title: Single B-cell deconvolution of peanut-specific antibody responses in allergic patients.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pathology, Stanford University, Stanford, Calif.
authors.forename: Ramona A
authors.initials: RA
authors.lastname: Hoh
authors.affiliation: Department of Pathology, Stanford University, Stanford, Calif.
authors.forename: Shilpa A
authors.initials: SA
authors.lastname: Joshi
authors.affiliation: Department of Pathology, Stanford University, Stanford, Calif; Biomedical Informatics Training Program, Stanford University, Stanford, Calif.
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Pathology, Stanford University, Stanford, Calif.
authors.forename: Chen
authors.initials: C
authors.lastname: Wang
authors.affiliation: Department of Pathology, Stanford University, Stanford, Calif.
authors.forename: Krishna M
authors.initials: KM
authors.lastname: Roskin
authors.affiliation: Department of Pathology, Stanford University, Stanford, Calif.
authors.forename: Ji-Yeun
authors.initials: JY
authors.lastname: Lee
authors.affiliation: Department of Pathology, Stanford University, Stanford, Calif.
authors.forename: Tho
authors.initials: T
authors.lastname: Pham
authors.affiliation: Department of Pathology, Stanford University, Stanford, Calif.
authors.forename: Tim J
authors.initials: TJ
authors.lastname: Looney
authors.affiliation: Department of Pathology, Stanford University, Stanford, Calif.
authors.forename: Katherine J L
authors.initials: KJL
authors.lastname: Jackson
authors.affiliation: Department of Pathology, Stanford University, Stanford, Calif.
authors.forename: Vaishali P
authors.initials: VP
authors.lastname: Dixit
authors.affiliation: Department of Pathology, Stanford University, Stanford, Calif.
authors.forename: Jasmine
authors.initials: J
authors.lastname: King
authors.affiliation: Department of Pediatrics, Stanford University, Stanford, Calif.
authors.forename: Shu-Chen
authors.initials: SC
authors.lastname: Lyu
authors.affiliation: Department of Pediatrics, Stanford University, Stanford, Calif.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Jenks
authors.affiliation: Division of Allergy and Clinical Immunology, School of Medicine, Johns Hopkins University, Baltimore, Md.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Hamilton
authors.affiliation: Department of Pediatrics, Stanford University, Stanford, Calif.
authors.forename: Kari C
authors.initials: KC
authors.lastname: Nadeau
authors.affiliation: Department of Pathology, Stanford University, Stanford, Calif. Electronic address: sboyd1@stanford.edu.
authors.forename: Scott D
authors.initials: SD
authors.lastname: Boyd
mesh_terms: D055516:2S Albumins, Plant / Q000276:immunology*; D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000276:immunology*; D052179:Antigens, Plant / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D002648:Child; D002675:Child, Preschool; D003888:Desensitization, Immunologic; D005260:Female; D006023:Glycoproteins / Q000276:immunology*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007073:Immunoglobulin E / Q000235:genetics; D007074:Immunoglobulin G / Q000235:genetics; D008297:Male; D008565:Membrane Proteins; D009154:Mutation; D021183:Peanut Hypersensitivity / Q000276:immunology* / Q000628:therapy; D010940:Plant Proteins / Q000276:immunology*; D055815:Young Adult
chemical_list: D055516:2S Albumins, Plant; D000485:Allergens; D052179:Antigens, Plant; C500889:Ara h 1 protein, Arachis hypogaea; C500890:Ara h 2 allergen, Arachis hypogaea; D006023:Glycoproteins; D007074:Immunoglobulin G; D008565:Membrane Proteins; D010940:Plant Proteins; D007073:Immunoglobulin E
keywords: B cell; IgE; IgG(4); Peanut; allergen specific; allergy; antibody; antigen specific; high-throughput DNA sequencing; immunotherapy; repertoire; somatic mutation