育异常。目前正在对这些小鼠进行详细的细胞和功能表征。然而，关于T细胞发育，我们没有观察到SOCS 4缺陷在胸腺中产生T细胞的任何不利影响，表明与其他SOCS家族分子的潜在冗余。我们目前正在评估SOCS 4缺陷型T细胞的功能方面。为了检查强制SOCS 4表达是否会影响T细胞，我们还产生了T细胞特异性SOCS 4转基因小鼠。在这里，我们发现SOCS 4过表达抑制T细胞的发育和分化。具体地说，我们发现SOCS 4的组成型表达损害外周T细胞的存活和稳态，使幼稚T细胞数量显着减少，细胞凋亡显着增加。了解SOCS 4在T细胞中的下游作用仍然是本研究的主要目的，我们希望进一步了解SOCS 4如何干扰T细胞发育和分化的机制。与在胸腺细胞和T细胞中高度表达的SOCS 1、SOCS 3和SOCS 4不同，我们发现Cish在静息T细胞中仅以低水平表达。值得注意的是，我们发现Cish表达通过TCR刺激而不是通过细胞因子信号传导上调，这与SOCS 1和SOCS 3表达的调节形成对比。这些结果表明Cish和其他SOCS家族成员在控制T细胞免疫应答中的不同作用。此前，Cish已被报道抑制gc细胞因子引起的STAT 5磷酸化。然而，为什么Cish表达是由TCR信号传导而不是细胞因子信号传导诱导的，我们还不清楚。我们现在已经产生了Cish转基因小鼠，其在人CD 2启动子/增强子的控制下表达FLAG标记的Cish cDNA。我们没有发现任何重大的变化，胸腺细胞的发展或T细胞稳态的存在下，增加Cish表达，表明Cish确实影响T细胞功能的稳态条件下。此外，我们也没有发现Cish过表达对细胞因子受体表达或信号传导的任何影响。为了确定Cish的确切下游靶点，我们目前正在进行利用Cish缺陷或Cish过表达T细胞的实验，并且我们正在绘制与野生型T细胞相比其活化和分化的差异。总之，我们期望SOCS家族成员的表达和功能的综合分析将为我们提供一个清晰的画面，细胞因子信号是如何控制在T细胞在其发展和分化。

To understand the roles of SOCS family molecules in controlling cytokine signaling in T cells, we utilized a multi-pronged approach to alter the expression of SOCS molecules and assess their effects during T cell development and differentiation. We generated a series of genetically engineered mice that either lack or overexpress individual SOCS family molecules. Using these tools, among others, we made significant advances in assessing the role of SOCS3, which has been considered a major downstream suppressor of the IL-6 signaling pathway. Interestingly, stimulation by gc cytokines such as IL-7 and IL-15 also induced expression of SOCS3, so that we suspected a potential role for SOCS3 in suppressing gc cytokine signaling. Indeed, genetically engineered mice that overexpress SOCS3 in T cells suppressed IL-6 signaling as expected, but we found that SOCS3 also substantially inhibited downstream signaling of the gc cytokines IL-2, IL-4, and IL-7. Because IL-2 is a critical mediator of Foxp3+ Treg cell generation, we further assessed the generation of Foxp3+ Treg cells in SOCS3 transgenic mice. As expected, we observed a substantial defect in Foxp3+ Treg cell development in the thymus of SOCS3 transgenic mice. Moreover, IL-2 signaling in Foxp3+ Treg cells of SOCS3-transgenic mice markedly decreased STAT5 phosphorylation when stimulated in vitro. Finally, in vitro differentiation of naive CD4 T cells into Foxp3+ Treg cells by TCR activation in the presence of IL-2 and TGF-beta showed dramatically impaired generation of Treg cells. Collectively, these results revealed a previously unappreciated role of SOCS3 in controlling gc cytokine signaling and consequently in suppressing cellular processes that depend on gc cytokines such as IL-2. In addition to SOCS3, we have been focusing on SOCS4, because we found it is highly expressed in immature thymocytes, suggesting a potential role in the maturation process of T cells. To assess its requirement in thymopoiesis, we generated SOCS4-deficient mice utilizing a gene-trap ES cell system, and we verified the absence of SOCS4 expression by real-time reverse transcription PCR. Gross phenotypic analysis of these mice did not show abnormalities in their development. Detailed cellular and functional characterization of these mice are currently under progress. Regarding T cell development, however, we did not observe any adverse effect of SOCS4 deficiency in producing T cells in the thymus, indicating potential redundancy with other SOCS-family molecules. We are currently assessing the functional aspects of SOCS4-deficient T cells. To examine if enforced SOCS4 expression would affect T cells, we also generated T cell-specific SOCS4-transgenic mice. Here, we found that SOCS4 overexpression suppresses the development and differentiation of T cells. Specifically, we found that constitutive expression of SOCS4 impaired peripheral T cell survival and homeostasis so that naive T cell numbers were significantly reduced, and apoptosis was markedly increased. Understanding the downstream effects of SOCS4 in T cells remains the major aim of this study, and we hope to gain further mechanistic insights on how SOCS4 interferes with T cell development and differentiation. Unlike SOCS1, SOCS3, and SOCS4 which are highly expressed in both thymocytes and T cells, we found that Cish is expressed only at low levels in resting T cells. Notably, we found that Cish expression upregulated by TCR stimulation and not by cytokine signaling, which contrasts to the regulation of SOCS1 and SOCS3 expression. These results suggested distinct roles for Cish and other SOCS family member in controlling T cell immune responses. Previously, Cish had been reported to inhibit STAT5 phosphorylation by gc cytokines. However, why Cish expression is induced by TCR signaling, and not by cytokine signaling, was unclear to us. We have now generated Cish-transgenic mice that express a FLAG-tagged Cish cDNA under the control of the human CD2 promoter/enhancer. We did not find any major changes in thymocyte development or T cell homeostasis in the presence of increased Cish expression, indicating that Cish does affect T cell function under steady-state condition. Moreover, we also did not find any effects of Cish overexpression on cytokine receptor expression or signaling. To identify the exact downstream targets of Cish, we are currently performing experiments that utilize Cish-deficient or Cish-overexpressing T cells, and we are mapping differences in their activation and differentiation compared to wildtype T cells. Altogether, we expect that the comprehensive analysis of SOCS family member expression and function will provide us a clear picture of how cytokine signaling is controlled in T cells during their development and differentiation.