Food allergic reactions are an increasing cause of morbidity and mortality worldwide, and are triggered byallergen-specific IgE antibodies bound to mast cell and basophil effector cells. The molecular features thatmake some human IgE antibodies pathogenic have been difficult to study from polyclonal sera that containmixtures of unknown numbers and proportions of antibody types. Similarly, the mechanisms by which IgG4 andIgG1 antibodies elicited by oral immunotherapy (OIT) can protect patients from allergic reactions would beclarified by studying defined antibodies of known sequence from patients following treatment. We will study the functional activity of defined peanut allergen-specific human IgE, IgG4 and IgG1 monoclonalantibodies (mAbs) isolated from symptomatic allergic patients compared to sensitized but non-allergic controls,and from patients post-treatment who vary in their response to OIT. Cultured primary human mast cells will beused as effector cells, and will be studied with three assays extending from early cell activation (Calcium influx),through effector release, to later cytokine secretion. IgE clones will be evaluated for their allergen specificity,binding affinity, and epitope recognition, and tested in combinations to determine which species can sensitizemast cells. Blocking of mast cell activation by mAb IgG4 and IgG1 will be tested, and Fc mutations used todetermine whether Fc? receptors contribute to decreases in mast cell effector functions. Finally, IgE and otherantibody isotypes isolated from patient gastrointestinal tract biopsies will be compared to antibodies isolatedfrom the blood, to determine whether there is enrichment for potentially pathogenic IgE in the mucosal siteswhere allergic reactions occur. These studies are likely to have a significant impact on basic and translational human allergy research, asthey will use a fully human, but well-defined experimental system to identify the molecular features ofpathogenic IgE clones in the blood and GI tracts of allergic patients, and evaluate potential IgG4 and IgG1-mediated mechanisms of protection following OIT. Improved understanding of IgE clone affinities, epitopereactivities, and combinations that result in mast cell sensitization could enable better diagnostic andprognostic testing in allergic patients, while more knowledge about the criteria leading to protective IgG4 andIgG1 could guide therapeutic trials and the development of improved immunotherapy regimens.