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

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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.