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转导中的作用，我们利用一系列基因工程小鼠来改变SOCS蛋白的丰度、时机和对T细胞发育和分化的调节。最近，我们总结了一项关于SOCS3及其细胞因子特异性的研究，发现与我们目前的观点相反，SOCS3不仅抑制gp130家族的细胞因子信号转导，而且还抑制GC家族的细胞因子，包括IL-2、IL-4和IL-7。我们先前的研究表明，在IL-7和IL-2等GC细胞因子的刺激下，T细胞可诱导SOCS3的表达，提示SOCS3在抑制GC细胞因子信号转导中具有潜在的作用。事实上，在T细胞中过表达SOCS3的基因工程小鼠，除了抑制迄今为止被认为是抑制SOCS3的主要目标的IL-6信号外，还有效地抑制了T细胞中的IL-2、IL-4和IL-7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，转SOCS3基因的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，TCR激活的初始CD4T细胞在体外分化为Foxp3+Treg细胞显示iTreg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细胞因子信号转导中先前未被认识到的作用，并因此在抑制依赖于GC细胞因子的细胞过程中，如IL-2。这项研究最近发表在《欧洲免疫学杂志》上。我们现在正在跟进这项研究，通过研究SOCS1和SOCS3之间在抑制GC细胞因子信号方面的潜在冗余。正如我们发表的研究中所描述的，IL-7信号上调SOCS1和SOCS3的表达。目前尚不清楚这两个分子的共同表达是否是有效抑制细胞因子信号所必需的，也不清楚联合去除这两个分子是否会产生比单独缺失SOCS1或SOCS3更明显的效果。由于SOCS3缺乏是胚胎致死的，而且SOCS1缺乏是围产期致命的，我们目前正在杂交SOCS1和SOCS3小鼠以产生SOCS1/3双花小鼠，我们的目标是通过T细胞特异性Cre转基因来删除SOCS1/3的表达。我们希望利用这个小鼠模型进一步深入了解SOCS1/3活性的分子途径。除了SOCS3，我们发现了解非经典SOCS分子的作用是很重要的。因此，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度表达。SOCS4在胸腺细胞中的高度调控表达提示，SOCS4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，我们利用基因陷阱ES细胞系统建立了SOCS4缺陷小鼠(SOCS4-KO小鼠)，并通过实时逆转录聚合酶链式反应和Western印迹分析证实了SOCS4的缺失。SOCS4-KO小鼠的大体表型分析没有显示其发育异常。对于胸腺T细胞的发育，我们也没有观察到SOCS4缺乏的任何不良影响，使CD4和CD8T细胞的谱系承诺和成熟保持不变。这些结果表明，SOCS4与其他SOCS家族分子之间存在潜在的冗余，这一点还有待研究。此外，是否SOCS4对于细胞因子介导的效应性T细胞的产生是必需的还需要评估，我们目前正在分析这种SOCS4缺陷的T细胞的功能。总之，了解SOCS4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SOCS4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相交。最后，我们还在研究SoCS家族的另一个重要成员--CISH的作用，它被认为控制T细胞中的细胞因子和抗原受体信号。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。此外，与细胞因子刺激上调SOCS1和SOCS3的诱导不同，TCR刺激显著上调了CISH的表达，而细胞因子信号转导却没有上调。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。虽然已有报道称CISH可抑制Gc细胞因子对STAT5的磷酸化，但我们尚不清楚为什么CISH的表达是由TCR信号而不是细胞因子信号诱导的。我们假设，在TCR和细胞因子受体信号的串扰中，CISH扮演了以前未被认识到的角色，我们怀疑CISH可能在TCR介导的细胞因子受体信号的脱敏中发挥作用。为了识别和定位CISH的下游靶点，我们目前正在进行实验，利用缺乏CISH的T细胞或被迫表达CISH的T细胞。因此，绘制细胞因子信号在存在或不存在情况下的差异图谱将使我们进一步了解CISH在抑制细胞因子受体信号转导中的作用。综上所述，我们期望对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如何控制细胞因子信号的负调控循环。

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.