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

为了理解SOCS家族分子在控制T细胞中细胞因子信号传导中的作用，我们利用一系列基因工程小鼠来改变SOCS蛋白在T细胞发育和分化时的丰度、时序和调节。最近，我们总结了一项关于SOCS 3及其细胞因子特异性的研究，揭示了与我们目前的观点相反，SOCS 3不仅抑制gp 130家族的细胞因子的信号传导，而且抑制gc家族的细胞因子，包括IL-2，IL-4和IL-7。我们先前记录了由gc细胞因子（如IL-7和IL-2）刺激T细胞诱导SOCS 3的表达，这表明SOCS 3在抑制gc细胞因子信号传导中的潜在作用。事实上，在T细胞中过表达SOCS 3的基因工程小鼠除了抑制IL-6信号传导之外，还有效地抑制了T细胞中的IL-2、IL-4和IL-7信号传导，IL-6信号传导迄今为止被认为是SOCS 3抑制的主要靶标。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，当在体外刺激时，SOCS 3转基因Foxp 3 + Treg细胞中的IL-2信号传导显示出显著降低的STAT 5磷酸化。最后，在IL-2和TGF-β存在下，TCR活化的初始CD 4 T细胞体外分化为Foxp 3 + Treg细胞显示iTreg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，并因此在抑制依赖于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。这项工作最近发表在《欧洲免疫学杂志》上。我们现在通过询问SOCS 1和SOCS 3在抑制gc细胞因子信号传导中的潜在冗余来跟进这项研究。如我们发表的研究所述，IL-7信号转导上调SOCS 1和SOCS 3的表达。目前尚不清楚这两种分子的共表达是否是有效抑制细胞因子信号传导所必需的，以及两种分子的组合消融是否会诱导比仅缺失SOCS 1或SOCS 3不同的效果。由于SOCS 3缺陷是胚胎致死性的，并且由于SOCS 1缺陷是围产期致死性的，因此我们目前正在使SOCS 1和SOCS 3 floxed小鼠杂交以产生SOCS 1/3双floxed小鼠，并且我们的目标是使用T细胞特异性Cre转基因来删除SOCS 1/3表达。我们期望利用这种小鼠模型进一步了解SOCS 1/3活性的分子途径。除了SOCS 3之外，我们发现了解非经典SOCS分子的作用也很重要。因此，我们一直专注于SOCS 4，因为我们发现它在未成熟的胸腺细胞中高度表达。SOCS 4在胸腺细胞中的高度调节表达表明在T细胞的发育中具有潜在的作用。因此，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（SOCS 4-KO小鼠），并且我们通过实时逆转录PCR和Western印迹分析验证了SOCS 4表达的缺乏。SOCS 4-KO小鼠的总体表型分析未显示其发育异常。关于胸腺T细胞发育，我们也没有观察到SOCS 4缺乏的任何不利影响，因此CD 4和CD 8 T细胞谱系定型和成熟保持不变。这些结果表明SOCS 4与其他SOCS家族分子的潜在冗余，这仍有待研究。此外，SOCS 4是否是必需的干扰素介导的效应T细胞的产生需要进行评估，我们目前正在分析这种SOCS 4缺陷型T细胞的功能方面。总而言之，了解SOCS 4在T细胞中的作用和需求仍然是本研究的主要目的，我们希望进一步了解SOCS 4是否以及如何与T细胞发育和分化中的细胞因子信号通路交叉。最后，我们还研究了SOCS家族的另一个重要成员Cish的作用，Cish被提议控制T细胞中的细胞因子和抗原受体信号传导。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。此外，Cish表达通过TCR刺激而不是通过细胞因子信号传导显著上调，这与通过细胞因子刺激上调的SOCS 1和SOCS 3的诱导形成对比。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。虽然据报道Cish通过gc细胞因子抑制STAT 5磷酸化，但我们不清楚为什么Cish表达将由TCR信号传导而不是由细胞因子信号传导诱导。我们假设Cish在TCR与细胞因子受体信号传导的串扰中具有以前未被认识的作用，并且我们怀疑Cish可能在TCR介导的细胞因子受体信号传导的脱敏中发挥作用。为了鉴定和定位Cish的下游靶点，我们目前正在进行利用Cish缺陷的T细胞或被迫表达Cish的T细胞的实验。因此，在存在或不存在Cish的情况下绘制细胞因子信号传导的差异将使我们能够进一步理解Cish在抑制细胞因子受体信号传导中的作用。总之，我们期望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画面，如何在T细胞的发育和分化过程中的细胞因子信号的负调控回路控制。

To understand the role of SOCS family molecules in controlling cytokine signaling in T cells, we utilized a series of genetically engineered mice to alter the abundance, timing and regulation of SOCS proteins upon T cell development and differentiation. Most recently, we concluded a study on SOCS3 and its cytokine specificity to reveal that, contrary to our current view, SOCS3 does not only suppress the signaling of cytokines of the gp130 family but also cytokines of the gc family, including IL-2, IL-4 and IL-7. We previously documented that T cell stimulation by gc cytokines, such as IL-7 and IL-2, induced the expression of SOCS3, suggesting a potential role for SOCS3 in suppressing gc cytokine signaling. Indeed, genetically engineered mice that overexpress SOCS3 in T cells potently suppressed IL-2, IL-4, and IL-7 signaling in T cells, in addition to suppressing IL-6 signaling which so far had been considered the major target of SOCS3 suppression. 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 SOCS3-transgenic Foxp3+ Treg cells showed markedly decreased STAT5 phosphorylation when stimulated in vitro. Finally, the in vitro differentiation of TCR-activated naive CD4 T cells into Foxp3+ Treg cells in the presence of IL-2 and TGF-beta showed dramatically impaired generation of iTreg 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. This work has been recently published in the European Journal of Immunology. We are now following up on this study by asking potential redundancy between SOCS1 and SOCS3 in suppressing gc cytokine signaling. As described in our published study, IL-7 