PROJECT ABSTRACT Defining how allergen-specific, memory Th2 cells develop has the potential to change our therapeutic approach to allergic asthma, the most common asthma subtype. Th2 cells are a dominant source of type 2 cytokines IL-4, IL-5, and IL-13 that orchestrate allergic inflammation in murine models and humans with allergic asthma. In addition, allergen-specific, memory Th2 cells persist in vivo, driving recurrent allergic inflammation upon allergen re-exposure. We recently showed that tissue-resident memory Th2 cells (Th2 Trm) that durably persist in the lungs are a transcriptionally distinct memory Th2 cell subset that is critical for orchestrating recurrent allergic airway inflammation (Rahimi et al., Journal of Experimental Medicine, 2020). The objective of this proposal is to define novel mechanisms regulating the development of Th2 Trm in a murine model of allergic asthma. In the lungs, effector T cells require interactions with antigen presenting cells (APC) and cognate antigen for Trm development. In counterbalance, mechanisms limiting terminal T cell differentiation and death in peripheral tissues, such as T cell inhibitory receptors and Tregs, are required for Trm development. During allergic inflammation, the specific APC presenting antigen to effector Th2 cells and the mechanisms regulating Th2 Trm development have not been delineated. To define the APC interacting with effector Th2 cells within the lungs, we took advantage of the fact that the Th2 cell-derived cytokines IL-4 and IL-13 induce APC to express the chemokines CCL17 and CCL22, which attract Th2 cells and regulatory T cells (Tregs) via the chemokine receptor CCR4. Using a novel Ccl17/Ccl22 reporter mouse, we show that during allergic inflammation, a subpopulation of type 2 conventional dendritic cells (cDC2) specifically upregulate Ccl17 and Ccl22 expression. Furthermore, we demonstrate that the DC activation marker PD-L2 defines Ccl17/Ccl22-expressing cDC2. We hypothesize that PD-L2+ cDC2 provide unique signals, including CCL17 and CCL22, that regulate Th2 cell activation and Th2 Trm development. We further hypothesize CCR4 expression in both Th2 cells and Tregs regulates the development of Th2 Trm. In this application, we propose to develop 2 novel murine models to define cDC2-Th2 cell interactions within the lungs in vivo. Specifically, we propose to develop PD-L2+ cDC reporter/deleter mice (Aim 1) and Ccr4 flox mice (Aim 2). We delineate a research plan to address fundamental biology concerning cDC2-Th2 cell crosstalk regulating allergic immunity and the development of Th2 Trm in vivo in a model of allergic asthma. The goal of this R03 award is to assist my lab in building new experimental tools to extend work performed during my K08 award and position me to become an independent, R01-funded investigator with expertise in the mechanisms driving Th2 immunity in allergic asthma.