Post-Transcriptional Regulation of Interleukin-7 Receptor Expression

在本研究中，我们研究了白细胞介素-7受体（IL-7R）表达的两个组成部分，即IL-7Ra和gc链的表达和调控机制。虽然我们可以确认这两个受体亚基的表达在转录水平上受到很大程度的调节，但我们也发现了IL-7R表达的一个新的调控层，涉及转录后的选择性剪接事件。具体来说，我们发现了一种新的gc-链剪接异构体，它是通过跳过包含整个gc-链跨膜结构域的进化保守外显子而产生的。结果，新的异构体产生了一个新的mRNA转录本，我们预测这将导致开放阅读框的移码突变和gc链的可溶性形式的产生。事实上，使用新开发的酶联免疫吸附试验（ELISA），我们能够在小鼠血清中检测到高水平的可溶性gc链。为了评估可溶性gc链是否确实是替代剪接的产物，接下来，我们通过用替代剪接产物中对应的独特序列的肽免疫兔子，产生了能够特异性识别替代剪接产物的抗体。事实上，我们能够在正常小鼠血清中检测到这些替代剪接产物，更重要的是，在患有自身免疫性疾病的小鼠血清中检测到更高水平的剪接产物。这些数据表明分泌型gc-chain的产生是一个正常的生理过程，可溶性gc-chain表达的增加可能与疾病状态有潜在的相关性。可溶性gc链是否作为免疫激活的结果产生，或者它们是否通过调节细胞因子的可用性发挥免疫调节作用仍有待确定。然而，利用重组表达和纯化的可溶性gc-链的初步数据显示，在可溶性gc-链存在的情况下，IL-7信号传导可以被有效阻断，这表明可溶性gc-链可以干扰gc-链细胞因子信号传导，可溶性gc-链可能在调节细胞因子响应性方面发挥迄今未知的作用。由于我们认为可溶性gc链的表达可能是T细胞中的一个重要机制，接下来我们希望评估这种新的异构体是否也在人类T细胞中表达。实时聚合酶链反应（Real - time polymerase chain reaction， PCR）和ELISA分析显示，可溶性gc-chain在人T细胞中也有表达，在正常人血清中也有表达，这表明这种可溶性gc-chain的产生是一种具有潜在重要意义的进化保守机制。综上所述，在本部分项目中，我们在小鼠和人淋巴细胞中都发现了一种新的gc-chain剪接异构体，并提出了一种新的调节gc-chain表达的机制，以及通过转录后机制调节gc-chain细胞因子的反应性。与在所有淋巴细胞群体中观察到的gc链表达相反，IL-7Ra链的表达是细胞特异性的，根据单个细胞的发育和激活状态进行动态调节和微调。在这方面，首先，我们评估了IL-7Ra在胸腺细胞和外周T细胞中的表面和信使RNA （mRNA）表达。与以前的报告一致（Chong et al. 2003）。免疫：18:4750)，除CD4+CD8+ DP胸腺细胞外，在所有胸腺细胞群中均检测到细胞表面IL-7Ra的表达，并通过实时PCR检测其与IL-7Ra mRNA表达水平密切相关。外周血中，B细胞表面不表达IL-7R蛋白和IL-7Ra信息，而CD4+和CD8+ T细胞IL-7R蛋白和mRNA表达均呈阳性。这些结果支持了IL-7Ra表达主要受转录机制调控的观点。然而，对小鼠T细胞中IL-7R转录本的详细分析令人惊讶地揭示了一种以前未知的替代剪接产物的存在。这种替代剪接产物是由于另一种内含子剪接供体位点的使用，它产生的mRNA转录物比全长IL-7Ra转录物长约100 bp。然而，选择性剪接也会导致开放阅读框的帧移位，从而导致IL-7R表达的可溶性形式的产生。到目前为止，还没有关于IL-7R的可溶性形式或另一种剪接异构体在小鼠中的报道。然而，在人类中，通过选择性剪接产生可溶性IL-7R是一个有充分记录的观察结果，然而，这是整个跨膜结构域外显子跳变的产物。重要的是，这种外显子6跳过的替代剪接产物在小鼠中不存在。尽管如此，ELISA证实在正常小鼠血清中存在可溶性IL-7R，我们目前正在产生新的抗体，这些抗体应该与内含子保留和选择性剪接产生的可溶性IL-7R蛋白特异性反应。因此，即使可溶性IL-7R产生的确切机制在小鼠和男性之间可能有所不同，但可溶性IL-7R的产生在进化上是保守的，可能在免疫调节中具有长期的影响。事实上，最近的数据显示，获得性免疫缺陷综合征（AIDS）患者的血清IL-7R水平较高，而细胞表面IL-7R水平较低，如果IL-7R的表达在转录后被调节以产生另一种剪接形式，情况可能就是如此。在系统水平上循环IL-7R蛋白水平升高和个体细胞水平上IL-7Ra表达降低的生物学意义是有趣的，我们希望在不久的将来解决这些问题。综上所述，我们的主要成果和发现可以总结如下：小鼠和人淋巴细胞gc-链新剪接异构体的鉴定可溶性gc链在细胞因子信号传导中的功能分析及其在炎症和自身免疫性疾病中的潜在作用。3. 代gc-链转基因小鼠，研究可溶性gc-链4缺失或过表达的影响。小鼠IL-7Ra链新剪接异构体的鉴定

In this study, we addressed the expression and regulatory mechanisms of either component of the interleukin-7 receptor (IL-7R) expression, namely the IL-7Ra and the gc-chain. While we could confirm that expression of both receptor subunits are largely regulated on a transcriptional level, we also identified a new layer of regulation in IL-7R expression which involves the post-transcriptional event of alternative splicing. Specifically, we discovered a novel splice isoform of the gc-chain that was generated by skipping an evolutionally conserved exon harboring the entire transmembrane domain of the gc-chain. As a result, the novel isoform produces a new mRNA transcript that we predicted to result in a frameshift mutation of the open reading frame and in the production of a soluble form of the gc-chain. In fact, using a newly developed enzyme-linked immunosorbent assay (ELISA), we were able to detect high levels of soluble gc-chains in mice serum. To assess whether soluble gc-chains were indeed the products of alternative splicing, next we generated antibodies that would specifically recognized the alternative splice product by immunizing rabbits with peptides corresponding to a unique sequence within the alternative splice product. Indeed, we were able to detect these alternative splice products in normal mice sera and importantly, at much higher levels in mice with autoimmune diseases. These data indicate that the production of secreted gc-chain is a normal physiological process and that increased expression of soluble gc-chain could potentially correlates with disease status. Whether soluble gc-chains are produced as a consequence of immune activation or whether they play immunregulatory roles by regulating cytokine availability have to be still determined. Preliminary data using recombinantly expressed and purified soluble gc-chains, however, revealed that IL-7 signaling could be effectively blocked in the presence of soluble gc-chains indicating that soluble gc-chain could interfere with gc-chain cytokine signaling and that soluble gc-chain might play a hitherto unknown role in