PROJECT SUMMARY Food allergy (FA) affects 8% of children and 5% of adults in the U.S., and 30% of those have clinical reactivity to multiple foods. In 2 independent phase 1 clinical trials, we showed that simultaneous oral desensitization (D) to multiple food allergens (multi-OIT) is safe and feasible, and can be achieved in 6-9 months with anti-IgE adjunctive therapy. Polarization of naïve T cells into IL-4-secreting Th2 cells is the first step leading to allergic responses. Thus, understanding how modulation of T cell responses can lead to D or sustained unresponsiveness (SU) during successful OIT is critical. We propose to monitor T cells using innovative technologies in: (1) each of the cohorts proposed in Project 1 (i.e., multi-FA participants (n=60) treated with multi-OIT +/- omalizumab or dupilumab who develop D [defined as a positive food challenge reaction after a 6 week withdrawal of OIT] vs. SU [defined as a negative food challenge reaction after withdrawal of OIT] to the respective allergens in their multi-OIT);; (2) long term follow up studies of >240 participants on OIT;; and (3) GI biopsies obtained over time in OIT participants). In Project 3, we will investigate whether changes in participants’ T cell subpopulations can identify markers predictive of clinical outcomes. We particularly will focus on changes in allergen-specific Th2 cells in those who exhibit favorable responses to OIT. By characterizing and quantifying the modulation of T cell phenotype and function associated with various multi-OIT outcomes, we will identify T cell signatures of SU in multi-FA participants. Our main hypotheses are that successful multi-OIT will: (1) reprogram total and allergen-specific Th2 cells to Th1 and/or Treg subtype, (2) replace allergen-specific Th2 cells by Th1 and Treg subtype, and/or (3) expand allergen-specific clones with diverse phenotype and function, potentially overriding the effects of Th2 cells. We speculate that stable epigenetic changes in IL4, IL10, IFNγ and/or FOXP3 genes mediate the anticipated shift from Th2 phenotype, contributing to SU. To test these hypotheses, we propose to: (Aim 1) Characterize the immunophenotypic and functional changes induced by multi-OIT in total and allergen- specific T cells;; (Aim 2) Use MHC class II multimers to sort allergen- (peanut/milk/cashew) specific single cells and perform targeted RNA-seq to investigate their molecular signatures and clonal ancestry at single cell resolution;; and (Aim 3) Quantify epigenetic changes (i.e., methylation of CpG islands) in key genes (i.e., FOXP3, IL4, IFNγ, IL10) to assess possible links between gene methylation, and thus expression of these genes, and favorable OIT clinical outcomes. If we achieve these aims, we expect our results will both provide new insights into the mechanisms underlying successful clinical outcomes in multi-OIT and improve understanding of the immune changes that can contribute to...