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

我们研究的主要目的是了解细胞因子信号通路的调控机制，并评估负调控分子（如SOCS）在这些过程中的作用。虽然细胞因子信号传导的主流观点认为它对T细胞的功能和存活至关重要，但我们最近取得了一系列意想不到的发现，揭示了T细胞的细胞因子独立存活，我们甚至报道了细胞因子对T细胞分化有害的情况。在这方面，我们对小肠上皮上皮内淋巴细胞（iel）中的CD4+CD8aa+ T细胞产生了兴趣，这些细胞来源于传统的CD4 T细胞，在肠道中募集后分化为CD4+CD8aa+ T细胞。CD4 T细胞表型转化为CD4+CD8aa+ T细胞是通过转录重编程介导的，这与CD4谱系特异性转录因子ThPOK的缺失和CD8谱系特异性转录因子Runx3d的获得有关。有趣的是，这种重编程似乎独立于稳态gc细胞因子信号传导，因为我们证明il -7缺乏和il -15缺乏都不会导致CD4+CD8aa+ T细胞分化减弱（Li C. et al., 2022, Cell Mol Immunol.）。相反，我们发现缺乏这些稳态gc细胞因子导致CD4+CD8aa+ T细胞的频率和数量显著增加，这表明细胞因子信号传导对它们的产生是有害的。然而，在缺乏IL-7和/或IL-15的情况下，CD4+CD8aa+ T细胞生成增加的分子机制尚不清楚。最近，我们发现与矮子相关的转录激活因子Runx3d是限制CD4+CD8aa+ IELs丰度的关键因素，我们发现Runx3d的缺乏导致CD4+CD8aa+ IELs的急剧增加。由于Runx3d的表达位于gc细胞因子信号的下游，这些结果表明细胞因子信号可以通过Runx3d控制CD4+CD8aa+ IEL分化（Li C., 2023, Cell Death Diff）。因此，我们发现了一种由稳态gc信号负调控的CD4+CD8aa+ IEL分化和表型获取的新转录回路。然而，在CD4+CD8aa+ iel中，哪些细胞信号驱动和维持这种细胞因子/ runx3d介导的途径仍有待评估。具体来说，目前尚不清楚SOCS表达是否参与了这一过程，我们认为这是可能的，因为SOCS家族分子可以阻止CD4 T细胞中的细胞因子信号传导，从而促进其分化。我们目前正在评估IELs中SOCS的表达，以评估这些问题。另一项正在进行的关于SOCS分子作用的研究是质疑SOCS4的作用，SOCS4仍然是SOCS家族成员，在未成熟胸腺细胞中大量表达，但在成熟T细胞中表达较少。SOCS4的这种发育调控表达表明，SOCS4在胸腺T细胞生成中可能发挥作用。为了研究其在胸腺发育中的作用和需求，我们之前利用基因诱捕ES细胞系统培养了SOCS4缺陷小鼠，并通过实时反转录PCR验证了SOCS4 mRNA表达缺失。令人失望的是，评估socs4缺陷小鼠胸腺中的T细胞发育并没有发现socs4缺陷的任何不利影响。在没有SOCS4的情况下，我们没有观察到胸腺细胞数量、阳性选择和谱系分化的显著变化。在T细胞发育中缺乏任何可识别的影响是否由于与其他socs家族分子的潜在冗余目前尚不清楚。然而，我们最近建立了新的小鼠模型来解决这个问题。其中，我们将SOCS4- ko等位基因引入HY TCR转基因小鼠，利用单特异性表达TCR的胸腺细胞，测试SOCS4在TCR信号传导和细胞因子信号传导中的作用。SOCS4与TCR信号通路的潜在联系来自我们最近的观察，即SOCS4缺陷T细胞对TCR信号表现出更高的反应性，因此我们发现有必要测试SOCS4对TCR信号通路的影响。在这里，我们发现gc家族细胞因子信号在socs4缺陷胸腺细胞和T细胞中保持不变，但TCR信号的强度明显增加。为什么SOCS4会干扰TCR-而不是细胞因子受体信号，目前我们还不清楚。还需要评估缺乏SOCS4是否会干扰tcr诱导的T细胞激活和分化，我们的目标是在未来的研究中评估评估SOCS4缺陷T细胞的功能方面。在研究SOCS4的同时，我们也在研究SOCS1调控gc信号的机制。SOCS1的表达在T细胞发育过程中受到动态调控，未成熟胸腺细胞中SOCS1的表达量最大，而SOCS1在正选择过程中被下调。我们之前已经证明，未成熟CD4、CD8双阳性胸腺细胞中的SOCS1对于阻止预选择胸腺细胞中的细胞因子信号传导至关重要。然而，在胸腺细胞发育过程中，是什么以特定阶段的方式驱动SOCS1表达尚不清楚。我们一直在使用SOCS1报告小鼠解决这个问题，其中人类CD4 cDNA在停止磁带后插入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，我们已经使用这些小鼠在体内和体外不同刺激条件下监测SOCS1的转录。此外，我们最近的研究表明，SOCS3在抑制胸腺细胞gc细胞因子信号传导方面具有部分冗余作用，因此我们目前正在培养SOCS1-floxed、SOCS3-floxed的CD4-Cre小鼠，以检验T细胞中主要SOCS家族分子之间的冗余性。我们的目标是利用这些小鼠模型来更好地了解细胞因子受体信号在稳态和炎症条件下的调节机制。

The main objective of our study is to understand the regulatory mechanisms of cytokine signaling pathways, and to assess the role of negative regulatory molecules, such as SOCS, in these processes. While the prevailing view on cytokine signaling posits that it is essential for T cell function and survival, we recently made a series of unexpected findings that revealed cytokine independent survival of T cells, and we even reported cases where cytokines are detrimental for the differentiation of T cells. In this regard, we have become interested in CD4+CD8aa+ T cells among intraepithelial lymphocytes (IELs) in the small intestine epithelium, which are derived from conventional CD4 T cells that differentiate into CD4+CD8aa+ T cells upon their recruitment in the gut. The phenotypic conversion of CD4 T cells into CD4+CD8aa+ T cells is mediated by transcriptional reprogramming that is associated with the loss of the CD4 lineage-specifying transcription factor ThPOK and the acquisition of the CD8 lineage-specifying transcription factor Runx3d. Interestingly, such reprogramming appeared to be independent of homeostatic gc cytokine signaling, as we demonstrated that neither IL-7-deficiency nor IL-15-defiency resulted in diminished differentiation of CD4+CD8aa+ T cells (Li C. et al., 2022, Cell Mol Immunol.). Instead, we found that the lack of these homeostatic gc cytokines resulted in markedly increased frequencies and numbers of CD4+CD8aa+ T cells, indicating that cytokine signaling is detrimental for their generation. The molecular mechanism of increased CD4+CD8aa+ T cell generation in the absence of IL-7 and/or IL-15, however, has been unclear. Most recently, we identified the runt-related transcriptional activator Runx3d as a key factor that limits the abundance of CD4+CD8aa+ IELs, and we showed that the lack of Runx3d results in a dramatic increase of CD4+CD8aa+ IELs. Because the expression of Runx3d is downstream of gc