During the last few of years, we have intensely focused our research on the mechanism of gc-chain expression during T cell development and activation. In addition to transcriptionally controlled mechanisms, we discovered a novel post-transcriptional pathway of gc-chain expression that leads to the generation of soluble gc-chains. We detected soluble gc proteins in significant amounts in both normal human and mouse serum, and we have investigated its function under both homeostatic and autoimmune conditions. Soluble gc (sgc) is generated by exon skipping and a frameshift in the open reading frame by alternative splicing. To further assess its role in vivo, we generated sgc transgenic mice that overexpress soluble gc in T cells. These sgc Tg mice expressed high levels of sgc in serum 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, sgc Tg mice displayed a significant increase in autoimmune reaction as shown by a faster, stronger and more lasting clinical disease score. The underlying mechanism for such an enhanced reaction turned out to be increased generation of pro-inflammatory Th17 cells, and we are currently in the process of identifying the exact mechanism for this enhanced reactivity. In line of this research, we focused on cytokine receptor regulation in DP thymocytes because these cells expressed the highest level of sgc among all tested cells. Thus, we considered assessing the role of DP cell specific nuclear factors, such as RORgt, a logical step for understanding how cytokine receptor expression is regulated. Consequently, first we assessed the requirement of RORgt for IL-7Ra and gc-chain expression in DP cells. We found that IL-7Ra expression still to be suppressed in RORgt-deficient DP thymocytes but then found that surface gc-chain levels were significantly increased in the same cells. Moreover, Western blot analyses showed an increase in total gc-chain protein levels that was accompanied with increased molecular weight of immunoprecipitated gc-chain. Importantly, however, gc mRNA levels remained unaffected by the absence of RORgt which suggested that RORgt affects gc-chain expression over a novel post-transcriptional mechanism.Since gc-chain is known to be glycosylated, we considered the possibility that the increased molecular weight would be a result of hyper-glycosylations. There are two major forms of glycosylation: N-linked and O-linked. Using endoglycosidases, we asked whether gc-chain in RORgt-deficient DP cells is hyper-glycosylated. If so, endoglycosidase treatment should reduce the molecular weight of gc-chains from RORgt-deficient cells. Interestingly, O-glycosidase treatment did not affect gc molecular weight, but endoglycosidase F (endo F) treatment significantly reduced its size. These data suggest that RORgt alters the glycosylation signature of the gc-chain, potentially by affecting N-linked glycosylation. The biological function of differentially glycosylated gc-chain is completely unknown. Also, the physiological cues that can induce different gc glycosylation in vivo is not known, and we are currently addressing these questions. Furthermore, we have started investigating the effects of other transcription factors that are highly expressed in DP thymocytes and that are potentially involved in cytokine receptor expression. Preliminary findings suggest that both IL-7Ra and gc-chain expression is regulated by multiple factors in both transcriptional and post-transcription mechanism whose importance requires the development of further in vivo models to assess their requirement and function in T cell development and activation.
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