Protein O-GlcNAcylation in Regulatory T Cell Function

项目总结 调节性T(Treg)细胞以FOXP3的表达为特征，是一种独特的T淋巴细胞谱系 控制免疫自身耐受性和组织动态平衡的物质。这些细胞与许多 人类疾病，如自身免疫性疾病、炎症、器官移植排斥反应和一些感染 和癌症。生物医学界对靶向Treg细胞治疗已经产生了浓厚的兴趣 免疫治疗；然而，显著的障碍，例如Treg细胞功能的异质性和不稳定性， 需要克服，才能实现临床可行性。这项拟议的研究旨在理解一部小说 控制Treg细胞稳定性和功能的分子机制。我们的初步数据发现， 由T细胞受体(TCR)激活的新型O-连接N-乙酰氨基葡萄糖(O-GlcNAc)蛋白修饰 信号转导，对于Treg细胞的抑制功能是不可或缺的。O-GlcNAc的Treg细胞特异性敲除 转移酶(OGT)是一种催化O-GlcN酰化的酶，它不影响Treg细胞的谱系特征，但 导致小鼠发生侵袭性自身免疫综合征。O-GlcN酰化修饰和 稳定FOXP3蛋白和转录分析显示IL-2/STAT5信号转导受到抑制 OGT缺陷的Treg细胞。因此，我们假设TCR启动的蛋白质O-GlcN酰化调节 Foxp3和STAT5，从而连接TCR和IL-2信号通路，调节Treg细胞的动态平衡和 效应器功能。在目标1中，我们假设O-GlcN酰化缺失会破坏FOXP3蛋白的稳定性，从而消除 Treg细胞的抑制功能，并将Treg细胞转化为效应性T细胞。我们将确定是否 O-GlcN酰化反应抵消泛素化以稳定FOXP3蛋白，从而保持Treg的特性。通过 产生FOXP3突变小鼠，在其中O-GlcN酰化被特异性取消，我们将测试FOXP3 O-GlcN酰化是Treg细胞抑制活性所必需的。在目标2中，我们建议检查这种影响 STAT5 O-GlcN酰化对Treg细胞抑制功能的影响我们将检验O-GlcN酰化的假设 对照IL-2刺激的Treg细胞中STAT5的激活和FOXP3和STAT5的协同作用 蛋白O-GlcN酰化对Treg细胞功能的影响在目标3中，我们将确定TCR之间的分子联系 和Treg细胞的O-GlcN酰化。我们将检验TCR信号促进蛋白质O- GlcN酰化和Treg细胞抑制活性以钙/CaMKII依赖的方式。最后，通过不偏不倚 O-GlcNAc蛋白质组学，我们将表征TCR调节的蛋白质O-GlcN酰化图谱并确定潜在的 O-GlcNAc介导的Treg细胞功能的互补机制拟议的研究将刺激 开辟了一条治疗的新途径，旨在激活和维持Treg细胞进行免疫治疗。

PROJECT SUMMARY Regulatory T (Treg) cells, characterized by the expression of FOXP3, are a distinct lineage of T lymphocytes that control immunological self-tolerance and tissue homeostasis. These cells are associated with a multitude of human diseases, such as autoimmune disease, inflammation, organ transplant rejection, and some infections and cancers. The biomedical community has developed an intense interest in targeting Treg cells for immunotherapy; however, significant barriers, for example Treg-cell functional heterogeneity and instability, need to be surmounted in order to achieve clinical feasibility. The proposed study aims to understand a novel molecular mechanism that control the stability and function of Treg cells. Our preliminary data have found that a novel O-linked N-Acetylglucosamine (O-GlcNAc) protein modification, activated by T-cell receptor (TCR) signaling, is indispensable for the suppressive function of Treg cells. Treg cell-specific knockout of O-GlcNAc transferase (OGT), the enzyme catalyzing O-GlcNAcylation, does not affect Treg cell lineage specification but results in the development of an aggressive autoimmune syndrome in mice. O-GlcNAcylation modifies and stabilizes FOXP3 protein and transcriptomic analyses have revealed that IL-2/STAT5 signaling is suppressed in OGT-deficient Treg cells. Therefore, we hypothesize that TCR-initiated protein O-GlcNAcylation modulates FOXP3 and STAT5, thereby linking TCR and IL-2 signaling pathways, to regulate Treg cell homeostasis and effector function. In Aim 1, we hypothesize that loss of O-GlcNAcylation destabilizes FOXP3 protein, eliminating the suppressive function of Treg cells and converting Treg cells into effector T cells. We will determine whether O-GlcNAcylation counteracts ubiquitination to stabilize FOXP3 protein, thus maintaining the Treg identity. By generating FOXP3 mutant mice, in which O-GlcNAcylation is specifically abolished, we will test whether FOXP3 O-GlcNAcylation is required for the suppressive activity of Treg cells. In Aim 2, we propose to examine the effect of STAT5 O-GlcNAcylation on Treg cell suppressive function. We will test the hypothesis that O-GlcNAcylation controls IL-2-stimulated STAT5 activation in Treg cells and FOXP3 and STAT5 act cooperatively to mediate the effect of protein O-GlcNAcylation in Treg cell function. In Aim 3, we will identify the molecular link between TCR and O-GlcNAcylation in Treg cells. We will test the hypothesis that TCR signaling promotes protein O- GlcNAcylation and Treg cell suppressive activity in a Ca2+/CaMKII-dependent manner. Lastly, through unbiased O-GlcNAc proteomics, we will characterize TCR-regulated protein O-GlcNAcylation profile and identify potential complementary mechanisms for O-GlcNAc-mediated Treg cell function. The proposed study will stimulate the opening of a new avenue in therapeutics directed to activate and maintain Treg cells for immunotherapy.