Post-Transcriptional Regulation of Interleukin-7 Receptor Expression

由于IL-7受体由两条链组成，我们对这两条链的调控机制都很感兴趣，因此我们将项目进一步分为两个平行但互补的研究，即IL-7Ra部分和gc-chain部分。对于IL-7Ra链，我们和其他人之前已经表明，IL-7Ra的表达主要通过转录机制调节，但最近的数据表明，IL-7Ra表达的转录后调控在控制IL-7R的响应性中也起着重要作用。自从这个项目开始以来，一些报告揭示并强调了这一机制正在出现的重要性。具体来说，一系列全基因组关联研究（GWAS）发现，IL-7Ra基因的遗传变异与多发性硬化症（MS）风险增加有关。有趣的是，这种特殊的单核苷酸多态性与IL-7Ra转录物的选择性剪接异构体的产生有关，从而导致IL-7Ra受体蛋白的可溶性形式的产生。然而，这种可溶性IL-7Ra蛋白如何增加多发性硬化症的风险仍不得而知。重要的是，在正常的体内平衡条件下也发现了可溶性IL-7Ra蛋白，这些可溶性IL-7Ra蛋白的表达增加被认为与自身免疫有关。虽然这种可溶性形式已经在人类中被描述过，但还没有关于老鼠的报道。在当前项目的框架内，我们最近也在小鼠中发现了可溶性IL-7Ra链。这些可溶性受体是通过一种新的选择性剪接机制产生的，这种机制采用内含子保留而不是外显子剪接，就像人类一样。为了测试这种可溶性IL-7Ra在体内的作用，我们现在已经在T系细胞中产生了过表达可溶性IL-7Ra （sIL7Ra）的转基因小鼠。我们通过real - time PCR鉴定了转基因sIL7Ra的特异性表达，并完成了这些转基因T细胞表型和免疫功能表征的初步过程。可溶性IL7Ra转基因小鼠在胸腺细胞和外周LN细胞数量方面相对正常，但我们发现了激活/记忆表型细胞的积累。然而，与野生型细胞相比，体外刺激sIL7Ra Tg没有显示出任何增强的免疫反应性。IL-7依赖性幼稚T细胞和记忆T细胞体内稳态的详细分析目前正在进行中。gc-chain的正常表达对IL-7信号传导至关重要，但gc-chain的表达是如何被调节的仍不得而知。在过去的几年里，我们已经评估了T细胞发育和激活过程中gc-chain的表达，我们发现gc-chain的表达在转录和转录后机制中都受到积极调节。事实上，TCR刺激成熟T细胞诱导了gc表达的急剧但短暂的增加，同时IL-7Ra表达的下调。这种对gc和IL-7Ra表达在T细胞发育和激活中的动态调控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，我们在T系细胞中产生了组成性过表达gc的转基因（Tg）小鼠。这些gc转基因小鼠的初步分析显示，在胸腺细胞数量或外周T细胞稳态方面没有异常表型。然而，进一步的分析显示，gc转基因小鼠的记忆表型细胞显著积累，表明初始T细胞稳态受损。事实上，当评估IL-7诱导的STAT5磷酸化时，我们发现gc Tg T细胞在IL-7信号传导方面的效率明显较低。奇怪的是，将这种gc Tg引入IL-2缺陷的自身免疫性小鼠，改善了它们的疾病表型。我们目前正在评估这一观察的免疫学机制。此外，我们最近发现了一个选择性剪接的gc转录物，它编码可溶形式的gc，而牺牲了膜gc。我们在正常人和小鼠血清中检测到大量的可溶性gc蛋白，并研究了其在稳态条件下的功能。可溶性gc （sgc）是由跨膜结构域开放阅读框中的移码通过选择性剪接产生的。该可溶性蛋白是否具有相同的三维折叠特征以及与IL-7和IL-7Ra的结合亲和力尚不清楚。为了解决这一问题，我们成功地在细菌细胞和昆虫细胞中过表达了sgc，并利用圆二色性分析了这些蛋白的物理特性，发现它们与天然gc链蛋白的胞外结构域相当。这项工作是与马里兰大学的斯科特·t·沃尔什博士合作完成的。为了进一步评估其在体内的作用，接下来，我们产生了sgc转基因小鼠，这些小鼠组成性地过表达gc的选择性剪接异构体。这些sgc Tg小鼠在血清中表达高水平的sgc（比WT小鼠高34倍），它们的T细胞表现出增加的激活记忆表型百分比。重要的是，当sgc Tg小鼠在实验性自身免疫性脑脊髓炎（EAE）模型中受到免疫反应性攻击时，我们观察到自身免疫反应的显著增加，表现为更快、更强、更持久的临床疾病评分。然后，我们确定了促炎Th17细胞增加产生的这种增强反应的潜在机制。事实上，利用重组可溶性gc-chain，我们发现重组gc-chain作为诱饵受体结合IL-2，但不产生信号传导。我们认为可溶性gc链受体的表达可能是一种下调或终止gc细胞因子的新机制，从而调节免疫应答的范围和强度。

Since the IL-7 receptor is composed of two chains, and since we were interested in the regulatory mechanisms of both chains, the projected was further divided into two parallel but complementary studies which is the IL-7Ra part and gc-chain part. For the IL-7Ra chain, we and others have previously shown that IL-7Ra expression is mainly regulated over a transcriptional mechanism but recent data suggested that post-transcriptional regulation of IL-7Ra expression also plays an important role in controlling IL-7R responsiveness. Since initiation of this project, a number of reports have uncovered and emphasized the emerging importance of this mechanism. Specifically, a series of genome-wide association studies (GWAS) have found that a genetic variation in the IL-7Ra gene that is associated with increased risk for multiple sclerosis (MS). Interestingly, this particular single nucleotide polymorphism is linked to generation of an alternatively spliced isoform of IL-7Ra transcripts that results in the production of a soluble form of IL-7Ra receptor proteins. How such soluble IL-7Ra proteins would increase the risk for multiple sclerosis, however, remains unknown. Importantly, soluble IL-7Ra proteins are also found under normal homeostatic conditions, and it is the increased expression of these soluble IL-7Ra proteins that are proposed to be linked with autoimmunity. While such a soluble form had been described in humans, there has been no report on mice. In frame of this current project, we have recently identified soluble IL-7Ra chains also in mice. These soluble receptors were produced by a novel mechanism