Defining a stromal niche for type 2-like lung regulatory T cells

项目摘要 肺部疾病如急性肺损伤（ALI）和急性呼吸窘迫综合征（ARDS）是导致 全球发病率和死亡率的原因。调节性T细胞（TCFs）传统上被认为是至关重要的 全身免疫反应的负调节剂;然而，它们在组织如肺中的局部作用是 越来越受到重视，它们可以促进ALI和ARDS中的肺上皮再生。 发现组织驻留型Tbet（tTbet）的亚群表达转录因子T-bet、Gata 3、ROR β t和 Bcl 6，并分别增强了对Th 1，Th 2，Th 3/17和Tfh免疫“风味”的抑制。 尽管tTreg的功能已开始被了解，但肺tTreg是如何被调节、定位和调节的， 保持在它们各自的组织小生境中的细胞数目仍然未知。间充质基质细胞（MSC）是 免疫调节剂，并已强调在tTreg生物学中发挥作用。通过一个亚组的IL-33的产生 骨髓间充质干细胞促进内脏脂肪组织的扩张和维持，人骨髓间充质干细胞可诱导 来自体外常规CD 4 + T细胞的T细胞。我们的小组在肺内发现了一个基质细胞龛， 外膜成纤维细胞（AF），MSC亚群，调节2型效应淋巴细胞（例如ILC 2和Th 2细胞）， 部分通过分泌IL-33和胸腺基质淋巴细胞生成素（TSLP）。使用3D厚切片成像， 显示肺2型样tTGFs（即Gata 3 hi ST 2+，KLRG 1+）也定位于该小生境，表明AF可能 调节肺组织及其亚群。当与肺AF共培养时，淋巴调节性T细胞显着增加 在接触依赖性细胞中增殖、存活和2型样Treg标志物ST 2和KLRG 1的表达。 方式此外，AF优先支持ST 2 hi T细胞，如通过更高的增殖、存活性、 ST 2和KLRG 1的表达。使用CellphoneDB V2.0，我鉴定了细胞外基质（ECM）-整合素 配体-受体配对，如ICAM、VCAM和CD 49 d，可能介导AF和 你好在共培养系统中阻断所有三种后，我发现Treg增殖和ST 2 KLRG 1的表达。我假设AF调节肺Treg亚群的维持和分化， 优先支持在损伤后肺修复中起关键作用的2型样肺TCLs。这项建议 将定义2型肺结核的地形图及其在初治和炎症环境中的作用（目标1）， 确定外膜成纤维细胞在调节肺Treg亚群中的作用（目的2）。这项工作利用 小鼠炎症模型，以及基因消融以剖析肺中的局部2型tTreg应答。 将使用高维流式细胞术、先进成像和肺功能测量来量化 影响免疫细胞和肺功能恢复。这些目标的实现将阐明AF在以下方面的作用： 肺2型TTT的功能和调节，提供了新的机制洞察骨髓间充质干细胞的作用， 调节免疫亚群。本研究的完成为精密度的发展提供了基础 本发明涉及选择性调节肺组织Tcl 4亚群以影响多种肺部疾病的结果的治疗剂。

Project Summary Lung diseases such as acute lung injuries (ALI) and acute respiratory distress syndrome (ARDS) are leading causes of morbidity and mortality worldwide. Regulatory T cells (Tregs) are traditionally thought of as critical negative regulators of systemic immune responses; however, their local roles in tissues such as the lung are being increasingly appreciated, where they can promote lung epithelial regeneration in both ALI and ARDS. Subsets of tissue-resident Tregs (tTregs) are found to express transcription factors T-bet, Gata3, RORt, and Bcl6, and have enhanced suppression towards their Th1, Th2, Th3/17, and Tfh immune ‘flavors,’ respectively. Although tTreg function are beginning to be understood, how lung tTregs are regulated, positioned, and maintained within their respective tissue niches remains unknown. Mesenchymal stromal cells (MSCs) are immune regulators and have been highlighted to play a role in tTreg biology. Production of IL-33 by a subset of MSCs promotes the expansion and maintenance of visceral adipose tissue tTregs and human MSCs can induce Tregs from conventional CD4+ T cells in vitro. Our group identified a stromal cell niche within the lung where adventitial fibroblasts (AFs), an MSC subset, regulate type 2 effector lymphocytes (e.g. ILC2s and Th2 cells), in part via the secretion of IL-33 and thymic stromal lymphopoietin (TSLP). Using 3D thick section imaging, I have shown that lung type 2-like tTregs (i.e. Gata3hi ST2+, KLRG1+) also localize to this niche, indicating AFs may regulate lung tTregs and their subsets. When co-cultured with lung AFs, lymphoid Tregs significantly increased proliferation, survival, and expression of type 2-like Treg markers ST2 and KLRG1 in a contact-dependent manner. Additionally, AFs preferentially support ST2hi Tregs, as evidenced by higher proliferation, survivability, and ST2 and KLRG1 expression. Using CellphoneDB V2.0, I identified extracellular matrix (ECM)–integrin ligand–receptor pairings, such as ICAM, VCAM, and CD49d, that may mediate interactions between AFs and Tregs. Upon blocking all three in a co-culture system, I found a significant decrease in Treg proliferation and ST2 and KLRG1 expression. I hypothesize that AFs regulate the maintenance and differentiation of lung Treg subsets, preferentially supporting type 2-like lung Tregs, which play critical roles in post-injury lung repair. This proposal will define the topography of type 2 lung tTregs and their role in naïve and inflammatory settings (Aim 1) and determine the role of adventitial fibroblasts in the regulation of lung Treg subsets (Aim 2). This work utilizes murine models of inflammation, as well as genetic ablations to dissect the local type 2 tTreg response in the lung. High-dimensional flow cytometry, advanced imaging, and lung function measurements will be used to quantify impacts on immune cells and functional lung recovery. Completion of these aims will elucidate the role of AFs in the function and regulation of lung type 2 tTregs, providing novel mechanistic insight into the role MSCs play in regulating immune subsets. Completion of this study provides a foundation for the development of precision therapeutics to selectively regulate lung tissue Tregs subsets to impact the outcome of diverse lung diseases.