Post-Transcriptional Regulation of Interleukin-7 Receptor Expression

Division Of Basic Sciences - Nci

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Abstract

We previously identified alternative splicing of gc and IL-7Ra pre-mRNAs as a major mechanism to control the abundance of these cytokine receptors in developing thymocytes and in activated T cells. We found that TCR stimulation can upregulate alternative splicing of gc, and we also identified that thymic differentiation of immature thymocytes is associated with distinct amounts of alternatively spliced gc pre-mRNA. In case of IL-7Ra, however, it is less clear how alternative splicing is controlled upon T cell activation and differentiation. In humans, the alternative splicing of IL-7Ra pre-mRNA had been previously reported, but until our recent study (Won HY et al., 2020, Cellular & Molecular Immunology), the existence of alternatively spliced IL-7Ra in mice had not been known. In human T cells, IL-7Ra pre-mRNA is alternatively spliced by omitting exon 6, which encodes the entire transmembrane region. The cloning of IL-7Ra mRNA species from mouse T cells showed that the mouse immune system utilized a distinct mechanism from human T cells, so that intron-retention, instead of exon exclusion, generates splice isoforms of IL-7Ra mRNA. Whether this is the only mechanism to generate alternative splice products of IL-7Ra remains to be examined. Nonetheless, we are also aware of the fact that the alternative splice product will possibly result in the production of soluble IL-7Ra proteins, and we aim to examine whether such soluble IL-7Ra proteins are involved in controlling T cell immunity and T cell differentiation. In course of our study examining the biological effects of soluble IL-7Ra proteins, we previously found that soluble IL-7Ra proteins can pre-associate with gc proteins in the absence of ligands. Our work demonstrated that such direct interaction of gc and IL-7Ra proteins is not unique to the IL-7Ra but also observed for other cytokine proprietary receptors of the gc family, such as the IL-2Rb. SPR analysis of the binding affinities between these cytokine receptor pairs revealed a dramatic preference for IL-7Ra/gc association compared to IL-2Rb/gc association. Under this scenario, we predicted that all gc molecules would be sequestered by IL-7Ra, thus limiting its availability for other gc family receptors, including IL-2Rb. Such a pre-association model of cytokine receptors puts forward a new layer of control in cytokine signaling, which we predicted to impact cytokine signaling during T cell activation and differentiation. To test this hypothesis, we first used fluorescent beads to quantify the absolute number of gc molecules on cell surface of T cells. We found that IL-7Ra proteins outnumbered gc proteins at a ratio of four to one on the 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 documented that the availability of gc proteins is limited, and that IL-7 signaling is curtailed in CD4 T cells because of the limited amounts of gc. These findings further suggested that signaling by cytokines other than IL-7, such as IL-2 and IL-4, would be heavily constrained in the presence of IL-7Ra proteins. To directly demonstrate that IL-7Ra can act as a negative regulator of IL-2 or IL-4 signaling, we next analyzed cytokine receptor signaling in Foxp3+ Treg cells. While most T cells depend on IL-7 for their survival, it is well established that Foxp3+ Treg cells require IL-2 for their development and homeostasis. Importantly, Treg cells express uniquely low amounts of IL-7Ra but it has not been known why this would be the case and whether it is necessary to suppress IL-7Ra expression for Foxp3+ Treg cell function. In our recent study (Waickman AT et al., 2020, iScience), we have now reported that Foxp3 is a transcriptional suppressor of IL-7Ra expression, and that IL-7Ra downregulation is necessary to maximize IL-2 signaling in Foxp3+ Treg cells. Specifically, overexpression of the extracellular domain of IL-7Ra induced the pre-association and sequestration of gc proteins with IL-7Ra, thus dramatically inhibiting IL-2 signaling in Foxp3+ Treg cells. We found that such gc sequestration can be also observed in the generation of IL-17-producing Th17 cells. Here, we found that the deletion of IL-7Ra increased the availability of gc while overexpression of IL-7Ra significantly constrained gc availability so that the abundance of IL-7Ra controlled the efficiency of Th17 cell differentiation. Collectively, our results unveiled a previously unappreciated crosstalk of IL-7R and IL-2R where the abundance of IL-7Ra controls the signaling of IL-2R. Finally, we have initiated a series of new studies where we expanded the scope of our studies to the epigenetic aspect of IL-7Ra expression, in parallel to our current studies. We recently identified the chromatin remodeling factor Ikaros (Ikfz1) as a strong candidate to control the accessibility of IL-7Ra gene locus and to control IL-7Ra expression during the early T cell development in the thymus and in the survival/homeostasis of peripheral T cells. To further understand how Ikaros contributes to cytokine receptor regulation in general and specifically to IL-7Ra expression, we have generated a series of Ikfz1-conditional knock out mice, and also generated Ikfz1 transgenic mice. Analyzing the T cell development and differentiation of these mice in context of IL-7R expression is currently under investigation.