signaling upregulates the expression of both SOCS1 and SOCS3. It is currently unclear whether the co-expression of these two molecules is necessary for effective suppression of cytokine signaling, and whether the combined ablation of both molecules would induce a distinct effect than the deletion of only SOCS1 or SOCS3. Because SOCS3-deficiency is embryonic lethal and because SOCS1-deficiency is perinatal lethal, we are currently in the process of intercrossing SOCS1 and SOCS3 floxed mice to generate SOCS1/3 double floxed mice, and we aim to delete the SOCS1/3 expression using a T cell specific Cre transgene. We expect to gain further insight into the molecular pathways of SOCS1/3 activity, utilizing this mouse model. In addition to SOCS3, we found it important to understand the role of non-classical SOCS molecules. As such, we have been focusing on SOCS4, because we found it is highly expressed in immature thymocytes. The highly regulated expression of SOCS4 in thymocytes suggested a potential role in the development of T cells. Thus, to assess its requirement in thymopoiesis, we generated SOCS4-deficient mice utilizing a gene-trap ES cell system (SOCS4-KO mice), and we verified the absence of SOCS4 expression by real-time reverse transcription PCR and Western blot analysis. Gross phenotypic analysis of SOCS4-KO mice did not show abnormalities in their development. Regarding the T cell development thymus, we also did not observe any adverse effect of SOCS4 deficiency so that CD4 and CD8 T cell lineage commitment and maturation remained unaltered. These results suggested a potential redundancy of SOCS4 with other SOCS-family molecules, which remains to be examined. Also, whether SOCS4 is required for the cytokine-mediated generation of effector T cells need to be assessed, and we are currently analyzing the functional aspects of such SOCS4-deficient T cells. Altogether, understanding the role and requirement for SOCS4 in T cells remains a major aim of this study, and we hope to gain further mechanistic insights on whether and how SOCS4 intersect with cytokine signaling pathways in T cell development and differentiation. Finally, we are also investigating the role of another prominent member of the SOCS family, Cish, which is proposed to control both cytokine and antigen receptor signaling in T cells. 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. Moreover, Cish expression was dramatically upregulated by TCR stimulation and not by cytokine signaling, which contrasts to the induction of SOCS1 and SOCS3 that are upregulated by cytokine stimulation. These results suggested distinct roles for Cish and other SOCS family member in controlling T cell immune responses. While Cish is reported to inhibit STAT5 phosphorylation by gc cytokines, it is unclear to us why Cish expression would be induced by TCR signaling, and not by cytokine signaling. We hypothesize a previously unappreciated role for Cish in the crosstalk of TCR versus cytokine receptor signaling, and we suspect that Cish could play a role in TCR-mediated desensitization of cytokine receptor signaling. To identify and map the downstream targets of Cish, we are currently performing experiments that utilize T cells that are deficient for Cish or T cells that are forced to express Cish. Thus, mapping differences in cytokine signaling in the presence or absence of Cish will allow us the further understand the role of Cish in suppressing cytokine receptor signaling. Altogether, we expect that the comprehensive analysis of SOCS family member expression and function will provide us a clear picture of how the negative regulatory loop of cytokine signaling is controlled in T cells during their development and differentiation.