title: Allergen specificity of IgG(4)-expressing B cells in patients with grass pollen allergy undergoing immunotherapy.
abstract: BACKGROUND
  Serum IgG(4) responses to allergen immunotherapy are well documented as blocking allergen binding to receptor-bound IgE on antigen-presenting cells and effector cells, but the molecular characteristics of treatment-induced IgG(4), particularly in relation to expressed antibody, are poorly defined.
  
  
  OBJECTIVE
  We aimed to clone and express recombinant IgG(4) from patients receiving grass pollen immunotherapy using single B cells to obtain matched heavy- and light-chain pairs.
  
  
  METHODS
  IgG(4)(+) B cells were enriched from blood samples taken from 5 patients receiving grass pollen immunotherapy. Matched heavy- and light-chain variable-region sequences were amplified from single IgG(4)(+) B cells. Variable regions were cloned and expressed as recombinant IgG(4). Binding analysis of grass pollen-specific IgG(4) was performed by using surface plasmon resonance. Functional assays were used to determine IgE blocking activity. In a separate experiment grass pollen-specific antibodies were depleted from serum samples to determine the proportion of grass pollen-specific IgG(4) within total IgG(4).
  
  
  RESULTS
  Depletion of grass pollen-specific antibodies from serum led to a modest reduction in total IgG(4) levels. Matched heavy- and light-chain sequences were cloned from single IgG(4)(+) B cells and expressed as recombinant IgG(4). We identified an IgG(4) that binds with extremely high affinity to the grass pollen allergen Phl p 7. Furthermore, we found that a single specific mAb can block IgE-mediated facilitated allergen presentation, as well as IgE-mediated basophil activation.
  
  
  CONCLUSIONS
  Although increases in IgG(4) levels cannot be wholly accounted for within the allergen-specific fraction, allergen immunotherapy might result in the production of high-affinity allergen-specific blocking IgG(4).
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom. louisa.james@kcl.ac.uk
authors.forename: Louisa K
authors.initials: LK
authors.lastname: James
authors.forename: Holly
authors.initials: H
authors.lastname: Bowen
authors.forename: Rosaleen A
authors.initials: RA
authors.lastname: Calvert
authors.forename: Tihomir S
authors.initials: TS
authors.lastname: Dodev
authors.forename: Mohamed H
authors.initials: MH
authors.lastname: Shamji
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Beavil
authors.forename: James M
authors.initials: JM
authors.lastname: McDonnell
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Durham
authors.forename: Hannah J
authors.initials: HJ
authors.lastname: Gould
mesh_terms: D000328:Adult; D000485:Allergens / Q000276:immunology*; D000595:Amino Acid Sequence; D000915:Antibody Affinity; D000918:Antibody Specificity; D052179:Antigens, Plant; D001402:B-Lymphocytes / Q000276:immunology*; D002135:Calcium-Binding Proteins / Q000276:immunology*; D003888:Desensitization, Immunologic; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood*; D008875:Middle Aged; D008969:Molecular Sequence Data; D006109:Poaceae / Q000276:immunology*; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology / Q000628:therapy*
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D002135:Calcium-Binding Proteins; D007074:Immunoglobulin G; C468525:Phl p 7 allergen