Role of E3 Ubiquitin ligase RNF133 in T cell function and tolerance

项目总结/摘要 CD 4 + T细胞是适应性免疫反应的主要调节者，并且是免疫系统中自身耐受性的破坏。 CD 4+细胞导致许多自身免疫性疾病。理解T细胞耐受的机制 将提供新的重要见解，在推进我们的知识信号和遗传控制的T 辅助（TH）细胞耐受性程序，可能对炎症性疾病具有治疗意义。 最近，我们已经认识到GRAIL在免疫耐受中的重要作用。尽管 GRAIL在T细胞耐受性中的关键作用，GRAIL敲除（KO）小鼠不会自发发展 早期自身免疫，这表明与GRAIL密切相关的蛋白质可能有助于控制 炎症的早期发作。有趣的是，在五个GRAIL同源物中，RNF 133显示出最高的 在耐受性T细胞中表达，表明RNF 133沿着GRAIL可能有助于T细胞的建立。 细胞耐受性;然而，其在T细胞中的作用尚未研究。RNF 133 KO体外活化的CD 4 + T细胞和 在致耐受性条件下的体内试验显示出增强的增殖和细胞因子（白细胞介素（IL）-17）水平 和IL-21）产生，表明RNF 133在控制T细胞增殖中的潜在作用。 TH（TH 17和T滤泡（Tfh））细胞反应。事实上，RNF 133 KO小鼠的IgG和IgG 1水平升高， 在血清中的TH 17和Tfh细胞在外周淋巴组织中的百分比， 随着年龄的增长，在5-6个月大时自身免疫症状增加。此外，RNF 133表达在 调节性T细胞（Tcells）对于维持其抑制功能以及稳定性和预防 它们不能获得致病性TH 17表型，这表明RNF 133的功能是控制致病性TH 细胞反应。基于此，我们假设RNF 133可能是一个重要的检查点分子， 维持免疫耐受性和预防炎症的发生和发展。 在目标1中，我们建议确定负责调节和功能的分子机制， RNF 133在T细胞耐受中的作用及体内T细胞耐受研究 模型在目标2中，我们将确定RNF 133在Tfh细胞耐受性中的作用和潜在机制， 好.此外，我们将评估RNF 133控制抗体介导的自身免疫的机制。 在目的3中，我们将确定RNF 133调节Tcl 3和TH 17细胞的机制 编程和稳定性。这一发现的生理意义将在一个实验中进行评估。 变态反应性脑脊髓炎（EAE）模型。我们将采用酵母双杂交筛选和反相蛋白 阵列测定以鉴定RNF 133的确切靶标，其决定其在TH编程中的功能。 拟议的研究将提供新的重要洞察表征的机制 潜在的T细胞耐受性，这将导致药理学方法的发展，以促进 在自身免疫方面的耐受状态。

PROJECT SUMMARY/ABSTRACT CD4+ T cells are the master regulators of adaptive immune responses, and a breakdown of self-tolerance in CD4+ cells leads to many autoimmune diseases. Understanding of the mechanisms underlying T cell tolerance will provide new significant insight in advancing our knowledge on the signaling and genetic controls of T helper (TH) cell tolerance programs that may have therapeutic implications for inflammatory diseases. Recently, we have acknowledged the essential role of GRAIL in immunological tolerance. Despite the critical role of GRAIL in T cell tolerance, GRAIL knockout (KO) mice do not spontaneously develop autoimmunity at early age, suggesting that closely-related protein(s) to GRAIL could contribute to control of the early onset of inflammation. Interestingly, among the five GRAIL homologs, RNF133 shows the highest expression in tolerant T cells, suggesting that RNF133 along with GRAIL could contribute to establishment of T cell tolerance; however its role in T cells has not been studied. RNF133 KO CD4+ T cells activated in vitro and in vivo under tolerogenic conditions exhibited enhanced level of proliferation and cytokine (interleukin (IL)-17 and IL-21) production compared to wild-type (WT) T cells, indicating the potential role of RNF133 in controlling TH (TH17 and T follicular (Tfh)) cell responses. In fact, RNF133 KO mice have elevated levels of IgG and IgG1 in the sera and percentage of TH17 and Tfh cells in the peripheral lymphoid tissues as early as 12 weeks of age followed by rise of autoimmune symptoms at 5-6 months of age. Moreover, RNF133 expression in regulatory T cells (Tregs) is essential to maintain their suppressive function, as well as stability and prevents them from acquiring pathogenic TH17 phenotype, suggesting that RNF133 functions to control pathogenic TH cell responses. Based on this, we hypothesize that RNF133 may be an important checkpoint molecule in maintaining immunological tolerance and in preventing the onset and development of inflammation. In Aim 1, we propose to determine the molecular mechanisms responsible for regulation and function of RNF133 in T cell tolerance by utilizing conditional gene knockdown approaches and in vivo T cell tolerance models. In Aim 2, we will determine the role of RNF133 in Tfh cell tolerance and underlying mechanisms as well. In addition, we will assess the mechanisms whereby RNF133 controls antibody-mediated autoimmunity. In Aim 3, we will determine the mechanism(s) by which RNF133 regulates Tregs and TH17 cell programming and stability. The physiological significance of this finding will be assessed in an experimental allergic encephalomyelitis (EAE) model. We will employ yeast two-hybrid screening and reverse phase protein array assay to identify the exact target(s) of RNF133, which determines its function in TH programming. The proposed research will provide new significant insight into characterization of mechanisms underlying T cell tolerance that will lead to development of pharmacological approaches to promote the tolerance state in terms of autoimmunity.