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

我们通过建立一套新的实时聚合酶链反应（PCR）方法来启动这项研究，该方法可以特异性和定量地报告SOCS家族每个成员的信使RNA （mRNA）表达。然后，我们利用C57BL/6小鼠的各个器官，对SOCS分子表达进行了全面的组织分布分析。与之前的报道一致，我们证实了SOCS1在胸腺中高度表达，特别是在发育中的胸腺细胞中，这表明它在T细胞的发育和分化中起着关键作用。令人惊讶的是，另外两个与免疫细胞相关的SOCS家族成员，即SOCS3和CISH的mRNA表达仅在胸腺和脾脏中低水平表达，而在肺和肾中分别高水平表达。这些组织中如此高水平的SOCS3和CISH表达的生物学意义目前尚不清楚。然而，这些数据表明，与SOCS1不同，SOCS3和CISH在T细胞发育过程中具有不同的发育表达模式，并且它们的表达通常维持在基础水平，仅在细胞因子信号诱导下才会被诱导。事实上，当我们用IL-7或IL-4等细胞因子刺激纯化的CD4+或CD8+ T细胞时，我们观察到SOCS3和CISH表达的强烈诱导，这可能限制了这些细胞因子的进一步信号传导。对于SOCS家族的其他成员，我们观察到不同组织的不同表达模式，这表明每个SOCS成员可能对不同的细胞具有特定的功能。例如，我们在大脑中观察到SOCS7的高度特异性表达，而在肾脏、活体和肺中检测到高水平的SOCS2表达，但没有在免疫组织中检测到。然而，最有趣的是，我们发现SOCS4 mRNA在胸腺中富集，特别是在胸腺细胞中高度表达，这与SOCS1的表达模式非常相似。为了进一步研究SOCS4的表达是否像SOCS1一样受到发育调节，我们对胸腺细胞亚群进行了电子分类和纯化，发现SOCS4 mRNA在未成熟的CD4+CD8+双阳性（DP）胸腺细胞中高度上调，但在成熟的CD4+单阳性（SP）和CD8+ SP胸腺细胞以及成熟的T细胞中下调。Western blot分析进一步表明，SOCS4蛋白表达与SOCS4 mRNA表达相关，SOCS4蛋白在未成熟DP胸腺细胞中高表达，而在成熟胸腺细胞和外周T细胞中低表达。文献表明，T细胞发育过程中，正向选择T细胞受体（TCR）信号下调SOCS1的表达。为了评估SOCS4的表达是否也受TCR信号的控制，接下来我们分离成熟T细胞和未成熟DP胸腺细胞，体外刺激TCR，以确定其对SOCS4 mRNA表达的影响。与SOCS1一样，板结合的抗TCR抗体刺激和TCR信号的药理学模拟都强烈下调了SOCS4 mRNA的表达。这些数据表明，SOCS4可能是SOCS1的潜在冗余分子，可保护未成熟胸腺细胞免受过早获得细胞因子信号。我们目前正在培育t系特异性SOCS4转基因小鼠和SOCS4条件敲除（KO）小鼠，以评估SOCS4过表达和条件缺失对免疫稳态和激活的影响。最终，我们希望产生SOCS1缺陷和SOCS4缺陷的双KO小鼠，这将使我们能够解决SOCS1和SOCS4潜在的冗余作用，并确定SOCS4在T细胞分化和激活过程中限制细胞因子信号传导的具体作用。然而，除了SOCS1与SOCS4的冗余作用外，我们还对SOCS1与其他SOCS分子的潜在串扰和功能冗余感兴趣。我们特别对SOCS3非常感兴趣，因为我们发现SOCS3的表达在T细胞发育过程中不受影响，但受到细胞因子信号传导的强烈调节。由于我们发现SOCS3 mRNA在胸腺细胞和静息T细胞中都有显著的组成性表达，我们进一步考虑了在免疫稳态过程中维持SOCS3表达对于抑制细胞因子信号传导至关重要的可能性。为此，我们培养了在未成熟胸腺细胞和成熟T细胞中特异性删除Socs3的小鼠。为此，我们将SOCS3捆绑小鼠与cd8双阳性增强子驱动的Cre转基因小鼠杂交，目前我们正在对这些小鼠进行分析。初步数据表明，这些小鼠的胸腺细胞发育和T细胞稳态相对正常，尽管CD8 T细胞数量略有增加。这些T细胞的功能分析及其进一步的表型分析目前正在进行中。最后，为了研究SOCS3在T细胞中过表达的影响，我们培育了SOCS3转基因小鼠，这些小鼠在所有T细胞中特异性表达SOCS3。初步数据表明，细胞因子信号通路在这些细胞中严重受损，细胞因子信号通路的减弱影响了T细胞的发育和稳态。重要的是，SOCS3先前涉及抑制STAT3介导的免疫反应，并且根据最近关于白细胞介素17分泌CD4+辅助性T细胞（Th17细胞）及其对STAT3信号的需求的发现，我们认为利用这些SOCS3转基因小鼠在Th17细胞分化和激活的背景下测试自身免疫性疾病模型（如实验性自身免疫性脑脊髓炎（EAE））是很重要的。综上所述，主要的发现和成就可以总结如下：SOCS家族分子表达的综合分析及组织分布SOCS4作为一种潜在的新型免疫调节SOCS分子的鉴定T系特异性缺失SOCS3 - 4的条件敲除小鼠的产生和表征。人CD2迷你盒控制下过表达SOCS3转基因小鼠的产生和特性研究。5. T细胞中SOCS分子表达调控机制分析

We initiated this study by establishing a new set of real time-polymerase chain reaction (PCR) assays that specifically and quantitatively report messenger RNA (mRNA) expression of each members of the SOCS family. Then, using various organs from C57BL/6 mice, we performed a comprehensive tissue distribution analysis of SOCS molecules expression. In agreement with prior reports, we confirmed that SOCS1 was highly expressed in the thymus and specifically in developing thymocytes indicating its critical role in T cell development and differentiation. Surprisingly, mRNA expression of the other two immune cell-related SOCS family members, namely SOCS3 and CISH, were found to be expressed only in low levels in thymus and spleens whereas they were found in high levels in lung and kidney, respectively. The biological significance of such high level SOCS3 and CISH expression in these tissues is currently not clear to us. However, these data suggest that, in contrast to SOCS1, SOCS3 and CISH have distinct developmental expression patterns during T cell development, and that their expression is normally maintained at a basal level only to be induced upon cytokine signaling. In fact, when we stimulated purified CD4+ or CD8+ T cells with cytokines such as IL-7 or IL-4, we observed a strong induction of both SOCS3 and CISH expression which presumably limits further signaling by these cytokines. As for other members of the SOCS family, we observed different patterns of expression depending on the tissues which collectively suggested that each individual SOCS member might have a specific function for different cells. For example, we observed a highly specific expression of SOCS7 in the brain whereas high levels of SOCS2 expression were detected in kidney, live and lung but in no immune tissues. Most interestingly, however, we found SOCS4 mRNA to be enriched in the thymus and particularly to be highly expressed in thymocytes which closely resembled the expression pattern of SOCS1. To further investigate whether SOCS4 expression is developmentally regulated as SOCS1, we electronically sorted and purified thymocyte