Previously, we reported the alternative splicing of gc pre-mRNA as a new mechanism to downregulate surface gc protein expression and to produce a new bioactive molecule in the form of soluble gc proteins. Notably, we found that increased expression of sgc resulted in impaired generation and differentiation of thymic NKT cells, which critically depend on IL-15 signaling. Interestingly, generation of IL-2-dependent Foxp3 T regulatory cells or IL-7-dependent CD8SP thymocytes were not affected, indicating distinct sensitivity of different gc cytokine receptors to sgc proteins. Altogether, these results demonstrated a previously unappreciated role for sgc in downregulating surface gc expression and in dampening gc cytokine signaling in thymocytes. Realizing such impact of alternative splicing on cytokine receptor expression, we shifted our focus to other receptors of the gc cytokine family. Among others, we found soluble IL-7Ra proteins in serum of both human and mice suggesting a potential role for these protein products in controlling T cell immunity. 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 marked contrast, so far, soluble IL-7Ra proteins has not been reported in mice. In fact, there is no molecular evidence reported for an alternative IL-7Ra mRNA splice isoform in mice, so that soluble IL-7Ra has been considered not evolutionally conserved. However, here we found that mice do express soluble IL-7Ra. By establishing a sensitive ELISA assay to serum IL-7Ra proteins, we could detect soluble IL-7Ra proteins in serum of wildtype mice. 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. Cloning of IL-7Ra mRNA species from mouse T cells showed that alternative splicing of IL-7Ra pre-mRNA utilized a distinct mechanism from human T cells in that is use intron-retention, instead of exon exclusion, for alternative splicing. Thus, we identified the molecular basis of soluble IL-7Ra proteins in mice. Nonetheless, we do not exclude the possibility of membrane protein shedding to produce soluble IL-7Ra proteins, and we are also investigating other post-transcriptional mechanisms of IL-7Ra production. Whether soluble IL-7Ra proteins are involved in controlling T cell immunity and T cell differentiation is an important question that is currently under investigation. In parallel to these studies, we used fluorescent beads to quantify the absolute number of gc cytokine receptors on cell surface of T cell subsets with the aim to gain better understanding of the expression under steady state condition and during T cell activation/differentiation. We found that IL-7Ra proteins outnumbered gc molecules at a ratio of four to one on surface on 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 the availability of gc proteins is limited for association with IL-7Ra, and consequently IL-7 signaling is curtailed 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. To understand the quantitative effects of cytokine receptor availability and signaling, we have generated a series of new experimental models that include enforced expression of gc or IL-7Ra proteins on T cells. The downstream biological effects of altering gc availability is currently under investigation.
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