cytokine signaling, these results suggest that cytokine signaling could control CD4+CD8aa+ IEL differentiation through Runx3d (Li C., 2023, Cell Death Diff). Thus, we identified a new transcriptional circuitry of CD4+CD8aa+ IEL differentiation and phenotype acquisition that is negatively controlled by homeostatic gc signaling. However, it remains to be assessed what cellular signals drive and maintain this cytokine/Runx3d-mediated pathway in CD4+CD8aa+ IELs. Specifically, it is unclear whether SOCS expression could have been involved in this process, which we consider likely, because SOCS family molecules could prevent cytokine signaling in CD4 T cells to promote their differentiation. We are currently in the process of assessing SOCS expression in IELs to assess these issues. Another ongoing study on the role of SOCS molecules is to interrogate the role of SOCS4 which remains a poorly characterized SOCS family member that is abundantly expressed in immature thymocytes but less so on mature T cells. Such developmentally regulated expression of SOCS4 suggested a potential role for SOCS4 in thymic T cell generation. To examine its role and requirement in thymopoiesis, we previously generated SOCS4-deficient mice utilizing a gene-trap ES cell system and verified the absence of SOCS4 mRNA expression by real-time reverse transcription PCR. Disappointingly, assessing the T cell development in the thymus of SOCS4-deficient mice did not reveal any adverse effect of SOCS4-deficiency. We failed to observe significant changes in thymocyte numbers, positive selection, and lineage differentiation in the absence of SOCS4. Whether the lack of any discernible effects in T cell development would be due to potential redundancy with other SOCS-family molecules is currently not clear to us. However, we have recently established new mouse models to address this question. Among others, we introduced the SOCS4-KO allele into HY TCR transgenic mice to test the role of SOCS4 in TCR signaling and cytokine signaling using monospecific TCR expressing thymocytes. A potential connection of SOCS4 with the TCR signaling pathway came from our recent observation that SOCS4-deficient T cells showed higher responsiveness to TCR signaling, so that we found it necessary to test the effect of SOCS4 on TCR signaling. Here, we found that gc family cytokine signaling in SOCS4-deficient thymocytes and T cells remained unaltered but the magnitude of TCR signaling evidently increased. Why SOCS4 would interfere with TCR- instead of cytokine receptor signaling is currently unclear to us. It also needs to be assessed whether the lack of SOCS4 interferes with TCR-induced activation and differentiation of T cells, and we aim to assess assessing the functional aspects of SOCS4-deficient T cells in our future studies. In parallel to our studies on SOCS4, we have been studying the mechanistic aspect of SOCS1 controlled regulation of gc signaling. SOCS1 expression is dynamically regulated upon T cell development, and immature thymocyte express the largest amounts of SOCS1 which is downregulated upon positive selection. We have previously demonstrated that SOCS1 in immature CD4, CD8 double-positive thymocytes is critical to prevent cytokine signaling in pre-selection thymocytes. However, it was unclear what drives SOCS1 expression in a stage-specific manner in developing thymocytes. We have been addressing this question using 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 have been using these mice to monitor SOCS1 transcription under different stimulatory conditions both in vivo and in vitro. Additionally, our recent studies suggested a partially redundant role of SOCS3 in the suppression of gc cytokine signaling in thymocytes, so that we are currently in the process of generating SOCS1-floxed, SOCS3-floxed CD4-Cre mice to examine the redundancy among major SOCS family molecules in T cells. We are aiming to employ these mouse models to gain better understanding on the regulatory mechanisms of cytokine receptor signaling under homeostatic and inflammatory conditions.