Post-Transcriptional Regulation of Interleukin-7 Receptor Expression

点击翻译按钮获取中文摘要

The functional IL-7 receptor is composed of the IL-7 proprietary IL-7Ra and the common gamma chain (gc), which is shared with other cytokines. In the last few years, our efforts have been focused on understanding the regulation of the IL-7Ra and gc expression, and specifically on the post-transcriptional mechanisms of their expression during T cell development and differentiation. We previously reported that both mouse and human T cells produce an alternative splice product of gc that resulted in the generation and secretion of soluble gc proteins (Hong C et el., 2014, Immunity). Whether such an alternative splice mechanism is also present for IL-7Ra, specifically in the mouse model, has been unclear. In humans, it is reported that alternative splice products of IL-7Ra pre-mRNA are generated and they encode for soluble IL-7Ra proteins. In mice, however, soluble IL-7Ra proteins have not been reported, and there is no molecular evidence available for an alternative IL-7Ra mRNA splice isoform. In humans, soluble IL-7Ra is produced by alternative splicing that skips exon 6, which encodes the entire transmembrane region. We failed to detect such alternative transcripts in mice, suggesting that -if mice would generate IL-7Ra splice products- that the mechanism for alternative splicing would differ between humans and mice. By molecular cloning and sequencing of IL-7Ra mRNA species from mouse T cells, we recently showed that they indeed generate pre-mRNA splice isoforms. However, the alternative splicing of IL-7Ra pre-mRNA in mice utilized a distinct mechanism from that in human T cells (Won HY et al., 2020, Cell Mol Immunol). Specifically, mouse T cells utilized intron-retention, instead of exon exclusion, for alternative splicing, but this splice isoform of the IL-7Ra pre-mRNA still encoded for potential soluble IL-7Ra proteins (Won HY et al., 2020, Cell Mol Immunol). Whether such soluble IL-7Ra proteins are indeed secreted and if they would control T cell immunity and T cell differentiation are important questions that are currently under investigation. Nonetheless, these results affirm the importance of alternative splicing in regulating IL-7 receptor expression, and we are currently in the process of identifying the mechanistic details of these processes. Collectively, these results indicate that the abundance of surface cytokine receptors can be controlled in many layers, and not only by transcriptional control as conventionally considered. As an additional mechanism that controls the availability of cytokine receptors, we also focused on analyzing the role of cytokine receptor pre-association. Our previous work demonstrated a direct interaction of gc proteins with proprietary cytokine receptors of the gc family, such as the IL-7Ra (Hong C. et al., 2014, Immunity). The biological significance of such cytokine receptor pre-association was further assessed using surface plasmon resonance assays where we quantified the binding kinetics of gc to IL-7Ra versus IL-2Rb. Here, we found that gc preferentially bound to IL-7Ra compared to IL-2Rb, so that IL-7Ra would sequester gc from IL-2Rb and impair IL-2 receptor signaling. Based on these findings, we predicted that the downregulation of IL-7Ra would promote IL-2 receptor signaling while forced IL-7Ra would suppress IL-2 signaling. Such a negative regulatory mechanism of IL-2R signaling by IL-7Ra would be even more important when the availability of gc would be limited so that IL-2Rb need to compete with IL-7Ra for binding to gc. Indeed, when we used fluorescent beads to generate standard curves and then quantified the absolute number of gc cytokine receptors on cell surface of T cells, we found that IL-7Ra proteins outnumbered gc molecules at a ratio of four to one on surface on resting naive CD4 T cells. Other gc family cytokine receptors, such as the IL-2Rb, were expressed at significantly lower numbers than gc. These results document that the availability of gc proteins is limited under steady-state conditions, and they also indicate that IL-7 signaling is curtailed in CD4 T cells because of limiting amounts of gc and not IL-7Ra. To further understand the quantitative effects of cytokine receptor availability and signaling, we have generated a series of new experimental models that include forced expression of gc or IL-7Ra proteins on T cells. Specifically, we examined the effect of IL-7Ra on Foxp3+ Treg cells whose generation depends on IL-2R signaling. Foxp3+ Treg cells express uniquely low amounts of IL-7Ra among CD4 T cells, but it has not been clear why. We found that the downregulation of IL-7Ra expression is directly mediated by the transcription factor Foxp3. and that the loss of IL-7Ra is an integral feature of the Foxp3 differentiation program. These observations made us to propose that IL-7Ra would interfere with IL-2R signaling because IL-7Ra would sequester gc. In agreement, we found that forced expression of IL-7Ra profoundly impaired the in vitro differentiation of Foxp3+ Treg cells and impaired IL-2 signaling. Thus, these results unveil another layer of controlling cytokine receptor signaling in a post-transcriptional level. We are currently testing the implications of gc sequestration in the generation and effector function of other T cell subsets, such as IL-17-producing Th17 cells, and we are also in the process of developing new in vivo models to demonstrate the biological significance of our findings.