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.
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