PROJECT SUMMARY Patients with severe uncontrolled asthma represent a distinct endotype with persistent airway inflammation and remodeling refractory to corticosteroid treatment. In this Project 3 (Vijayanand, PI), we propose to investigate how airway remodeling events in severe asthma are driven by this poorly characterized CD4+ T cell subset – cytotoxic/pro-inflammatory CD4+ TRM cells. In the context of lung cancer and viral infections, our laboratory has recently shown the importance of both TRM cells and cytotoxic CD4+T cells in immune responses. In collaboration with Drs. Broide and Croft, Project 1 and 2, we are fully geared to explore the role of this novel cytotoxic TRM subset in driving airway remodeling. Our long-standing collaboration with the University of Southampton, UK, will enable continued access to airway specimens from patients with severe asthma enrolled in the WATCH study (Southampton, UK) that currently consists of >500 subjects (Asthma Clinical Core B). In Aim 1, we will define the functional role of cytotoxic CD4+ TRM cells in driving airway remodeling using in vitro cell culture models of asthmatic airway structural cells. We will first determine the molecules expressed and released by cytotoxic/pro-inflammatory CD4+ TRM cells; then, we will test if products released by cytotoxic CD4+ TRM cells induce remodeling features in airway structural cells. We will individually test the activity of molecules released by cytotoxic TRM cells, such as TNF, LIGHT, to narrow down targets that are significant drivers of remodeling events. We will also determine if cytotoxic TRM cells can cause pyroptosis and/or necroptosis, i.e., inflammatory cell death of airway structural cells, which can, in turn, promote remodeling. In Aim 2, we will perform unbiased enhancer profiling studies in cytotoxic CD4+ TRM cells to define transcription factors that play an important role in the differentiation and function of these cells. Then, we will knock down candidate transcription factors identified in T cells and determine their effects on the differentiation and function of cytotoxic TRM cells in vitro. Overall, studies in this program will improve our understanding of how cytotoxic CD4+ TRM cells are generated and how they interact with airway structural cells to drive remodeling events in severe asthma.