of alternative splicing employing intron-retention rather than exon-splicing, as is the case for humans. To test the role of such soluble IL-7Ra in vivo, we have now generated transgenic mice that overexpress soluble IL-7Ra (sIL7Ra) in T lineage cells. We have identified specific expression of transgenic sIL7Ra by real time PCR, and we have finished the initial process of phenotyping and characterizing the immune function of these transgenic T cells. Soluble IL7Ra transgenic mice were relatively normal in terms of thymocyte and peripheral LN cell numbers, but we identified an accumulation of activated/memory phenotype cells. In vitro stimulation of sIL7Ra Tg however did not show any enhanced immune reactivity compared to wildtype cells. Detailed analysis of IL-7 dependent naive and memory T cell homeostasis in vivo is currently under process. Normal expression of gc-chain is critical for IL-7 signaling but how gc-chain expression is regulated has remained largely unknown. During the last couple of years, we have assessed gc-chain expression during T cell development and activation, and we found that gc-chain is expression is actively regulated over both transcriptional and post-transcriptional mechanisms. In fact, TCR stimulation of mature T cells induced a dramatic but transient 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. Further analysis, however, revealed a significant accumulation of memory phenotype cells in gc transgenic mice indicating the naive T cell homeostasis was impaired. Indeed, when assessing for IL-7 induced STAT5 phosphorylation, we found that gc Tg T cells were significantly less efficient in IL-7 signaling. Curiously enough, introducing such a gc Tg into IL-2 deficient autoimmune mice ameliorated their disease phenotype. We are currently assessing the immunological mechanism of this observation. Additionally, we have recently identified an alternatively spliced gc 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 and we have investigated its function under homeostatic conditions. Soluble gc (sgc) is generated by a frameshift in the open reading frame of the transmembrane domain by alternative splicing. Whether this soluble protein has the same three dimensional folding characteristics and binding affinity to IL-7 and IL-7Ra was not known. To address this issue, we have successfully overexpressed sgc in bacterial cells and also in insect cells, and analyzed the physical characteristics of these proteins using circular dichroism and found them comparable to the extracellular domain of native gc-chain protein. This work was done in collaboration with Dr. Scott T. Walsh at the University of Maryland. To further assess its role 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) and their T cells showed an increased percentage of activated memory phenotype. Importantly, when sgc Tg mice were challenged in an experimental autoimmune encephalomyelitis (EAE) model for immune reactivity, we observed a significant increase in autoimmune reaction as shown by a faster, stronger and more lasting clinical disease score. We then identified the underlying mechanism for such an enhanced reaction in the increased generation of pro-inflammatory Th17 cells. In fact, using recombinant soluble gc-chain, we identified that recombinant gc-chain acts as decoy receptor to bind IL-2 but did not result in signaling. We propose that expression of soluble gc-chain receptor could present a novel mechanism to downregulate or terminate gc cytokines and thus regulate the extend and magnitude of an immune response.