Post-Transcriptional Regulation of Interleukin-7 Receptor Expression

关于GC链表达的传统观点认为，GC链蛋白是在恒定水平上表达的，它们的表达在T细胞激活或分化过程中不受调节。然而，我们自己的数据表明，GC链的表达在转录和转录后水平上都受到积极的调控。事实上，我们发现在未成熟的DP胸腺细胞中表面GC表达下调，但在T细胞发育过程中进一步成熟为CD8SP或CD4SP胸腺细胞时上调。此外，在外周T细胞中，TCR刺激诱导GC表达显著增加，同时伴随着IL-7ra表达的下调。这种动态调节在GC和IL-7ra表达中对T细胞发育和激活的生物学意义尚不完全清楚。因此，为了评估GC链调控的意义，我们培育了在T系细胞中结构性过表达GC的转基因(TG)小鼠。对这些GC转基因小鼠的初步分析显示，胸腺细胞数量或外周T细胞动态平衡没有异常表型。然而，我们观察到CD8SP胸腺细胞的百分比和数量在统计学上显著增加，这支持T细胞发育的动力学信号模型，该模型认为胸腺内GC细胞因子信号在CD8谱系承诺中起关键作用。在这一点上，GC链的结构性过表达有利于CD8系T细胞的发育，表明GC蛋白的表达是潜在的有限的，而GC的过度表达导致GC细胞因子信号的增加。事实上，在GC转基因T细胞中，IL-7诱导的细胞内磷酸化STAT5水平显著高于非转基因对照T细胞。我们目前正在研究这种GC过度表达在免疫激活条件下和自身免疫中的作用。虽然GC表达的主动调节主要是一种转录事件，但我们之前已经证明，GC也会产生一种选择性剪接转录本，以膜GC为代价编码一种可溶形式的GC。我们在正常人和小鼠血清中都检测到了大量的可溶性GC蛋白，但它在体内的功能仍然不清楚。可溶性GC(SGC)蛋白与膜GC蛋白的不同之处在于，GC的整个跨膜区和胞内区都被9个氨基酸的新表位交换，该表位是在选择性剪接过程中通过开放阅读框中的移码而产生的。这种SGC是否具有与IL-7和IL-7ra相同的三维折叠特性和结合亲和力尚不清楚。为了解决这个问题，我们已经成功地在细菌细胞和昆虫细胞中过表达了SGC，我们的目标是评估它的结构和与IL-7和IL-7ra的结合亲和力。这项工作目前正在与马里兰大学的斯科特·T·沃尔什博士合作进行。为了评估SGC在体内的作用，接下来，我们产生了SGC转基因小鼠，这些小鼠结构性地过度表达选择性剪接的GC亚型。这些SGC转基因小鼠在血清中表达高水平的SGC(比WT小鼠高34倍)，但对中枢和次级免疫器官的初步分析没有显示T细胞发育或动态平衡的任何变化。使用这些SGC转基因小鼠，我们随后评估了SGC在自身免疫实验模型中的作用。实验性自身免疫性脑脊髓炎(EAE)是一种公认的人类多发性硬化症的小鼠模型，用特定的抗原肽攻击正常B6小鼠会导致强烈的自身免疫反应，导致神经系统炎症和瘫痪。有趣的是，SGC转基因小鼠比野生型对照小鼠表现出更强的自身免疫反应，疾病起病更早，疾病评分更高，自发缓解延迟。因此，SGC显然正在影响自身免疫反应的过程。导致如此强烈的自身免疫反应的细胞机制目前正在研究中。此外，在人类中，可溶性GC链的表达是否也与自身免疫有关尚不清楚。为了解决这个问题，我们发起了与NINDS的Bibi Bielekova博士的合作。在这里，我们的目的是评估SGC在MS患者和健康对照组中的表达，以测试SGC表达与自身免疫的潜在相关性。此外，由于可溶性IL-7Ra(sIL-7Ra)的表达先前已被认为与MS有关，我们还计划评估sIL7-Ra在相同样本中的表达。可溶性IL-7ra蛋白在人类中已被描述，但在小鼠中未被描述。最近，我们在小鼠中也发现了一种可溶性的IL-7ra，它是通过一种新的选择性剪接机制产生的，该机制使用内含子保留而不是报道的外显子剪接来产生可溶性的人IL-7ra。为了测试这种可溶性IL-7ra在体内的作用，我们最近培育了在T细胞中过表达可溶性IL-7ra的转基因小鼠，目前我们正在对这些转基因T细胞的表型和免疫功能进行鉴定。总之，我们已经确认可溶性IL-7受体是一种新的免疫调节机制，控制T细胞的激活和效应器功能，我们期待这些数据应用于翻译和临床研究，以更好地治疗和理解免疫性疾病。

The conventional view on gc-chain expression has been that gc-chain proteins are expressed at constant levels and that their expressions are not modulated during T cell activation or differentiation. Our own data, however, indicated that gc-chain expression is actively regulated at both transcriptional and post-transcriptional levels. Indeed, we found that surface gc expression is downregulated in immature DP thymocytes but upregulated upon further maturation into CD8SP or CD4SP thymocytes during T cell development. Furthermore, in peripheral T cells, TCR stimulation induced a dramatic increase in gc expression with concomitant downregulation of IL-7Ra expression. The biological importance of such dynamic regulations in both gc and IL-7Ra expression in T cell development and activation is not fully understood. Consequently, to assess the significance of gc-chain regulation, we generated transgenic (Tg) mice that constitutively overexpress gc in T lineage cells. Initial analysis of these gc transgenic mice showed no aberrant phenotype regarding thymocytes numbers or peripheral T cell homeostasis. However, we observed a statistically significant increase in CD8SP thymocyte percentage and numbers, which is in support of the kinetic signaling model of T cell development that proposes a critical role for intrathymic gc cytokine signaling in CD8 lineage commitment. In this regard, constitutive overexpression of gc-chain favors CD8 lineage T cell development indicating that gc protein expression is potentially limited and that overexpression of gc results in increased gc cytokine signaling. In fact, IL-7-induced intracellular phosphor-STAT5 levels were significantly increased in gc transgenic T cells compared to non-transgenic control T cells. We are currently investigating the effect of such gc