Post-Transcriptional Regulation of Interleukin-7 Receptor Expression

功能性IL-7受体由IL-7专有的IL-7 Ra和与其他细胞因子共享的共同γ链（gc）组成。在过去的几年里，我们的努力一直集中在了解IL-7 Ra和gc表达的调控，特别是在T细胞发育和分化过程中其表达的转录后机制。我们先前报道了小鼠和人T细胞都产生gc的选择性剪接产物，其导致可溶性gc蛋白的产生和分泌（Hong C等人，2014，Immunity）。这种选择性剪接机制是否也存在于IL-7 Ra中，特别是在小鼠模型中，尚不清楚。在人类中，据报道产生IL-7 Ra前mRNA的可变剪接产物，并且它们编码可溶性IL-7 Ra蛋白。然而，在小鼠中，尚未报道可溶性IL-7 Ra蛋白，并且没有可用于替代IL-7 Ra mRNA剪接同种型的分子证据。在人类中，可溶性IL-7 Ra通过选择性剪接产生，其跳过编码整个跨膜区的外显子6。我们未能在小鼠中检测到这种选择性转录，这表明-如果小鼠会产生IL-7 Ra剪接产物-人类和小鼠之间的选择性剪接机制会有所不同。通过对小鼠T细胞IL-7 Ra mRNA种类的分子克隆和测序，我们最近表明它们确实产生前mRNA剪接异构体。然而，小鼠中IL-7 Ra前mRNA的选择性剪接利用与人T细胞中不同的机制（Won HY等人，2020，Cell Mol Immunol）。具体地，小鼠T细胞利用内含子保留而不是外显子排除进行可变剪接，但是IL-7 Ra前mRNA的这种剪接同种型仍然编码潜在的可溶性IL-7 Ra蛋白（Won HY等人，2020，Cell Mol Immunol）。这种可溶性IL-7 Ra蛋白是否真的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受体表达中的重要性，我们目前正在确定这些过程的机制细节。总的来说，这些结果表明，表面细胞因子受体的丰度可以在许多层中进行控制，而不仅仅是通过常规考虑的转录控制。作为控制细胞因子受体可用性的另一种机制，我们还重点分析了细胞因子受体预结合的作用。我们先前的工作证明了gc蛋白与gc家族的专有细胞因子受体如IL-7 Ra的直接相互作用（Hong C.例如，2014，Immunity）。使用表面等离子体共振测定进一步评估这种细胞因子受体预缔合的生物学意义，其中我们定量了gc与IL-7 Ra相对于IL-2 Rb的结合动力学。在这里，我们发现，与IL-2 Rb相比，gc优先与IL-7 Ra结合，因此IL-7 Ra会将gc与IL-2 Rb隔离并削弱IL-2受体信号传导。基于这些发现，我们预测下调IL-7 Ra将促进IL-2受体信号传导，而强制性IL-7 Ra将抑制IL-2信号传导。当gc的可用性受到限制使得IL-2 Rb需要与IL-7 Ra竞争结合gc时，IL-7 Ra对IL-2 R信号传导的这种负调节机制将更加重要。事实上，当我们使用荧光珠产生标准曲线，然后定量T细胞的细胞表面上的gc细胞因子受体的绝对数量时，我们发现IL-7 Ra蛋白在静息的幼稚CD 4 T细胞表面上以4：1的比例超过gc分子。其他gc家族细胞因子受体，如IL-2 Rb，表达的数量明显低于gc。这些结果证明了gc蛋白的可用性在稳态条件下是有限的，并且它们还表明，由于gc而不是IL-7 Ra的量有限，IL-7信号传导在CD 4 T细胞中被削减。为了进一步了解细胞因子受体可用性和信号传导的定量作用，我们已经产生了一系列新的实验模型，包括在T细胞上强制表达gc或IL-7 Ra蛋白。具体地，我们检查了IL-7 Ra对Foxp 3 + Treg细胞的作用，Foxp 3 + Treg细胞的产生依赖于IL-2 R信号传导。Foxp 3 + Treg细胞在CD 4 T细胞中表达独特的低量IL-7 Ra，但原因尚不清楚。我们发现IL-7 Ra表达的下调是由转录因子Foxp 3直接介导的。并且IL-7 Ra的缺失是Foxp 3分化程序的一个整体特征。这些观察结果使我们提出IL-7 Ra会干扰IL-2 R信号传导，因为IL-7 Ra会螯合gc。一致地，我们发现IL-7 Ra的强制表达深刻地损害Foxp 3 + Treg细胞的体外分化并损害IL-2信号传导。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传导的另一层。我们目前正在测试gc螯合在其他T细胞亚群（如产生IL-17的Th 17细胞）的产生和效应子功能中的意义，我们也在开发新的体内模型以证明我们发现的生物学意义。

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.