Post-Transcriptional Regulation of Interleukin-7 Receptor Expression

传统上，GC的表达被认为是发育设定的，不受T细胞的分化或激活状态的影响。我们以前的工作表明，情况并非如此，GC的表达在T细胞发育和动态平衡过程中受到动态控制。利用新建立的小鼠模型，如可溶性GC(SGC)转基因小鼠，我们发现GC可获得性或功能的改变可以控制胸腺不变NKT细胞(iNKT细胞)的产生和先天CD8T细胞的分化。因此，控制细胞因子受体信号的不是GC本身的表达，而是GC表面表达的量。在这方面，有趣的是，与未成熟的CD4-CD8双阴性(DN)或CD4、CD8单阳性(SP)胸腺细胞相比，未成熟的CD4+CD8+双阳性(DP)胸腺细胞表达独特的低数量的GC。为了了解这一观察背后的分子基础，我们研究了转录因子RORgt的作用，该转录因子在未成熟的DP细胞中唯一且高表达。我们推测，RORgt的特异性表达可能与抑制DP胸腺细胞中GC的表达有关。因此，RORgt的缺失可能会使GC的表达恢复到在糖尿病肾病或SP胸腺细胞中发现的数量。情况正是如此，因为我们发现缺乏RORgt的DP胸腺细胞上调了GC表面的表达，与成熟T细胞上发现的类似数量相同。已知RORgt可诱导未成熟DP细胞表达抗凋亡因子Bclxl。因此，我们想知道在未成熟的DP细胞中，Bclxl的表达是否足以抑制GC的表达。为此，我们培育了表达BclXL转基因的RORgt缺陷小鼠，我们发现BclXL足以下调DP胸腺细胞上GC的表达。这些结果揭示了一种新的细胞因子受体表达调控途径，该途径可由促生存因子如Bclxl调控。这些结果还表明，表面细胞因子受体的表达可以在多个层面上进行控制，而不是像通常所接受的那样，简单地通过转录控制来进行。在这方面，我们也一直在分析细胞因子受体预关联在GC细胞因子信号转导中的作用。我们以前的工作证明了GC与GC家族的其他专有受体，如IL-7Ra和IL-2Rb直接相互作用。对这些细胞因子受体对之间的结合亲和力的详细分析表明，与IL-2Rb/GC结合相比，IL-7Ra/GC结合具有显著的偏好。如果是这样的话，我们预测所有的GC分子将被隔离并预先与IL-7Ra结合，从而限制了它对包括IL-2Rb在内的其他GC家族受体的可获得性。这种细胞因子受体的预关联模型提出了一个新的控制细胞因子信号的层，我们发现证明它在T细胞激活和分化中的作用是很重要的。为此，我们使用荧光微珠来定量T细胞表面GC细胞因子受体的绝对数量。我们发现，在静息状态的CD4T细胞表面，IL-7ra蛋白与GC分子的数量之比为4：1。另一方面，其他GC细胞因子受体如IL-4Ra或IL-2Rb的表达明显低于GC。这些结果表明，GC蛋白的可获得性是有限的，并且由于GC而不是IL-7ra的数量有限，CD4T细胞上的IL-7信号被抑制。为了进一步了解细胞因子受体可获得性和信号转导的定量效应，我们现在已经建立了一系列新的实验模型，其中包括增强T细胞上GC或IL-7ra蛋白的表达。改变GC可用性的下游生物学效应目前正在调查中。最后，为了进一步了解选择性剪接对细胞因子受体表达的影响，我们重点研究了GC细胞因子家族中其他受体的选择性剪接。由于我们在人和小鼠的血清中都发现了可溶性的IL-7ra蛋白，我们推测这些蛋白产物在控制T细胞免疫方面具有潜在的作用。在人类中，先前已经描述了可溶性IL-7Ra，并且已知可溶性IL-7Ra蛋白是通过IL-7Ra前体mRNA的选择性剪接而产生的。然而，在小鼠中，还没有可溶的IL-7ra蛋白的报道，也没有分子证据表明在小鼠中存在另一种IL-7ra mRNA剪接异构体。因此，我们观察到小鼠血清中确实含有可溶的IL-7ra蛋白，这是令人惊讶的。在人类中，可溶性的IL-7ra是通过选择性剪接产生的，它省略了编码整个跨膜区的外显子6。然而，我们无法在小鼠身上检测到这种替代转录本，这表明产生可溶性IL-7ra蛋白的机制在人类和小鼠之间不同。事实上，从小鼠T细胞中克隆的IL-7Ra mRNA物种表明，IL-7Ra Pre-mRNA的选择性剪接利用了与人类T细胞不同的机制，即使用内含子保留而不是外显子排除进行选择性剪接。这是否是产生可溶性IL-7ra蛋白的唯一机制还需要研究，我们不排除膜蛋白脱落产生可溶性IL-7ra蛋白的可能性。这种可溶性IL-7ra蛋白是否真的参与控制T细胞免疫和T细胞分化是目前正在研究的一个重要问题。

Conventionally, gc expression has been thought to be developmentally set and not affected by the differentiation or activation status of T cells. Our previous works have shown that this is not that case, and that gc expression is dynamically controlled during T cell development and homeostasis. Using newly established mouse models, such as the soluble gc (sgc) transgenic mice, we found that alteration in gc availability or function can control the generation of thymic invariant NKT cells (iNKT cells) and the differentiation of innate type CD8 T cells. Thus, it is not the expression of gc per se but also the quantity of gc surface expression that controls cytokine receptor signaling. In this regard, it was interesting to learn that immature CD4+CD8+ double-positive (DP) thymocytes express uniquely low amounts of gc compared to immature CD4-CD8- double-negative (DN) or CD4, CD8 single-positive (SP) thymocytes. To understand the molecular basis behind this observation, we addressed the role of the transcription factor RORgt which is uniquely and highly expressed in immature DP cells. We hypothesized that the specific expression of RORgt would be associated with suppression of gc expression in DP thymocytes. Thus, deletion of RORgt could possibly revert gc expression to the amounts found in DN or SP thymocytes. This was precisely the case, because we found that RORgt-deficient DP thymocyte upregulated gc surface expression to comparable amounts found on mature T cells. RORgt is known to induce expression of the anti-apoptotic factor Bcl-xL in immature DP cells. Consequently, we wished to know