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

我们设计了一种多管齐下的方法来了解SOCS家族分子在T细胞中的作用和需求。我们培育了一系列缺乏或过表达SOCS4家族分子的基因工程小鼠，我们一直在分析SOCS表达改变如何影响T细胞功能。其中，我们一直关注SOCS4，因为我们发现它在未成熟胸腺细胞中高度表达，这表明它在T细胞成熟过程中具有潜在的作用。为此，我们利用基因诱捕ES细胞系统产生SOCS4缺陷小鼠，并通过实时反转录PCR验证了SOCS4不表达。对这些小鼠的总体分析没有显示出它们的发育异常。这些小鼠的详细表型和功能特征目前正在进行中。关于T细胞的发育，我们没有观察到缺乏SOCS4对胸腺中T细胞的产生有任何不利影响。然而，socs4缺陷T细胞的功能分析尚未完成，有待进一步的结果。为了检验强制SOCS4表达是否会影响T细胞，我们还生成了T细胞特异性SOCS4转基因小鼠。我们发现SOCS4过表达抑制T细胞发育和T细胞活化。具体来说，我们发现SOCS4的组成性表达损害了外周T细胞的存活和稳态，使初始T细胞数量显著减少，凋亡明显增加。了解SOCS4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SOCS4如何干扰T细胞发育和分化的机制。与SOCS4并行，我们还评估了SOCS3在T细胞发育和稳态中的表达。除了SOCS1， SOCS3是SOCS家族中唯一具有KIR结构域的分子。因此，提出了SOCS3和SOCS1的冗余角色。为了正式检验这种可能性，我们目前正在生成在T细胞中删除SOCS1和SOCS3的SOCS1/SOCS3条件KO小鼠。一旦生成，我们计划将其T细胞表型和功能与SOCS1或SOCS3单缺陷小鼠的T细胞表型和功能进行比较。此外，为了评估SOCS1和SOCS3是否能在抑制细胞因子信号传导方面发挥协同作用，我们培育了SOCS3转基因小鼠，其中转基因由人CD2迷你盒驱动。socs3转基因小鼠的整体T细胞发育与野生型小鼠相当，除了CD8SP胸腺细胞和外周CD8 T细胞数量的选择性损失（约减少50%）。这些数据与细胞因子对CD8谱系承诺和体内平衡的需求一致，它们表明SOCS3也在CD4/CD8谱系选择中发挥作用。为了直接确定SOCS3对细胞因子受体信号转导的影响，我们通过评估下游STAT6、STAT3、STAT5和STAT1的激活情况，分别检测了SOCS3转基因T细胞中IL-4、IL-6、IL-7和ifn - γ信号转导。有趣的是，我们发现SOCS3对所有细胞因子都有广泛的抑制作用，即使它们专有受体的表面表达没有改变。这些结果表明，与SOCS1类似，SOCS3也会干扰细胞因子受体信号，并可能利用直接抑制JAK激活的机制。与SOCS1、SOCS3和SOCS4在胸腺细胞和T细胞中高表达不同，我们发现Cish仅在静息T细胞中低水平表达。值得注意的是，Cish表达对TCR信号的反应也不同，因为它的表达是由TCR刺激上调的，而不是由细胞因子信号上调的。这与SOCS1和SOCS3的表达调控形成对比。这些结果表明Cish和其他SOCS家族成员在控制T细胞免疫反应中具有不同的作用。在T细胞中，Cish曾被报道抑制gc细胞因子对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。因此，为了进一步评估Cish的作用，我们在人类CD2迷你盒的控制下产生了表达flag标记的Cish cDNA的Cish转基因小鼠。在Cish表达增加的情况下，我们没有发现胸腺细胞发育或T细胞稳态发生任何重大变化，这表明Cish在稳态条件下确实影响T细胞功能。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传导有任何影响。为了确定Cish的确切下游靶点，我们目前正在进行利用Cish缺陷或过表达的T细胞的实验，并且我们正在绘制它们与野生型T细胞相比在激活和分化方面的差异。

We have devised a multi-pronged approach to understand the roles and requirements of SOCS family molecules in T cells. We have generated a series of genetically engineered mice that either lack or overexpress SOCS4 family molecules, and we have been analyzing how T cell functions are affected by altered expression of SOCS. Among others, we have been focusing on SOCS4, because we found it highly expressed in immature thymocytes, suggesting a potential role in the maturation process of T cells. To this end, we generated SOCS4-deficient mice utilizing a gene-trap ES cell system to produce SOCS4 deficient mice, and we verified the absence of SOCS4 expression by real-time reverse transcription PCR. Gross analysis of these mice did not show abnormalities in their development. Detailed phenotypic and functional characterization of these mice are currently under progress. Regarding T cell development, we did not observe any adverse effect of SOCS4 deficiency in producing T cells in the thymus. However, functional assays of SOCS4-deficient T cells have not been finished and awaits further results. To examine if enforced SOCS4 expression would affect T cells, we also generated a T cell-specific SOCS4 transgenic mouse. Here we found that SOCS4 overexpression suppresses T cell development and T cell activation. 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 apoptotis 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 how SOCS4 interferes with T cell development and differentiation. In parallel to SOCS4, we also assessed expression of SOCS3 during T cell development and homeostasis. In addition to SOCS1, SOCS3 is the only other SOCS family molecule with a KIR domain. Thus, redundant roles for SOCS3 and SOCS1 had been proposed. To formally examine this possibility, we are currently in the process of generating SOCS1/SOCS3-conditional KO mice that have deleted both SOCS1 and SOCS3 in T cells. Once generated, we plan to compare their T cell phenotype and function to those of SOCS1 or SOCS3 single deficient mice. Moreover, to assess whether SOCS1 and SOCS3 can exert synergistic effects on inhibiting cytokine signaling, we generated SOCS3 transgenic mice where the transgene is driven by a human CD2 mini-cassette. Overall T cell development in SOCS3-transgenic mice was comparable to wild type mice, except for a selective loss (about 50% reduction) of CD8SP thymocytes and peripheral CD8 T cell numbers. These data agree with a cytokine requirement for CD8 lineage commitment and homeostasis, and they demonstrate that SOCS3 also plays a role in CD4/CD8 lineage choice. To directly determine the effect of SOCS3 on cytokine receptor signaling, we examined IL-4, IL-6, IL-7 and IFN-gamma signaling in SOCS3-transgenic T cells by assessing downstream STAT6, STAT3, STAT5 and STAT1 activation, respectively. Interestingly, we found a broad inhibitory effect of SOCS3 on all cytokines that was tested, even as surface expression of their proprietary receptors was not altered. These results suggested that SOCS3, similar to SOCS1, interferes with cytokine receptor signaling, and that it potentially utilizes a mechanism that involves direct inhibition of JAK activation. Unlike SOCS1, SOCS3 and SOCS4 which are highly expressed in thymocytes and T cells, we found that Cish is expressed only at low levels in resting T cells. Notably, Cish expression also differed in its response to TCR signaling because its expression was 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 repsonses. In T cells, Cish was previously reported to inhibit STAT5phosphorylation by gc cytokines. However, why Cish expression is induced by TCR signaling, and not by cytokine signaling, was unclear to us. Thus, to further assess the role for Cish, we generated Cish transgenic mice that express a FLAG-tagged Cish cDNA under the control of the human CD2 mini-cassette. 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 utilizes Cish-deficient or overexpressing T cells, and we are mapping differences in their activation and differentiation compared to wildtype T cells.