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

为了了解SOCS家族分子在控制T细胞细胞因子信号转导中的作用，我们采用多管齐下的方法改变SOCS分子的表达，并评估它们在T细胞发育和分化过程中的作用。我们产生了一系列基因工程小鼠，它们要么缺乏或过度表达单个SOCS家族分子。使用这些工具和其他工具，我们在评估SOCS3的作用方面取得了重大进展，SOCS3被认为是IL-6信号通路的主要下游抑制因子。有趣的是，IL-7和IL-15等GC细胞因子的刺激也诱导了SOCS3的表达，因此我们怀疑SOCS3在抑制GC细胞因子信号转导中的潜在作用。事实上，在T细胞中过表达SOCS3的基因工程小鼠如预期的那样抑制了IL-6信号，但我们发现SOCS3也实质上抑制了GC细胞因子IL-2、IL-4和IL-7的下游信号。由于IL-2是Foxp3+Treg细胞生成的关键介质，我们进一步评估了SOCS3转基因小鼠中Foxp3+Treg细胞的生成。正如预期的那样，我们在SOCS3转基因小鼠的胸腺中观察到Foxp3+Treg细胞发育的实质性缺陷。此外，SOCS3转基因小鼠Foxp3+Treg细胞中的IL-2信号在体外刺激时显著降低STAT5的磷酸化。最后，在IL-2和转化生长因子-β的存在下，通过TCR激活将原始的CD4T细胞在体外分化为Foxp3+Treg细胞，结果显示Treg细胞的生成显著受损。总而言之，这些结果揭示了SOCS3在控制GC细胞因子信号转导从而抑制依赖于GC细胞因子(如IL-2)的细胞过程中先前未被认识到的作用。除了SOCS3，我们一直关注SOCS4，因为我们发现它在未成熟的胸腺细胞中高表达，这表明它在T细胞的成熟过程中可能起到作用。为了评估其在胸腺生成中的需求，我们利用基因陷阱ES细胞系统产生了SOCS4缺陷小鼠，并通过实时逆转录聚合酶链式反应证实了SOCS4基因的缺失。对这些小鼠的大体表型分析没有显示它们的发育异常。这些小鼠的详细细胞和功能特征目前正在进行中。然而，在T细胞发育方面，我们没有观察到SOCS4缺乏对胸腺产生T细胞的任何不利影响，这表明可能存在与其他SOCS家族分子的冗余。我们目前正在评估SOCS4缺陷T细胞的功能。为了检验强制表达SOCS4是否会影响T细胞，我们还培育了T细胞特异性SOCS4转基因小鼠。在这里，我们发现SOCS4的过度表达抑制了T细胞的发育和分化。具体地说，我们发现SOCS4的结构性表达损害了外周T细胞的存活和动态平衡，导致初始T细胞数量显著减少，细胞凋亡率显著增加。了解SOCS4在T细胞中的下游效应仍然是本研究的主要目的，我们希望进一步从机制上了解SOCS4如何干扰T细胞的发育和分化。与在胸腺细胞和T细胞中高表达的SOCS1、SOCS3和SOCS4不同，我们发现仅在静息T细胞中低水平表达CISH。值得注意的是，我们发现CISH的表达受到TCR刺激而不是细胞因子信号的上调，这与SOCS1和SOCS3的表达调节相反。这些结果表明，CISH和其他SOCS家族成员在控制T细胞免疫反应中起着不同的作用。在此之前，已有报道称CISH可通过GC细胞因子抑制STAT5的磷酸化。然而，为什么CISH的表达是由TCR信号诱导的，而不是由细胞因子信号诱导的，我们还不清楚。我们现在已经产生了在人类CD2启动子/增强子的控制下表达标志标记的CISH基因的转基因小鼠。我们没有发现胸腺细胞发育或T细胞稳态的任何重大变化，这表明在稳态条件下，CISH确实影响T细胞的功能。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转导的任何影响。为了确定CISH的确切下游靶点，我们目前正在进行利用CISH缺乏或过度表达的T细胞的实验，我们正在绘制与野生型T细胞相比，它们的激活和分化方面的差异。总之，我们希望对SOCS家族成员的表达和功能的全面分析将为我们提供一个清晰的图景，说明T细胞在发育和分化过程中细胞因子信号是如何调控的。

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.