regulating cytokine responsiveness. Since we considered it likely that soluble gc-chain expression would be an important mechanism in T cells, next we wished to assess whether this novel isoform was also expressed human T cells. Real time polymerase chain reaction (PCR) analysis and ELISA showed that soluble gc-chain was also expressed by human T cells and we also found it expressed in normal human serum which altogether suggest that the generation of such a soluble form of gc-chain is an evolutionally conserved mechanism of potential critical importance. In conclusion, in this part of the project, we identified a novel splice isoform of gc-chain in both mouse and human lymphocytes, and we proposed a novel mechanism of regulating gc-chain expression as well as regulating gc-chain cytokine responsiveness by a post-transcriptional mechanism. In contrast to gc-chain expression which is observed in all lymphocyte populations, IL-7Ra chain expression is cell specific, dynamically regulated and fine tuned depending on the developmental and activation status of the individual cell. In this regard, first, we assessed both surface and messenger RNA (mRNA) expression of IL-7Ra in thymocytes and in peripheral T cells. In agreement with previous reports (Chong et al. 2003. Immunity 18:4750), cell surface IL-7Ra expression was detected in every thymocyte populations except in CD4+CD8+ DP thymocytes and this strictly correlated with IL-7Ra mRNA expression levels as determined by real time PCR. Also in the periphery, B cells didnt express surface IL-7R protein and IL-7Ra message while both CD4+ and CD8+ T cells were positive for IL-7R protein and mRNA expression. These results are in support of the notion that IL-7Ra expression is mostly regulated over a transcriptional mechanism. However, detailed analysis of IL-7R transcripts in mouse T cells, surprisingly, revealed the existence of a previously unknown alternative splice product. This alternative splice product resulted from an alternative use of an intronic splice donor site, which generated an approximately 100 bp longer mRNA transcript than the full length IL-7Ra transcript. Alternative splicing, however, also resulted in a frame shift of the open reading frame, which then would result in the production of a soluble form of the IL-7R expression. So far, there had been no reports on either a soluble form of IL-7R or an alternative splice isoform in mice. In humans, however, soluble IL-7R generation by alternative splicing is a well documented observation, which however is the product of exon skipping of the entire transmembrane domain. Importantly, such an alternative splice product by exon 6 skipping did not exist in mice. Nevertheless, ELISA confirmed that soluble IL-7R were present in normal mice serum, and we are currently in the process of generating new antibodies that should specifically react with soluble IL-7R proteins generated by intron retention and alternative splicing. Thus, even as the exact mechanisms of soluble IL-7R generation might differ between mice and men, the production of soluble IL-7R is evolutionally conserved and might have long ranging consequence in immune modulation. Indeed, recent data showed that Acquired Immune Deficiency Syndrome (AIDS) patients have high levels of serum IL-7R and decreased levels of cell surface IL-7R, which would be the case if IL-7R expression would be post-transcriptionally regulated to produce the alternative splice form. The biological meaning of increased levels of circulating IL-7R proteins on systemic levels and the decreased IL-7Ra expression on individual cell levels is intriguing, and we expect to address these issues in the very next future. In sum, our major achievements and findings can be summarized as follows: 1. Identification of a novel splice isoform of gc-chain in mice and in human lymphocytes 2. Functional analysis of soluble gc-chain in cytokine signaling and analyzing its potential role in inflammation and autoimmune disease. 3. Generation of gc-chain transgenic mice to study the effect of absent or overexpressing soluble gc-chain 4. Identification of a novel splice isoform of IL-7Ra chain in mice