How beta-catenin expands Foxp3+RORgammat+ Pro-inflammatory T-regulatory cells

摘要 调节性 T 细胞 (Treg) 的可塑性和功能多样性对于维持健康至关重要 免疫力，但它也被用来治疗疾病。我们已经描述了表达高水平 β- 的 Tregs 子集。 连环蛋白并促进炎症。这些 Tregs 的频率在炎症性肠病中增加 （和小鼠结肠炎）、结肠癌（和小鼠息肉病）以及多发性硬化症（和小鼠 EAE）。 Treg 中显性活性 β-catenin 的表达或 Tcf-1 的消除都会促进 Rorγt 的表达 Th17 谱系的经典转录因子，并促进 Th17 分化特征。在 在健康小鼠的肠系膜淋巴结（MLN）中，我们鉴定出至少 5 个转录上不同的激活 Tregs 簇。其中一个簇由 Rorγt 的表达定义，并具有突出的 Th17 差异化特征。在 Tcf-1 缺陷的 Tregs 中，Th17 分化谱扩展到所有其他 簇，并且 RORγt 簇的频率增加。尽管 Th17 较多，但 Tcf-1 缺乏 Tregs 就像抑制 T 细胞一样，但它们未能抑制炎症。这表明 Treg 的分叉 抑制功能并引出了一个问题，即它是否涉及 TCF-1 和 Foxp3 之间的合作。 我们发现，TCF-1 和 Foxp3 共同结合参与 T 细胞激活的基因的增强子元件， Th17分化。 β-连环蛋白在这些增强子处诱导新的可接近染色质区域，并且 上调相关基因的表达。此外，E-box结合蛋白HEB协同作用 TCF-1 和可能还有 Foxp3 来调节这些基因，因为 Treg 特异性消除了 TCF-1 和 Foxp3 HEB（但不是单独的每一种）可挽救与 β-连环蛋白相关的炎症和自身免疫病理 Tregs 中的激活。 HEB 始终调节 RORγt，抑制 Foxp3 和外周 Treg 发育。我们的 研究结果与 FoxP3 在上下文中直接激活或抑制转录的观点一致 依赖伙伴的方式。他们进一步暗示 β-连环蛋白、TCF-1 和 HEB 与 Foxp3 合作， 独立调节不同的 Treg 抑制活动。根据这些发现，我们假设， Wnt/β-catenin 信号传导通过控制 Treg 抑制功能和多样性来差异调节 访问 Foxp3 和 HEB 以选择 Tcf-1 结合的染色质位点。为了具体解决这个假设 目标 1 我们将确定 β-catenin 和 Tcf-1 在准备 Foxp3 结合的表观遗传景观中的作用 以及塑造 Treg 细胞类型多样性。在具体目标 2 中，我们将确定分泌型 Wnt 配体在 定义结肠浸润 Tregs 的功能特性。拟议的研究将阐明基本共同点 调节健康中 Treg 特性的 TF 之间的操作/拮抗作用，以及微环境如何 像 Wnts 这样的队列在疾病环境中利用它们。预期的发现有可能为新颖的研究提供信息 自身免疫和癌症的诊断和治疗工具。

ABSTRACT The plasticity and functional diversity of regulatory T cells (Tregs) is essential for the maintenance of healthy immunity but it is also exploited in disease. We have described a subset of Tregs that express high levels of β- catenin and promote inflammation. The frequency of these Tregs is increased in inflammatory bowel diseases (and mouse colitis), colon cancer (and mouse polyposis), as well as in multiple sclerosis (and mouse EAE). Expression of a dominant active β-catenin, or ablation of Tcf-1 in Tregs both foster expression of Rorγt the canonical transcription factor of the Th17 lineage, and promote a Th17 differentiation signature. In the mesenteric lymph nodes (MLN) of healthy mice, we identified at least 5 transcriptionally distinct activated clusters of Tregs. One of these clusters is defined by expression of Rorγt and has prominent Th17 differentiation characteristics. In Tcf-1 deficient Tregs the Th17 differentiation profile expanded to all other clusters, and the RORγt cluster increased in frequency. The Tcf-1 deficient Tregs in spite of being more Th17 like, suppressed T-cells, however they failed to suppress inflammation. This indicates a bifurcation of Treg suppressive functions and begs the question, of whether it involves a co-operation between TCF-1 and Foxp3. We have found that, TCF-1 and Foxp3 co-bind enhancer elements of genes involved in T cell activation and Th17 differentiation. β-catenin induces newly accessible chromatin regions at these enhancers and upregulates the expression of the associated genes. Moreover, the E-box binding protein HEB works together with TCF-1 and potentially also Foxp3 to regulate these genes, since Treg specific ablation of both TCF-1 and HEB (but not each one alone) rescues inflammatory and autoimmune pathologies associated with β-catenin activation in Tregs. Consistently, HEB regulates RORγt, represses Foxp3 and peripheral Treg development. Our findings are in line with the notion that FoxP3 directly activates or represses transcription, in a context- and partner-dependent manner. They further implicate β-catenin, TCF-1, and HEB in partnering with Foxp3 to independently regulate the diverse Treg suppressive activities. Based on these findings we hypothesize that, Wnt/β-catenin signaling differentially regulates Treg suppressive functions and diversity, by controlling access of Foxp3 and HEB to select chromatin sites bound by Tcf-1. To address this hypothesis in specific Aim 1 we will determine the role of β-catenin and Tcf-1 in preparing the epigenetic landscape for Foxp3 binding and in shaping Treg cell type diversity. In specific Aim 2 we will determine the role secreted Wnt ligands in defining the functional properties of colon infiltrating Tregs. The proposed studies will elucidate fundamental co- operations/antagonisms between TFs that regulate Treg properties in health, and how microenvironment queues like Wnts exploit them in disease settings. The expected findings have the potential to inform novel diagnostic and therapeutic tools for autoimmunity and cancer.