Post-Transcriptional Regulation of Interleukin-7 Receptor Expression

gc-chain家族细胞因子在T细胞的发育和分化中起着至关重要的作用。我们的目的是了解gc-chain在T细胞发育和激活过程中的表达机制。除了转录控制机制外，我们还发现了一种新的gc-链转录后表达途径，可导致可溶性gc-链的产生。我们在正常人和小鼠血清中检测到大量可溶性gc蛋白，并研究了其在稳态和自身免疫条件下的功能。可溶性gc （sgc）是由外显子跳跃和开放阅读框在选择性剪接时的移码产生的。为了评估其在体内的作用，我们产生了在T细胞中过表达可溶性gc的sgc转基因小鼠。这些sgc Tg小鼠在血清中表达高水平的sgc，其T细胞表现出激活记忆表型的百分比增加。重要的是，当sgc Tg小鼠在实验性自身免疫性脑脊髓炎（EAE）模型中受到免疫反应性挑战时，sgc Tg小鼠的自身免疫反应显著增加，表现为更快、更强、更持久的临床疾病评分。这种增强反应的潜在机制被证明是促炎Th17细胞的增加，我们目前正在确定这种增强反应的确切机制。可溶gc链的选择性剪接调控的分子机制目前尚不清楚。然而，我们发现未成熟的DP胸腺细胞和外周NK细胞表达高水平的替代剪接形式，因此我们考虑在这些细胞中高表达的核因子参与可溶性gc链的产生。重要的是，IL-7受体的IL-7特异性亚基也经历了选择性剪接，并且已经报道了选择性剪接导致可溶性IL-7Ra链蛋白的产生。值得注意的是，可溶性IL-7Ra蛋白与多发性硬化症风险增加有关，但其如何加剧病理尚不清楚。此外，在小鼠中，没有报道可溶性IL-7Ra蛋白，也没有分子证据表明存在另一种IL-7Ra剪接形式。我们建立了不同的系统来检测小鼠血清中可溶性IL-7R蛋白，但迄今为止还没有成功。我们的目标是在进一步的研究中探讨可溶性IL-7受体在小鼠模型中的作用。最后，我们研究了可能控制IL-7受体表达的核因子。我们发现并报道了锌指蛋白Gfi1控制CD8谱系T细胞中IL-7Ra的转录。然而，有趣的是，Gfi1的缺失或过表达并不影响CD4谱系细胞中IL-7R的表达。这些数据表明IL-7Ra在CD4和CD8 T细胞中的表达有不同的分子调控，这对理解CD4和CD8 T细胞中的IL-7有重要意义。值得注意的是，我们还发现了其他几个锌指蛋白控制IL-7Ra的表达，我们正在评估它们在转录和转录后水平上对IL-7R调控的影响。沿着这些路线，CD8 T细胞严重依赖于IL-7来维持体内平衡和生存，而CD4 T细胞相对独立于IL-7。IL-7受体的调控是否参与了这一过程是我们希望通过IL-7Ra转基因和IL-7转基因小鼠来解决的问题。总之，我们的研究揭示了IL-7R表达的复杂调控网络，我们认为理解这个网络对于理解T细胞的发育、分化和稳态很重要。

Cytokines of the gc-chain family play critical roles in T cell development and differentiation. We aimed to understand the mechanism of gc-chain expression during T cell development and activation. In addition to transcriptionally controlled mechanisms, we discovered a novel post-transcriptional pathway of gc-chain expression that leads to the generation of soluble gc-chains. We detected soluble gc proteins in significant amounts in both normal human and mouse serum, and we have investigated its function under both homeostatic and autoimmune conditions. Soluble gc (sgc) is generated by exon skipping and by a frameshift in the open reading frame upon alternative splicing. To assess its role in vivo, we generated sgc transgenic mice that overexpress soluble gc in T cells. These sgc Tg mice expressed high levels of sgc in serum and their T cells showed an increased percentage of activated memory phenotype. Importantly, when sgc Tg mice were challenged in an experimental autoimmune encephalomyelitis (EAE) model for immune reactivity, sgc Tg mice displayed a significant increase in autoimmune reaction as shown by a faster, stronger and more lasting clinical disease score. The underlying mechanism for such an enhanced reaction turned out to be increased generation of pro-inflammatory Th17 cells, and we are currently in the process of identifying the exact mechanism for this enhanced reactivity. The molecular mechanisms how alternative splicing of the soluble gc chain regulated is currently not known to us. However, we found that immature DP thymocytes and peripheral NK cells express high levels of the alternative splice form so that we consider the involvement of nuclear factors that are highly expressed in these cells in the generation of soluble gc chains. Importantly, the IL-7-specific subunit of the IL-7 receptor also undergoes alternative splicing, and it has been reported alternative splicing results in the generation of soluble IL-7Ra chain proteins. Notably, soluble IL-7Ra proteins are associated with increased risk for multiple sclerosis but how it exacerbates pathology is not clear. Moreover, in the mouse, soluble IL-7Ra proteins have not been reported and there is no molecular evidence for an alternative IL-7Ra splice form. We have set up different systems to detect soluble IL-7R proteins in mice serum but were not successful do far. We aim to pursue this issue in further studies to investigate the role of soluble IL-7 receptors in a mouse model. Finally, we investigated potential nuclear factors that could control IL-7 receptor expression. We discovered and reported that the zinc finger protein Gfi1 controls IL-7Ra transcription in CD8 lineage T cells. Interestingly, however, absence or overexpression of Gfi1 did not affect IL-7R expression in CD4 lineage cells. These data suggest distinct molecular regulation of IL-7Ra expression in CD4 versus CD8 T cells, which is of interest for understanding the IL-7 in CD4 and CD8 T cells. Notably, we also identified a couple of other zinc finger proteins that control IL-7Ra expression, and we are assessing their effect on IL-7R regulation on both transcriptional and post-transcriptional levels. Along these lines, CD8 T cells are critically dependent on IL-7 for homeostasis and survival while CD4 T cells are rather independent of IL-7. Whether IL-7 receptor regulation is involved in this process is a question that we aim to address using IL-7Ra transgenic and IL-7 transgenic mice. In sum, our studies reveal a complex regulatory network of IL-7R expression, and we consider the understanding of this network important to understand T cell development, differentiation and homeostasis.