Only a subset of patients with IgE-mediated food allergy develop clinical tolerance after oral immunotherapy (OIT), which has been attributed to the development of protective, functionally suppressive antibodies. However, those who lose tolerance are often seen to have an early transient increase in allergen- specific IgE. Previously, we have described the induction of allergen-specific memory B cells early in OIT, and these specific IgG B cells could serve as a reservoir of IgE memory via sequential class-switching. Therefore, the relative contribution of specific IgG B cells giving rise to protective IgG or IgA antibodies versus pathogenic IgE is critical to the development of immunological tolerance in OIT. Understanding the contributions of that B cell fate decision could lead to the development of new therapeutic modalities for allergic diseases. In OIT, we recently published that suppression of basophil sensitivity is a biomarker of tolerance. Functional suppression of basophils and not bulk levels of allergen-specific IgG is correlated with tolerance, suggesting that the antibody repertoire induced by OIT may be highly relevant to the development of tolerance. We have developed a highly specific fluorescent multimer to identify and characterize allergen-specific B cells in peanut OIT in order to define the clonal contributions of these B cells. Using these tools, we propose to track clonal evolution from the memory IgG B cells induced by OIT to the post-serum effector antibodies to explain how antibody evolution from specific IgG B cells influences clinical outcomes in OIT. Based on these findings, we hypothesize that allergen-specific memory IgG B cells can give rise to either protective or pathogenic antibodies that determine long-lived tolerance after OIT. Our approach combines the isolation of antigen-specific B cells for single-cell BCR sequencing, BCR repertoire analysis, and serum proteomics to dissect the clonality of serum antibodies to the peanut allergen, Ara h 2, over the course of OIT. We are uniquely positioned to conduct this study in that we have both serum and BCR repertoires from previously characterized OIT-treated patients. We will address our hypothesis in the following specific aims: (1) Evaluate the development of protective, functionally suppressive allergen-specific IgG in sustained tolerance after OIT; and (2) Evaluate the role of sequential switching in the loss of tolerance after OIT. We anticipate that the proposed studies will elucidate the connection between long-lasting clinical efficacy of OIT on a clonal level with protective and pathogenic antibodies, highlighting the critical role of memory allergen-specific IgG B cells and leading to new strategies for the treatment of food allergies. The development of techniques to track antigen-specific B cell fate, using BCR sequencing, proteomics and recombinant antibodies, will provide a powerful platform to further refine our understanding of how humoral immun...