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

在我们了解SOCS分子在T细胞中的作用的研究中，我们在评估SOCS3的作用方面取得了进展，SOCS3被认为是IL-6信号通路的主要下游抑制因子。有趣的是，除了IL-6的刺激外，gc细胞因子如IL-7和IL-15的刺激也能诱导T细胞中SOCS3的表达。因此，我们怀疑SOCS3在抑制gc细胞因子信号传导方面也有潜在的作用。我们在T细胞中产生并检测了过表达SOCS3的转基因小鼠，我们发现SOCS3显著抑制gc细胞因子IL-2、IL-4和IL-7的下游信号传导。这项研究最近发表[Luckey MA et al., 2020, Eur.]j . Immunol。]，在这里，我们记录了SOCS3在控制IL-2信号传导中的作用。由于IL-2是Foxp3+ Treg细胞发育的关键介质，我们首先评估了SOCS3转基因小鼠Foxp3+ Treg细胞的产生。在这里，我们观察到SOCS3转基因小鼠胸腺中Foxp3+ Treg细胞发育的实质性缺陷。此外，IL-2在体外刺激下，SOCS3转基因小鼠Foxp3+ Treg细胞中的IL-2信号通路显著降低STAT5磷酸化。最后，在IL-2和tgf - β存在下，通过TCR激活初始CD4 T细胞向Foxp3+ Treg细胞的体外分化，Foxp3+ Treg细胞的生成明显受损。总的来说，我们的研究结果表明，除了对IL-6和其他gp130家族细胞因子有抑制作用外，SOCS3还是gc细胞因子信号传导的有效负调节因子。因此，这些结果进一步表明，应该在包括gc细胞因子在内的更广泛的背景下考虑SOCS3的诱导及其在抑制细胞因子信号传导中的作用。由于SOCS3含有一个可以直接抑制JAK1和JAK3激酶的KIR结构域，我们目前正在研究SOCS3干扰gc细胞因子信号传导的详细机制，以及它是否利用相同的机制抑制IL-6信号传导。我们还研究了SOCS3在CD8记忆T细胞生成和iNKT细胞发育和功能中抑制其他gc细胞因子的作用。SOCS4仍然是SOCS家族中一个特征不明显的成员，它在未成熟胸腺细胞中大量表达，但在成熟T细胞中下调。SOCS4的这种发育调控表达提示其在T细胞成熟过程中具有潜在的作用。为了研究其在胸腺发育中的作用和需求，我们最近利用基因诱捕ES细胞系统培养了缺乏socs4的小鼠。我们通过实时反转录PCR验证了SOCS4 mRNA表达的缺失。socs4缺陷小鼠的总体表型分析未显示其发育异常，socs4缺陷小鼠按孟德尔比例出生。关于胸腺T细胞的发育，我们也没有观察到缺乏socs4的任何不良影响。是否缺乏任何可识别的影响是由于潜在的冗余与其他soc家族分子目前尚不清楚。为了解决这种可能性，我们从socs4缺陷小鼠和野生型小鼠中分离了初始CD8 T细胞，并进行了大量RNAseq分析。值得注意的是，我们没有发现其他SOCS家族成员的表达有任何显著差异，这表明在socs4缺陷T细胞中，不太可能存在上调其他SOCS分子表达的补偿作用。尽管如此，我们观察到缺乏SOCS4的T细胞中TCR反应性显著增加，这表明SOCS4可能与TCR信号通路交叉。奇怪的是，缺乏socs4的胸腺细胞和T细胞的细胞因子信号没有改变。缺乏SOCS4是否会干扰T细胞的激活和分化尚不清楚，我们目前正在评估缺乏SOCS4的T细胞的功能方面。为了检验强制表达SOCS4是否会影响T细胞中的TCR或细胞因子信号传导，我们还生成了T细胞特异性SOCS4转基因小鼠。在这里，我们发现SOCS4过表达抑制T细胞的发育和分化。具体来说，我们发现SOCS4的组成性过表达会损害外周T细胞的存活和稳态，从而使初始T细胞数量显著减少。沿着这些思路，T细胞凋亡在SOCS4转基因小鼠中显著增加。SOCS4如何干扰TCR或细胞因子信号从而导致T细胞发育和稳态受损的详细机制尚不清楚，解决这一问题仍然是本研究的主要目的。除了SOCS4和SOCS3，我们还继续研究了SOCS1调控的机制方面。虽然我们之前已经证明核因子ThPOK上调SOCS1（和SOCS3）的转录[Luckey MA et al., 2014, Nat. immuno1]。]， ThPOK仅在CD4 T细胞中表达，在CD8 T细胞和未成熟胸腺细胞中不表达。因此，目前尚不清楚SOCS1是如何在CD4、CD8双阳性胸腺细胞或细胞因子刺激的CD8 T细胞中诱导的。为此，我们获得了SOCS1报告小鼠，其中人类CD4 cDNA在STOP卡带后插入SOCS1基因。使用胸腺细胞特异性Cre转基因删除STOP磁带允许人类CD4报告基因的表达，并且我们能够利用这些小鼠在体内和体外不同条件下监测SOCS1的转录。目前，我们正在将这些SOCS1报告小鼠扩展到不同的疾病模型中，以更好地了解SOCS1在稳态和炎症条件下的调节机制。与SOCS1、SOCS3和SOCS4在胸腺细胞和T细胞中均高表达不同，我们发现SOCS家族成员Cish仅在静息T细胞中低水平表达。此外，Cish的表达受TCR刺激而非细胞因子信号通路的上调，而SOCS1和SOCS3的表达受细胞因子信号通路而非TCR信号通路的诱导。这些结果提示Cish和其他SOCS家族成员在控制T细胞免疫应答中具有不同的作用。此前，有报道称Cish可抑制gc细胞因子对STAT5的磷酸化。然而，TCR信号而非细胞因子信号诱导Cish的生物学意义尚不清楚。为了解决这个问题，我们在人类CD2启动子/增强子的控制下产生了Cish转基因小鼠，这些小鼠表达flag标记的Cish cDNA，因此Cish在所有T系细胞中都过表达。奇怪的是，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在稳态条件下不会影响T细胞功能。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，因此Cish转基因和野生型T细胞之间的gc信号传导仍然具有可比性。为了确定Cish的下游靶点，我们目前正在进行利用Cish缺陷或过表达Cish的T细胞的实验，我们正在绘制它们与野生型T细胞在激活和分化方面的差异。总之，我们期望对SOCS家族成员表达和功能的全面分析将为我们提供T细胞在发育和分化过程中如何控制细胞因子信号传导的清晰图像。

In our studies to understand the role of SOCS molecules in T cells, we made progress in assessing the effect of SOCS3 which has been considered a major downstream suppressor of the IL-6 signaling pathway. Interestingly, not only stimulation by IL-6 but also stimulation by gc cytokines, such as IL-7 and IL-15, also induced the expression of SOCS3 in T cells. Therefore, we suspected a potential role for SOCS3 in suppressing gc cytokine signaling also. We have generated and examined transgenic mice that