Comprehensive elucidation of signaling landscape effects on regulatory T cell phenotype and function

Project Summary Regulatory T cells (Tregs) play an essential role in maintaining immune homeostasis by preventing autoimmunity, modulating inflammation, supporting tissue repair, and ensuring balanced immune responses. These functions are orchestrated by cell-extrinsic signals that drive transcriptional programs necessary for Treg functional outputs. Recent studies have characterized the effects of various cell-extrinsic signals on Tregs one at a time. However, due to the diversity, scale, and context-dependent effects of cell-extrinsic signals, comprehensive understanding of how complex in vivo environmental signals shape Treg functions is lacking. A systematic understanding of how Tregs interpret cell-extrinsic signals will yield novel insights into basic mechanisms of immunoregulation, enable precise manipulation of Treg functions in pathological conditions, and inform the design of novel therapeutic strategies in settings of autoimmune and inflammatory diseases and cancer. This project aims to comprehensively characterize how cell-extrinsic signals shape Treg functions across distinct inflammatory environments. In Specific Aim 1, Perturb-seq will be used to, in one pooled experiment, profile transcriptional changes induced by genetic knockout of hundreds of Treg cell surface receptors in the context of systemic polytypic (type 1, 2, 3) autoimmunity and tissue inflammation (Aim 1a). Computational analyses of these data will identify shared and unique transcriptional programs elicited by diverse cell-extrinsic signals and categorize their functional relevance (Aim 1b). The effects of relevant newly identified signaling pathways on Tregs will be validated in follow-up experiments (Aim 1c). In Specific Aim 2, the context-dependent effects of Treg signaling pathways in archetypal inflammatory environments (type 1, 2, and 3) will be explored using mouse models of airway infections (influenza, Nippostrongylus brasiliensis, Aspergillus fumigatus). We will identify inflammatory environment driven variation in ligand and receptor expression for novel signaling pathways identified in Aim 1 (Aim 2a) and generate mice with Treg-specific receptor deficiencies to interrogate context- dependent effects of signaling pathways on Tregs in these infection models (Aim 2b). Context-dependent effects on Treg functional outputs will be assessed through analyses of immune responses to infection and tissue damage in these models (Aim 2c). Overall, this project will leverage innovative high-throughput approaches and diverse mouse models of infection to comprehensively elucidate how cell-extrinsic signaling environments influence Treg phenotype and function in context-dependent manners. The depth of understanding provided by these studies will facilitate development of clinical approaches for precise modulation of Tregs with significant therapeutic implications. Furthermore, this proposal is tailored to a physician-scientist in training as it focuses on a cell type with demonstrated clinical relevance and profiles cell-extrinsic signal effects that can be readily modulated therapeutically.