title: Influence of seasonal exposure to grass pollen on local and peripheral blood IgE repertoires in patients with allergic rhinitis.
abstract: BACKGROUND
  Previous studies of immunoglobulin gene sequences in patients with allergic diseases using low-throughput Sanger sequencing have limited the analytic depth for characterization of IgE repertoires.
  
  
  OBJECTIVE
  We used a high-throughput, next-generation sequencing approach to characterize immunoglobulin heavy-chain gene (IGH) repertoires in patients with seasonal allergic rhinitis (AR) with the aim of better understanding the underlying disease mechanisms.
  
  
  METHODS
  IGH sequences in matched peripheral blood and nasal biopsy specimens from nonallergic healthy control subjects (n = 3) and patients with grass pollen-related AR taken in season (n = 3) or out of season (n = 4) were amplified and pyrosequenced on the 454 GS FLX+ System.
  
  
  RESULTS
  A total of 97,610 IGH (including 8,135 IgE) sequences were analyzed. Use of immunoglobulin heavy-chain variable region gene families 1 (IGHV1) and 5 (IGHV5) was higher in IgE clonotypic repertoires compared with other antibody classes independent of atopic status. IgE repertoires measured inside the grass pollen season were more diverse and more mutated (particularly in the biopsy specimens) and had more evidence of antigen-driven selection compared with those taken outside of the pollen season or from healthy control subjects. Clonal relatedness was observed for IgE between the blood and nasal biopsy specimens. Furthermore in patients with AR, but not healthy control subjects, we found clonal relatedness between IgE and IgG classes.
  
  
  CONCLUSIONS
  This is the first report that exploits next-generation sequencing to determine local and peripheral blood IGH repertoires in patients with respiratory allergic disease. We demonstrate that natural pollen exposure was associated with changes in IgE repertoires that were suggestive of ongoing germinal center reactions. Furthermore, these changes were more often apparent in nasal biopsy specimens compared with peripheral blood and in patients with AR compared with healthy control subjects.
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom; Medical Research Council and Asthma UK Centre, Allergic Mechanisms in Asthma, London, United Kingdom. Electronic address: yu-chang.wu@kcl.ac.uk.
authors.forename: Yu-Chang B
authors.initials: YC
authors.lastname: Wu
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom; Medical Research Council and Asthma UK Centre, Allergic Mechanisms in Asthma, London, United Kingdom.
authors.forename: Louisa K
authors.initials: LK
authors.lastname: James
authors.affiliation: Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, New Haven, Conn.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Vander Heiden
authors.affiliation: Department of Pathology, Yale School of Medicine, New Haven, Conn.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Uduman
authors.affiliation: Medical Research Council and Asthma UK Centre, Allergic Mechanisms in Asthma, London, United Kingdom; Allergy and Clinical Immunology, National Heart and Lung Institute, Imperial College London, London, United Kingdom.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Durham
authors.affiliation: Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, New Haven, Conn; Department of Pathology, Yale School of Medicine, New Haven, Conn.
authors.forename: Steven H
authors.initials: SH
authors.lastname: Kleinstein
authors.affiliation: Institute of Cancer & Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Kipling
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom; Medical Research Council and Asthma UK Centre, Allergic Mechanisms in Asthma, London, United Kingdom.
authors.forename: Hannah J
authors.initials: HJ
authors.lastname: Gould
mesh_terms: D000328:Adult; D000485:Allergens / Q000276:immunology; D000915:Antibody Affinity / Q000235:genetics; D000916:Antibody Diversity / Q000235:genetics; D052179:Antigens, Plant / Q000276:immunology; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009154:Mutation / Q000235:genetics; D006109:Poaceae; D011058:Pollen / Q000276:immunology; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology*; D012621:Seasons; D055815:Young Adult
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D007143:Immunoglobulin Heavy Chains; D007073:Immunoglobulin E
keywords: Next-generation sequencing; allergic rhinitis; peripheral blood and nasal mucosal IgE repertoires