subpopulations and we found that SOCS4 mRNA is highly upregulated in immature CD4+CD8+ double positive (DP) thymocytes but then down regulated in mature CD4+ single positive (SP) and CD8+ SP thymocytes as well as in mature T cells. Western blot analysis further showed that SOCS4 protein expression correlated to SOCS4 mRNA expression in that SOCS4 protein expression was high in immature DP thymocytes but then downregulated in mature thymocytes and peripheral T cells. It is well documented that positive selecting T cell receptor (TCR) signal downregulates SOCS1 expression during T cell development. To assess whether SOCS4 expression is also under the control of TCR signals, next we isolated mature T cells and immature DP thymocytes and TCR stimulated them in vitro to determine its effect on SOCS4 mRNA expression. As in case of SOCS1, both plate-bound anti-TCR antibody stimulation as well as pharmacological mimics of TCR signals both strongly downregulated SOCS4 mRNA expression. These data suggest that that SOCS4 could be potentially a redundant molecule of SOCS1 that protects immature thymocytes from getting prematurely cytokine signaled. We are currently in the process of generating a T-lineage specific SOCS4 transgenic mouse and a SOCS4 conditional knock-out (KO) mouse to assess the effects of SOCS4 overexpression and conditional deletion on immune homeostasis and activation. Ultimately, we wished to generate SOCS1-deficient, SOCS4-deficient double KO mice, which will enable us to address potential redundant roles of SOCS1 and SOCS4 and also to determine the specific role of SOCS4 in limiting cytokine signaling during T cell differentiation and activation. In addition to a redundant role of SOCS1 with SOCS4, however, we are also interested in a potential cross-talk and functional redundancy of SOCS1 with other SOCS molecules. In particular, SOCS3 is of great interest to us as we found SOCS3 expression to be unaffected during T cell development but strongly modulated by cytokine signaling. Since we found SOCS3 mRNA to be constitutively expressed to a significant level in both thymocytes and resting T cells, we further considered the possibility that maintaining SOCS3 expression would be critical for dampening cytokine signaling during immune homeostasis. To this end, we generated mice where Socs3 was specifically deleted in immature thymocytes and in mature T cells. To do so, we crossed SOCS3 floxed mice with mice transgenic for a CD8-double positive enhancer drive Cre transgene, and we are currently in the process of analyzing these mice. The initial data suggest that thymocyte development and T cell homeostasis are relatively normal in these mice albeit with a slight increase in CD8 T cell numbers. Functional analysis of these T cells and their further phenotypic analyses are currently in process. Finally, to examine the effect of SOCS3 overexpression in T cells, we have generated SOCS3 transgenic mice that specifically express SOCS3 in all T lineage cells. Preliminary data suggest that cytokine signaling is severely impaired in these cells and that both T cell development and homeostasis were affected caused by diminished cytokine signaling. Importantly, SOCS3 has been previously implicated in suppressing STAT3-mediated immune responses, and in light of the recent findings on interleukin-17-secreting CD4+ helper T cells (Th17 cells) and their requirement for STAT3 signaling, we consider it important to utilize these SOCS3 transgenic mice to test autoimmune disease models such as experimental autoimmune encephalomyelitis (EAE) in context of Th17 cell differentiation and activation. In sum, the major findings and accomplishments can be summarized as follows: 1. Comprehensive analysis and tissue distribution of SOCS family molecule expression 2. Identification of SOCS4 as a potential and novel immune regulatory SOCS molecule 3. Generation and characterization of conditional knock-out mice with T lineage specific deletion of SOCS3 4. Generation and characterization of transgenic mice overexpressing SOCS3 under the control of a human CD2 mini-cassette. 5. Analysis of regulatory mechanisms that control SOCS molecule expression in T cells