Post-Transcriptional Regulation of Interleukin-7 Receptor Expression

IL-7信号严重依赖于gc细胞因子受体的表达，但gc在T细胞发育和分化过程中的表达分子机制尚不清楚。在我们对gc mRNA表达的研究过程中，我们发现了一种新的gc-chain表达的转录后途径，该途径导致可溶性和分泌gc-chain蛋白的产生。我们在正常人和小鼠血清中检测到大量的可溶性gc （sgc）蛋白，并随后研究了它们在稳态和自身免疫条件下的功能。为了评估sgc在体内的作用，我们制造了在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）模型中受到攻击时，sgc Tg小鼠表现出显著的自身免疫反应增加，表现为更快、更严重的临床疾病评分。另一方面，不能产生可溶性gc的基因工程小鼠免受炎症性自身免疫性疾病的影响，这表明可溶性gc决定了自身免疫性炎症的疾病严重程度。由于T细胞激活诱导可溶性gc的产生，而可溶性gc又诱导炎症细胞因子的表达，我们提出可溶性gc的产生是一种增强T细胞激活下游的促炎免疫反应的机制。与这一观点一致的是，在人类和小鼠的炎症条件下，可溶性gc都高度表达；可溶性gc在类风湿性关节炎患者的滑液中大量表达，在寄生虫攻毒小鼠中也高度诱导。由于sgc是促炎的，因此推测血清sgc表达的中和是否会改善炎症性疾病并抑制破坏性T细胞反应是很有趣的。sgc特异性单克隆抗体的产生和评估其对体内促炎反应的影响是我们计划追求的有趣问题，我们承认利用这种抗体作为治疗药物来抑制炎症和改善疾病的潜力。与可溶性gc受体平行，我们还观察到人和小鼠血清中存在IL-7Ra链。在人类中，IL-7Ra经历了选择性剪接，据报道，选择性剪接诱导了可溶性IL-7Ra链蛋白的产生。这一转录后事件解释了可溶性IL-7Ra在人血清中的存在。相比之下，在小鼠中，可溶性IL-7Ra蛋白尚未报道，也没有分子证据表明存在替代IL-7Ra剪接异构体。因此，我们在小鼠血清中发现的可溶性IL-7Ra蛋白的来源仍未得到解释。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，为了在转录水平上了解IL-7Ra和gc表达的调控机制，我们进行了一系列研究，探讨了转录因子在IL-7受体表达中的作用。我们之前报道过锌指蛋白Gfi1在CD8系T细胞中特异性地控制IL-7Ra转录，而在CD4系T细胞中不起作用。在目前的研究中，我们已经确定了许多其他转录因子，这些转录因子以分期和细胞特异性的方式控制IL-7Ra和gc细胞因子受体的表达。表征它们的作用和分子调控机制是本项目的主要目标。

IL-7 signaling is critically dependent on gc cytokine receptor expression but the molecular mechanism of gc expression during T cell development and differentiation are unknown. In course of our studies of gc mRNA expression, we discovered a novel post-transcriptional pathway of gc-chain expression that results in the generation of soluble and secreted gc-chain proteins. We detected such soluble gc (sgc) proteins in significant amounts in both normal human and mouse serum, and we subsequently investigated their function under both homeostatic and autoimmune conditions. To assess the role of sgc 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, sgc Tg mice displayed a significant increase of autoimmune reaction as shown by faster and more severe clinical disease scores. On the other hand, genetically engineered mice that are unable to produce soluble gc were protected from inflammatory autoimmune disease, suggesting that soluble gc determines the disease severity of autoimmune inflammation. Since T cell activation induces soluble production and because soluble gc in turn induces inflammatory cytokine expression, we propose that soluble gc production is a mechanism to reinforce pro-inflammatory immune responses downstream of T cell activation. In agreement with this perspective, soluble gc is highly expressed under inflammatory conditions in both humans and mice; soluble gc was abundantly expressed in synovial fluid of patients with rheumatoid arthritis and also highly induced in mice challenged with parasites. Because sgc is pro-inflammatory, it is interesting to speculate if neutralization of serum sgc expression would ameliorate inflammatory disease and dampen destructive T cell responses. Generation of sgc-specific monoclonal antibodies and assessing their effects on pro-inflammatory responses in vivo are interesting issues that we plan to pursuit, and we acknowledge the potential to utilize such antibodies as therapeutics to dampen inflammation and ameliorate disease. In parallel to soluble gc receptors, we also observed that IL-7Ra chains were present in serum of human and mice. In humans, the IL-7Ra undergoes alternative splicing, and it has been reported that alternative splicing induces the generation of soluble IL-7Ra chain proteins. This post-transcriptional event explains the presence of soluble IL-7Ra in human serum. In contrast, in the mouse, soluble IL-7Ra proteins have not been reported and there is no molecular evidence for an alternative IL-7Ra splice isoform. Thus, the origin of soluble IL-7Ra proteins as we discovered in mouse serum remains unaccounted for. We aim to pursue this issue in further studies to investigate the role of soluble IL-7 receptors in a mouse model. Finally, in an attempt to understand the regulatory mechanisms of IL-7Ra and gc expression at transcriptional level, we conducted a series of study addressing the role of transcription factors in IL-7 receptor expression. We previously reported that the zinc finger protein Gfi1 controls IL-7Ra transcription specifically in CD8 lineage T cells but not in CD4 lineage cells. In the current study, we have identified a number of other transcription factors that control IL-7Ra and gc cytokine receptor expression in a stage and cell-specific manner. Characterization of their role and molecular mechanism of regulation is a major goal of this project.