Post-Transcriptional Regulation of Interleukin-7 Receptor Expression

传统上，gc表达被认为是发育设定的，不受T细胞分化或活化状态的影响。我们以前的工作表明，这是不是这种情况下，和GC的表达是动态控制在T细胞的发展和稳态。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的可用性或功能的改变可以控制胸腺不变的NKT细胞（iNKT细胞）的产生和先天性CD 8 T细胞的分化。因此，控制细胞因子受体信号传导的不是gc本身的表达，而是gc表面表达的量。在这方面，有趣的是了解到，与未成熟的CD 4-CD 8-双阴性（DN）或CD 4，CD 8单阳性（SP）胸腺细胞相比，未成熟的CD 4 + CD 8+双阳性（DP）胸腺细胞表达独特的低量gc。为了理解这一观察结果背后的分子基础，我们讨论了在未成熟DP细胞中独特且高度表达的转录因子RORgt的作用。我们推测RORgt的特异性表达与DP胸腺细胞中GC表达的抑制有关。因此，RORgt的缺失可能会将gc表达恢复到DN或SP胸腺细胞中发现的量。这是确切的情况下，因为我们发现，RORgt缺陷DP胸腺细胞上调gc表面表达的成熟T细胞上发现相当的量。已知RORgt诱导抗凋亡因子Bcl-xL在未成熟DP细胞中的表达。因此，我们希望知道Bcl-xL表达是否足以抑制未成熟DP细胞中的gc表达。为此，我们产生了表达Bcl-xL转基因的RORgt缺陷小鼠，并且我们发现Bcl-xL足以下调DP胸腺细胞上的gc表达。这些结果揭示了一种新的细胞因子受体表达的调节途径，可以通过促生存因子，如Bcl-xL控制。这些结果还表明，表面细胞因子受体的表达可以在许多层中控制，而不是简单地通过转录控制，如传统上接受的。在这方面，我们也一直在分析的作用，细胞因子受体预协会在gc细胞因子信号。我们以前的工作证明了gc与gc家族的其他专有受体（如IL-7 Ra和IL-2 Rb）的直接相互作用。这些细胞因子受体对之间的结合亲和力的详细分析揭示了与IL-2 Rb/gc结合相比，IL-7 Ra/gc结合的显著偏好。如果是这种情况，我们预测所有gc分子将被隔离并预先结合到IL-7 Ra，从而限制其对其他gc家族受体包括IL-2 Rb的可用性。这种细胞因子受体的预缔合模型提出了一个新的控制细胞因子信号传导的层，我们发现证明其在T细胞活化和分化中的作用是重要的。为此，我们使用荧光珠来定量T细胞的细胞表面上的gc细胞因子受体的绝对数量。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。另一方面，其他gc细胞因子受体如IL-4 Ra或IL-2 Rb的表达数量显著低于gc。这些结果表明，gc蛋白的可用性是有限的，并且由于gc而不是IL-7 Ra的量有限，IL-7信号传导在CD 4 T细胞上被削减。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，我们现在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。目前正在研究改变气相色谱可用性的下游生物效应。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，我们集中研究了gc细胞因子家族中其他受体的选择性剪接。因为我们在人和小鼠的血清中发现了可溶性IL-7 Ra蛋白，我们假设这些蛋白产物在控制T细胞免疫中的潜在作用。在人类中，之前已经描述了可溶性IL-7 Ra，并且已知可溶性IL-7 Ra蛋白是通过IL-7 Ra前mRNA的选择性剪接产生的。然而，在小鼠中，尚未报道可溶性IL-7 Ra蛋白，并且没有分子证据可用于小鼠中的替代IL-7 Ra mRNA剪接同种型。因此，我们观察到小鼠血清中确实含有可溶性IL-7 Ra蛋白是令人惊讶的。在人类中，可溶性IL-7 Ra通过省略外显子6的选择性剪接产生，外显子6编码整个跨膜区。然而，我们无法在小鼠中检测到这种替代转录物，这表明产生可溶性IL-7 Ra蛋白的机制在人类和小鼠之间不同。事实上，从小鼠T细胞克隆IL-7 Ra mRNA种类显示，IL-7 Ra前mRNA的可变剪接利用了与人T细胞不同的机制，即对于可变剪接使用内含子保留而不是外显子排除。这是否是产生可溶性IL-7 Ra蛋白的唯一机制需要检查，我们不排除膜蛋白脱落产生可溶性IL-7 Ra蛋白的可能性。这种可溶性IL-7 Ra蛋白是否确实参与控制T细胞免疫和T细胞分化是目前正在研究的重要问题。

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.