Post-Transcriptional Regulation of Interleukin-7 Receptor Expression

我们之前发现gc和IL-7Ra前mrna的选择性剪接是控制发育中的胸腺细胞和活化的T细胞中这些细胞因子受体丰度的主要机制。我们发现TCR刺激可以上调gc的选择性剪接，我们还发现未成熟胸腺细胞的胸腺分化与不同数量的gc pre-mRNA的选择性剪接有关。然而，在IL-7Ra的情况下，人们不太清楚如何在T细胞激活和分化时控制选择性剪接。在人类中，IL-7Ra前mrna的选择性剪接之前已经有报道，但直到我们最近的研究（Won HY等，2020,Cellular & Molecular Immunology），才知道小鼠中IL-7Ra的选择性剪接存在。在人类T细胞中，IL-7Ra前mrna通过省略编码整个跨膜区域的外显子6来选择性剪接。从小鼠T细胞中克隆IL-7Ra mRNA物种表明，小鼠免疫系统使用与人类T细胞不同的机制，因此内含子保留而不是外显子排除产生IL-7Ra mRNA的剪接异构体。这是否是产生IL-7Ra替代剪接产物的唯一机制还有待研究。尽管如此，我们也意识到这样一个事实，即替代剪接产物可能会导致可溶性IL-7Ra蛋白的产生，我们的目的是研究这些可溶性IL-7Ra蛋白是否参与控制T细胞免疫和T细胞分化。在我们研究可溶性IL-7Ra蛋白生物学效应的过程中，我们之前发现可溶性IL-7Ra蛋白可以在没有配体的情况下与gc蛋白预结合。我们的工作表明，gc和IL-7Ra蛋白的这种直接相互作用不仅是IL-7Ra所特有的，而且在gc家族的其他细胞因子专有受体（如IL-2Rb）中也观察到。SPR分析这些细胞因子受体对之间的结合亲和力显示，与IL-2Rb/gc关联相比，IL-7Ra/gc关联具有显著的偏好。在这种情况下，我们预测所有gc分子将被IL-7Ra隔离，从而限制其对其他gc家族受体的可用性，包括IL-2Rb。这种细胞因子受体的预关联模型为细胞因子信号传导提供了一个新的控制层，我们预测它会影响T细胞活化和分化过程中的细胞因子信号传导。为了验证这一假设，我们首先使用荧光珠来量化T细胞表面gc分子的绝对数量。我们发现IL-7Ra蛋白在静止的初始CD4 T细胞表面以4:1的比例超过gc蛋白。另一方面，其他gc细胞因子受体，如IL-4Ra或IL-2Rb的表达量明显低于gc。这些结果证明gc蛋白的可用性是有限的，并且由于gc的数量有限，CD4 T细胞中的IL-7信号被削弱。这些发现进一步表明，在IL-7Ra蛋白存在的情况下，IL-7以外的细胞因子（如IL-2和IL-4）的信号传导将受到严重限制。为了直接证明IL-7Ra可以作为IL-2或IL-4信号的负调节因子，我们接下来分析了Foxp3+ Treg细胞中的细胞因子受体信号。虽然大多数T细胞依赖IL-7存活，但Foxp3+ Treg细胞的发育和稳态也需要IL-2。重要的是，Treg细胞表达的IL-7Ra含量很低，但目前尚不清楚为什么会出现这种情况，以及Foxp3+ Treg细胞功能是否需要抑制IL-7Ra的表达。在我们最近的研究中（Waickman AT et al., 2020, iScience），我们现在已经报道Foxp3是IL-7Ra表达的转录抑制因子，并且IL-7Ra下调是Foxp3+ Treg细胞中最大化IL-2信号传导所必需的。具体来说，细胞外区域IL-7Ra的过表达诱导gc蛋白与IL-7Ra的预关联和隔离，从而显著抑制Foxp3+ Treg细胞中的IL-2信号传导。我们发现这种gc隔离也可以在产生il -17的Th17细胞中观察到。在这里，我们发现IL-7Ra的缺失增加了gc的可用性，而IL-7Ra的过表达显著限制了gc的可用性，因此IL-7Ra的丰度控制了Th17细胞分化的效率。总的来说，我们的研究结果揭示了IL-7R和IL-2R之前未被认识到的串扰，其中IL-7Ra的丰度控制着IL-2R的信号传导。最后，我们启动了一系列新的研究，将我们的研究范围扩展到IL-7Ra表达的表观遗传学方面，与我们目前的研究平行。我们最近发现染色质重塑因子Ikaros （Ikfz1）是控制IL-7Ra基因位点的可及性和控制IL-7Ra在胸腺早期T细胞发育和周围T细胞生存/稳态过程中的表达的强有力的候选者。为了进一步了解Ikaros对细胞因子受体的总体调控以及对IL-7Ra表达的特异性调控，我们构建了一系列Ikfz1条件敲除小鼠，并构建了Ikfz1转基因小鼠。在IL-7R表达的背景下分析这些小鼠的T细胞发育和分化目前正在研究中。

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.