Post-Transcriptional Regulation of Interleukin-7 Receptor Expression

具有功能的IL-7受体由IL-7专有的IL-7ra和与其他细胞因子共享的公共伽马链(GC)组成。在过去的几年里，我们一直致力于了解IL-7ra和GC表达的调节，特别是在T细胞发育和分化过程中它们表达的转录后机制。我们之前曾报道，小鼠和人类T细胞都会产生GC的另一种剪接产物，导致产生和分泌可溶性GC蛋白(Hong C et el，2014，免疫学)。目前还不清楚IL-7ra是否也存在这种选择性剪接机制，特别是在小鼠模型中。据报道，在人类中，产生了IL-7Ra前-mRNA的选择性剪接产物，它们编码可溶性IL-7Ra蛋白。然而，在小鼠中，还没有关于可溶性IL-7ra蛋白的报道，也没有替代IL-7ra mRNA剪接异构体的分子证据。在人类中，可溶性IL-7ra是通过跳过外显子6的选择性剪接产生的，外显子6编码整个跨膜区。我们未能在小鼠身上检测到这种选择性转录本，这表明--如果小鼠产生IL-7ra剪接产物--选择性剪接的机制将在人类和小鼠之间有所不同。通过对小鼠T细胞中IL-7ra基因的克隆和测序，我们最近发现它们确实能产生前mRNA剪接异构体。然而，小鼠中IL-7Ra前-mRNA的选择性剪接使用了与人类T细胞不同的机制(Won HY等人，2020，Cell Mol Invol)。具体地说，小鼠T细胞利用内含子保留而不是外显子排除进行选择性剪接，但IL-7Ra前-mRNA的这种剪接亚型仍然编码潜在的可溶IL-7Ra蛋白(Won HY等人，2020，Cell Mol免疫)。这种可溶性的IL-7ra蛋白是否真的会分泌，以及它们是否会控制T细胞免疫和T细胞分化，是目前正在研究的重要问题。尽管如此，这些结果肯定了选择性剪接在调节IL-7受体表达方面的重要性，我们目前正在确定这些过程的机制细节。总而言之，这些结果表明，表面细胞因子受体的丰度可以在多个层面上控制，而不是像传统认为的那样，仅仅通过转录控制。作为控制细胞因子受体可获得性的另一种机制，我们还重点分析了细胞因子受体预关联的作用。我们以前的工作证明了GC蛋白与GC家族专有的细胞因子受体直接相互作用，如IL-7Ra(Hong C.等，2014，免疫)。通过表面等离子体共振分析进一步评估了这种细胞因子受体预结合的生物学意义，其中我们定量了GC与IL-7Ra和IL-2Rb的结合动力学。在这里，我们发现，与IL-2Rb相比，GC优先与IL-7Ra结合，因此IL-7Ra会将GC与IL-2Rb隔离，从而损害IL-2受体的信号转导。基于这些发现，我们预测，下调IL-7ra将促进IL-2受体信号转导，而强迫IL-7ra将抑制IL-2信号转导。当GC的可用性受到限制时，IL-2Rb需要与IL-7Ra竞争与GC结合，这种IL-2Ra对IL-2R信号的负调控机制将更加重要。事实上，当我们使用荧光珠产生标准曲线，然后量化T细胞表面GC细胞因子受体的绝对数量时，我们发现在静止的初始CD4T细胞上，IL-7ra蛋白与GC分子的表面数量之比为4：1。其他GC家族细胞因子受体，如IL-2Rb的表达明显低于GC。这些结果证明，在稳态条件下，GC蛋白的可获得性是有限的，它们还表明，由于GC而不是IL-7ra的数量限制，CD4T细胞中的IL-7信号被抑制。为了进一步了解细胞因子受体的可获得性和信号转导的定量效应，我们建立了一系列新的实验模型，包括强制表达T细胞上的GC或IL-7ra蛋白。具体地说，我们研究了IL-7ra对Foxp3+Treg细胞的影响，这些细胞的生成依赖于IL-2R信号。在CD4T细胞中，Foxp3+Treg细胞表达独特的低数量IL-7ra，但原因尚不清楚。我们发现IL-7ra的表达下调是由转录因子Foxp3直接介导的。而IL-7ra的缺失是Foxp3分化计划的一个重要特征。这些观察结果使我们推测IL-7ra可能会干扰IL-2R信号转导，因为IL-7ra会隔离GC。一致地，我们发现强制表达IL-7ra严重损害了Foxp3+Treg细胞的体外分化，并损害了IL-2信号转导。因此，这些结果揭示了在转录后水平控制细胞因子受体信号的另一层。我们目前正在测试GC隔离在其他T细胞亚群的生成和效应功能中的意义，例如产生IL-17的Th17细胞，我们还在开发新的体内模型，以证明我们的发现的生物学意义。

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.