Repeated exposure to low doses of an allergen prevents adverse reactions upon subsequent exposure to the same allergen. Allergen immunotherapy (AIT) has been effectively practiced to treat a broad spectrum of allergic diseases although examples of AIT in asthma are rare because of safety concerns. There is hence an unmet need for useful animal models for airway tolerance to common aeroallergens for future translation. Many studies suggest a critical time window in early life for achieving tolerance (Tol) to prevent development of allergic disease. We have established a new model of allergen-tolerance (Tol) initiated by repeated exposure of newborn mice to a low dose of the common household allergen, house dust mite (HDM). Using older mice, we previously demonstrated PPARg-mediated production of H2O2, a reactive oxygen species (ROS), from the mitochondria of lung CD11c+ cells in the context of inhaled Tol to the model allergen ovalbumin. Since ROS can be generated from mitochondria as well as from non-mitochondrial sources, using newborn MCAT transgenic mice that overexpress the enzyme catalase specifically in mitochondria to selectively deplete mitochondrial H2O2 (mt H2O2), we have recently published that Tol to HDM is abolished in MCAT mice. Functionally, we observed an impact of Tol in constraining numbers of conventional dendritic cells (cDCs) in the lung and correspondingly scRNA-seq data show a significant decrease in expression of the gene Marcksl1 implicated in cellular apoptosis and cell migration in Tol MCAT mice. Analysis of recently published human scRNA-seq data of endobronchial biopsies from asthmatic individuals subjected to segmental allergen challenge has revealed suppression of Marcksl1 expression relative to baseline in human lung cDC1s upon allergen challenge. At the other end of the spectrum, in studies of asthma, we and others have identified tissue resident memory (TRM) cells in the lungs of humans and mice. In our ongoing work we have found that the TRMs can be reactivated after a prolonged period of rest highlighting the importance of suppressing allergen sensitization in early life to minimize emergence of TRMs. These observations lead us to hypothesize that persistent exposure to low doses of allergens such as HDM programs lung cDCs to express Marcksl1 requiring mt H2O2 that is important for Tol establishment. Early life allergen Tol suppresses the development of TRMs to provide long-term protection from disease. We propose the following 3 aims, to address these hypotheses: Aim 1. To dissect the impact of cDC-specific deletion of Marcksl1 on loss of HDM-induced Tol. Aim 2. To determine ROS production in the presence or absence of Marcksl1 and the role of fatty acid oxidation in cDC1s in Tol induction. Aim 3. To analyze scRNA-seq data from mice and humans to understand DC subset-specific programming during Tol vs inflammation.