While T cell activation and differentiation are accompanied with significant changes in gc and IL-7Ra mRNA and protein expression, the molecular mechanisms that control expression of these receptor subunits remain mostly unclear. Previously, we reported that gc expression is highly induced in activated T cells, and we found that it substantially affected the magnitude of gc cytokine signaling. To understand the regulatory mechanism of gc expression, we started our analysis by assessing gc expression during T cell development in the thymus. Here we found that gc expression is highly upregulated on immature DN and mature SP thymocytes but significantly suppressed on pre-selection DP thymocytes. We found that deficiency of the transcription factor RORgt reversed this defect, and that gc expression was upregulated on DP thymocytes of RORgt-KO mice. These data suggested that RORgt is a suppressor of gc expression during T cell development. To directly demonstrate this point, we generated transgenic mice that express a murine RORgt cDNA under the control of human CD2 promoter, enhancer elements. We confirmed transgenic RORgt expression by intracellular staining and Western blot analysis. Interestingly, RORgt overexpression significantly changed T cell development in the thymus, because it skewed CD4, CD8 lineage commitment of positively selected thymocytes into CD8 cells. To confirm that the RORgt transgene is functional, we crossed the RORgt transgene into RORgt-deficient mice and generated RORgtKO-Tg mice which were restored in their T cell development and surface gc expression comparable to those of WT mice. Importantly, gc mRNA expression in immature DP cells was not affected, neither by the lack or overexpression of RORgt, indicating that gc expression is controlled by RORgt through post-transcriptional mechanisms. Mechanistic details of this post-transcriptional pathway are currently under investigation. Previously, we reported alternative splicing of gc pre-mRNA as a new mechanism to downregulate surface gc protein expression which also resulted in the production of bioactive soluble gc proteins. Because the soluble form of gc (sgc) is generated at the expense of membrane gc protein expression, sgc expression inversely correlates with the amount of surface gc expression. We have now identified DP thymocytes as a major source of soluble gc protein expression in the thymus that would create an overall suppressive milieu for gc cytokine signaling in the thymus. In agreement, we found that transgenic overexpression of sgc resulted in impaired generation and differentiation of thymic NKT cells, which critically depend on IL-15 signaling. Curiously, generation of IL-2-dependent Foxp3+ T regulatory cells or IL-7-dependent CD8SP thymocytes were not affected, indicating distinct responsiveness of different gc cytokine receptors to sgc proteins. Collectively, these results demonstrated a previously unappreciated role for sgc in downregulating surface gc expression and in dampening gc cytokine signaling in thymocytes. In addition to soluble gc receptors, we also found soluble IL-7Ra proteins in serum of human and mice. In humans, soluble IL-7Ra has been previously described, and it is known that they are produced by alternative splicing of the IL-7Ra pre-mRNA. In contrast, soluble IL-7Ra proteins had not been reported in mice, and there is no molecular evidence for an alternative IL-7Ra mRNA splice isoform in mice. Nonetheless, by setting up sensitive ELISA assays to IL-7Ra proteins, we could detect soluble IL-7Ra proteins in wild type mouse sera. In humans, soluble IL-7Ra is produced by alternative splicing that omits exon 6, which encodes the entire transmembrane region. However, we were unable to detect such alternative transcripts in mice, suggesting that the mechanisms to produce soluble IL-7Ra proteins differ between humans and mice. We do not exclude the possibility of membrane protein shedding, and we are also investigating other post-transcriptional mechanisms of IL-7Ra production. Finally, we used fluorescent beads to quantify the absolute number of gc cytokine receptors on cell surface of T cell subsets to gain better understanding of the expression under steady state condition and during T cell activation, differentiation. Importantly, we found that IL-7Ra proteins outnumbered gc molecules four to one on surface of resting naive CD4 T cells. On the other hand, other gc cytokine receptors such as IL-4Ra or IL-2Rb were expressed at significantly lower numbers than gc. These results indicated that gc availability is limited for association with IL-7Ra and consequently for IL-7 signaling, thus curtailing IL-7R signaling on CD4 T cells. However, this is not the case of other gc cytokines, such as for IL-2, IL-15 and IL-4, where gc expression and availability are not limiting to initiate cytokine receptor signaling. The biological effects of limited gc availability is under further investigation.
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