if Bcl-xL expression would be sufficient to suppress gc expression in immature DP cells. To this end, we generated RORgt-deficient mice that express a Bcl-xL transgene, and we found that Bcl-xL was sufficient to downregulate gc expression on DP thymocytes. These results reveal a new regulatory pathway of cytokine receptor expression that can be controlled by pro-survival factors, such as Bcl-xL. These results also suggest that surface cytokine receptor expression can be controlled in many layers and not simply by transcriptional control as conventionally accepted. In this regard, we have been also analyzing the role of cytokine receptor pre-association in gc cytokine signaling. Our previous work demonstrated a direct interaction of gc with other proprietary receptors of the gc family, such as IL-7Ra and IL-2Rb. Detailed analysis of the binding affinities between these cytokine receptor pairs revealed a dramatic preference for IL-7Ra/gc association compared to IL-2Rb/gc binding. If this would be the case, we predicted that all gc molecules would be sequestered and pre-bound to IL-7Ra, thus limiting its availability to other gc family receptors including IL-2Rb. Such a pre-association model of cytokine receptors puts forward a new layer of controlling cytokine signaling, and we found it important to demonstrate its role in T cell activation and differentiation. To this end, we used fluorescent beads to quantify 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. 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 revealed that the availability of gc proteins is limited, and that IL-7 signaling is curtailed on 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 now generated a series of new experimental models that include enforced expression of gc or IL-7Ra proteins on T cells. The downstream biological effects of altering gc availability is currently under investigation. Finally, to further understand the impact of alternative splicing on cytokine receptor expression, we focused on alternativesplicing of other receptors in the gc cytokine family. Because we found soluble IL-7Ra proteins in serum of both human and mice, we hypothesized a potential role for these protein products in controlling T cell immunity. In humans, soluble IL-7Ra has been previously described, and it is known that soluble IL-7Ra proteins are produced by alternative splicing of the IL-7Ra pre-mRNA. 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 mice. Thus, our observation that mice do contain soluble IL-7Ra proteins in serum was surprising. In humans, soluble IL-7Ra is produced by alternative splicing that omits exon 6, which encodes the entire transmembrane region. However, we were unable to detect such alternative transcripts in mice, suggesting that the mechanism to produce soluble IL-7Ra proteins differ between humans and mice. In fact, cloning of IL-7Ra mRNA species from mouse T cells showed that alternative splicing of IL-7Ra pre-mRNA utilized a distinct mechanism from human T cells in that is use intron-retention, instead of exon exclusion, for alternative splicing. Whether this is the only mechanism to generate soluble IL-7Ra proteins need to be examined, and we do not exclude the possibility of membrane protein shedding to produce soluble IL-7Ra proteins. Whether such soluble IL-7Ra proteins are indeed involved in controlling T cell immunity and T cell differentiation is an important question that is currently under investigation.