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
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