Single B-cell deconvolution of peanut-specific antibody responses in allergic patients

Background The frequencies, cellular phenotypes, epitope specificity, and clonal diversity of allergen-specific B cells in patients with food allergy are not fully understood but are of major pathogenic and therapeutic significance. Objective We sought to characterize peanut allergen-specific B-cell populations and the sequences and binding activities of their antibodies before and during immunotherapy. Methods B cells binding fluorescently labeled Ara h 1 or Ara h 2 were phenotyped and isolated by means of flow cytometric sorting from 18 patients at baseline and 13 patients during therapy. Fifty-seven mAbs derived from allergen-binding single B cells were evaluated by using ELISA, Western blotting, and peptide epitope mapping. Deep sequencing of the B-cell repertoires identified additional members of the allergen-specific B-cell clones. Results Median allergen-binding B-cell frequencies were 0.0097% (Ara h 1) or 0.029% (Ara h 2) of B cells in baseline blood from allergic patients and approximately 3-fold higher during immunotherapy. Five of 57 allergen-specific cells belonged to clones containing IgE-expressing members. Almost all allergen-specific antibodies were mutated, and binding to both conformational and linear allergen epitopes was detected. Increasing somatic mutation of IgG₄ members of a clone was seen in immunotherapy, whereas IgE mutation levels in the clone did not increase. Conclusion Most peanut allergen-binding B cells isolated by means of antigen-specific flow sorting express mutated and isotype-switched antibodies. Immunotherapy increases their frequency in the blood, and even narrowly defined allergen epitopes are recognized by numerous distinct B-cell clones in a patient. The results also suggest that oral immunotherapy can stimulate somatic mutation of allergen-specific IgG₄.