Asthma is the most common chronic lung disease. In most asthmatics, allergen-specific type-2 CD4 T cells orchestrate allergic airway inflammation by producing type-2 cytokines that ultimately drive asthma pathology. Studies suggest that a subset of long-lived memory Th2 cells that reside in the lung, known as tissue-resident memory Th2 cells (T2RM), play a significant role in driving asthma chronicity and persistence. Therapies aimed at depleting T2RM from the lung represent a novel approach to treating asthma, however factors that regulate the generation and persistence of lung-T2RMs remain poorly understood. I hypothesize that distinct and druggable molecular pathways control lung-T2RM survival and re-activation. This work aims to decipher molecular and cellular mechanisms that regulate the maintenance and re-activation of T2RM within the asthmatic lung niche. The host lab has recently developed a novel TCR transgenic (Tg) mouse line (1DER-CD5), containing a monoclonal population of house dust mite (HDM) specific CD4 T cells with high T2RM differentiation potential, which has been confirmed in preliminary experiments performed since joining the host lab. In WP1, I will perform single cell transcriptomic and proteomic analysis on 1DER-CD5 T cells in the lungs of mice in the memory phases of HDM-induced asthma to predict molecular and cellular networks that regulate lung-T2RM. In WP2, I will utilize targeted spatial transcriptomics, paired with confocal microscopy, to define the lung-T2RM niche and validate molecular and cellular interactions predicted in WP1. In WP3, I will use an in vivo CRISPR-screening approach with Cas9+ 1DER-CD5 T cells to pinpoint key druggable pathways that regulate lung-T2RM survival and re-activation. This work is expected to identify molecular pathways critical in regulating the persistence and re-activation of lung-T2RM, potentially leading to the development of novel therapies to deplete pathogenic T2RM from the airways of asthmatics.