overexpress SOCS3 in T cells, and we found that SOCS3 significantly suppressed the downstream signaling of the gc cytokines IL-2, IL-4 and IL-7. This study was recently published [Luckey MA et al., 2020, Eur. J. Immunol.], and here we documented a previously underappreciated role of SOCS3 in controlling IL-2 signaling. Because IL-2 is a critical mediator of Foxp3+ Treg cell development, we first assessed the generation of Foxp3+ Treg cells in SOCS3 transgenic mice. Here, 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 showed markedly decreased STAT5 phosphorylation when stimulated with IL-2 in vitro. Finally, the 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 Foxp3+ Treg cells. Collectively, our results revealed and documented that SOCS3 is a potent negative regulator of gc cytokine signaling, in addition to its suppressive effect on IL-6 and other gp130 family cytokines. Therefore, these results further suggest that the induction of SOCS3 and its role in inhibiting cytokine signaling should be considered in a broader context that also includes gc cytokines. Because SOCS3 contains a KIR domain that can directly inhibit JAK1 and JAK3 kinases, we are currently investigating the detailed mechanism how SOCS3 would interfere with gc cytokine signaling and whether it utilizes the same mechanism to suppress IL-6 signaling. We are also addressing the role of SOCS3 in the suppression of other gc cytokines in the context of CD8 memory T cell generation, and iNKT cell development and function. SOCS4 remains a poorly characterized member of the SOCS family, which is abundantly expressed in immature thymocytes but downregulated on mature T cells. Such developmentally regulated expression of SOCS4 suggested a potential role in the process of T cell maturation. To examine its role and requirement in thymopoiesis, we have recently generated SOCS4-deficient mice utilizing a gene-trap ES cell system. We were able to verify the absence of SOCS4 mRNA expression by real-time reverse transcription PCR. Gross phenotypic analysis of SOCS4-deficient mice did not show abnormalities in their development, and SOCS4-deficient mice were born at Mendelian ratio. Regarding the T cell development in the thymus, we also did not observe any adverse effect of SOCS4-deficiency. Whether the lack of any discernible effects is due to potential redundancy with other SOCS-family molecules is currently not clear to us. To address this possibility, we isolated naive CD8 T cells from SOCS4-deficient and wildtype mice and performed bulk RNAseq analysis. Notably, we did not find any significant differences in the expression of other SOCS family members, suggesting that it is unlikely that there is a compensatory effect of upregulating the expression of other SOCS molecules in SOCS4-deficient T cells. Nonetheless, we observed a significant increase in TCR responsiveness in SOCS4-deficient T cells, indicating that SOCS4 might intersect with the TCR signaling pathway. Curiously, cytokine signaling in SOCS4-deficient thymocytes and T cells was unaltered. Whether the lack of SOCS4 would interfere with the activation and differentiation