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细胞中的作用和要求。我们已经产生了一系列缺乏或过度表达SOCS 4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，表明在T细胞的成熟过程中的潜在作用。为此，我们利用基因捕获ES细胞系统产生SOCS 4缺陷小鼠，并通过实时逆转录PCR验证了SOCS 4表达的缺失。这些小鼠的大体分析未显示其发育异常。目前正在对这些小鼠进行详细的表型和功能表征。关于T细胞发育，我们没有观察到SOCS 4缺乏对胸腺中产生T细胞的任何不利影响。然而，SOCS 4缺陷型T细胞的功能测定尚未完成，并等待进一步的结果。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细胞发育和T细胞活化。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，并显着增加。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，我们希望获得SOCS 4如何干扰T细胞发育和分化的进一步机制见解。与SOCS 4平行，我们还评估了SOCS 3在T细胞发育和稳态期间的表达。除了SOCS 1，SOCS 3是唯一具有KIR结构域的其他SOCS家族分子。因此，已提出SOCS 3和SOCS 1的冗余作用。为了正式研究这种可能性，我们目前正在产生SOCS 1/SOCS 3条件性KO小鼠，这些小鼠在T细胞中缺失了SOCS 1和SOCS 3。一旦产生，我们计划将它们的T细胞表型和功能与SOCS 1或SOCS 3单缺陷小鼠进行比较。此外，为了评估SOCS 1和SOCS 3是否可以对抑制细胞因子信号传导发挥协同作用，我们产生了SOCS 3转基因小鼠，其中转基因由人CD 2微型盒驱动。SOCS 3转基因小鼠的总体T细胞发育与野生型小鼠相当，除了CD 8 SP胸腺细胞和外周CD 8 T细胞数量的选择性损失（约50%减少）。这些数据与CD 8谱系定型和稳态的细胞因子要求一致，并且它们证明SOCS 3也在CD 4/CD 8谱系选择中起作用。为了直接确定SOCS 3对细胞因子受体信号传导的影响，我们通过分别评估下游STAT 6、STAT 3、STAT 5和STAT 1活化来检查SOCS 3转基因T细胞中的IL-4、IL-6、IL-7和IFN-γ信号传导。有趣的是，我们发现SOCS 3对所有测试的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，SOCS 3，类似于SOCS 1，干扰细胞因子受体信号传导，并且它可能利用涉及直接抑制JAK活化的机制。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，Cish表达在其对TCR信号传导的响应方面也不同，因为其表达通过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细胞免疫应答中的不同作用。在T细胞中，Cish先前被报道通过gc细胞因子抑制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。因此，为了进一步评估Cish的作用，我们产生了Cish转基因小鼠，其在人CD 2微型盒的控制下表达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。

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.