overexpression under immune activating conditions and in autoimmunity. While the active regulation of gc expression is mostly a transcriptional event, we have previously shown that gc also produces an alternative splice transcript that encodes a soluble form of gc at the expense of membrane gc. We detected soluble gc proteins in significant amounts in both normal human and mouse serum but its function in vivo is still veiled. Soluble gc (sgc) proteins differ from membrane gc proteins in that the entire transmembrane domain and intracellular domain of gc have been swapped with a 9-amino acid novel epitope that is generated by a frameshift in the open reading frame during alternative splicing. Whether this sgc has the same three dimensional folding characteristics and binding affinity to IL-7 and IL-7Ra is not known. To address this issue, we have successfully overexpressed sgc in bacterial cells and in insect cells, and we aim to assess its structure and binding affinities to IL-7 and IL-7Ra. This work is in currently under progress in collaboration with Dr. Scott T. Walsh at the University of Maryland. To assess the role of sgc in vivo, next, we generated sgc transgenic mice that constitutively overexpress the alternatively spliced isoform of gc. These sgc Tg mice expressed high levels of sgc in serum (3 4 fold more compared to WT mice), but initial analysis of the central and secondary immune organs didnt show any changes in T cell development or homeostasis. Using these sgc transgenic mice, we then assessed the role of sgc in an experimental model of autoimmunity. Experimental autoimmune encephalomyelitis (EAE) is a well established mouse model for human multiple sclerosis, and challenging normal B6 mice with specific antigenic peptides results in a strong autoimmune reaction resulting in inflammation of the nervous system and paralysis. Interestingly, sgc Tg mice displayed a much stronger autoimmune reaction than wildtype control mice with an earlier onset of the disease, higher disease score and delayed spontaneous remission. Thus, sgc is clearly affecting the course of autoimmune reactions. The cellular mechanism that induces such fierce autoimmune reaction is currently under investigation. Additionally, whether soluble gc-chain expression is also linked to autoimmunity in humans is not known. To address this issue, we initiated a collaboration with Dr. Bibi Bielekova at NINDS. Here we aim to assess sgc expression in MS patients and healthy control subjects to test a potential correlation of sgc expression and autoimmunity. Furthermore, since soluble IL-7Ra (sIL-7Ra) expression had been previously implicated in MS, we also plan to assess sIL7-Ra expression in the same samples. Soluble IL-7Ra proteins had been described in humans but not in mice. Recently, we discovered a soluble form of IL-7Ra also in mice which is produced by a novel mechanism of alternative splicing employing intron-retention rather than exon-splicing as reported for generating soluble human IL-7Ra. To test the role of such soluble IL-7Ra in vivo, we have recently generated transgenic mice that overexpress soluble IL-7Ra in T lineage cells, and we are currently in the process of phenotyping and characterizing the immune function of these transgenic T cells. Collectively, we have identified soluble IL-7 receptors as a novel immunomodulatory mechanism that controls T cell activation and effector function, and we expect these data to be applied into translational and clinical research for better treatment and understanding of immunological diseases.