of T cells is yet unclear to us, and we are currently assessing the functional aspects of SOCS4-deficient T cells. To examine if the forced expression of SOCS4 would affect TCR or cytokine signaling in T cells, we also generated T cell-specific SOCS4-transgenic mice. Here, we found that SOCS4 overexpression suppressed the development and differentiation of T cells. Specifically, we found that constitutive overexpression of SOCS4 impaired peripheral T cell survival and homeostasis so that naive T cell numbers were significantly reduced. Along these lines, T cell apoptosis was markedly increased in SOCS4 transgenic mice. The detailed mechanisms how SOCS4 would interfere with TCR or cytokine signaling so that it would lead to impaired T cell development and homeostasis is not clear to us, and addressing this issue remains a major aim of this study. In addition to SOCS4 and SOCS3, we also continued our research on the mechanistic aspect of SOCS1 regulation. While we have previously shown that the nuclear factor ThPOK upregulates the transcription of SOCS1 (and SOCS3)[Luckey MA et al., 2014, Nat. Immunol.], ThPOK is only expressed in CD4 T cells and absent on CD8 T cells and immature thymocytes. Therefore, it has not been clear how SOCS1 is induced in CD4, CD8 double positive thymocytes or in cytokine stimulated CD8 T cells. To this end, we obtained SOCS1 reporter mice where a human CD4 cDNA is inserted into the SOCS1 gene after a STOP cassette. Deletion of the STOP cassette using thymocyte specific Cre transgenes permitted the expression of the human CD4 reporter, and we were able to utilize these mice to monitor SOCS1 transcription under different conditions both in vivo and in vitro. Currently, we are expanding our studies using these SOCS1 reporter mice into different disease models to gain better understanding of the regulatory mechanisms of SOCS1 under homeostatic and inflammatory conditions. Unlike SOCS1, SOCS3, and SOCS4 which are highly expressed in both thymocytes and T cells, we found that the SOCS family member Cish is expressed only at low levels in resting T cells. Moreover, Cish expression was upregulated by TCR stimulation and not by cytokine signaling which contrasts to SOCS1 and SOCS3 whose expression is induced by cytokine signaling but not by TCR signaling. 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, the biological significance of Cish induction by TCR signaling, and not by cytokine signaling, was unclear to us. To address this question, we generated Cish-transgenic mice that express a FLAG-tagged Cish cDNA under the control of the human CD2 promoter/enhancer so that Cish is overexpressed in all T lineage cells. Curiously, 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 not affect T cell function under steady-state condition. We also did not find any effects of Cish overexpression on cytokine receptor expression or signaling so that gc signaling remained comparable between Cish-transgenic and wildtype T cells. To identify the 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 how cytokine signaling is controlled in T cells during their development and differentiation.