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.
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