Rhinitis, sinusitis, and upper airway disease
Allergen specificity of IgG4-expressing B cells in patients with grass pollen allergy undergoing immunotherapy

https://doi.org/10.1016/j.jaci.2012.04.006Get rights and content

Background

Serum IgG4 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 IgG4, particularly in relation to expressed antibody, are poorly defined.

Objectives

We aimed to clone and express recombinant IgG4 from patients receiving grass pollen immunotherapy using single B cells to obtain matched heavy- and light-chain pairs.

Methods

IgG4+ 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 IgG4+ B cells. Variable regions were cloned and expressed as recombinant IgG4. Binding analysis of grass pollen–specific IgG4 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 IgG4 within total IgG4.

Results

Depletion of grass pollen–specific antibodies from serum led to a modest reduction in total IgG4 levels. Matched heavy- and light-chain sequences were cloned from single IgG4+ B cells and expressed as recombinant IgG4. We identified an IgG4 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.

Conclusion

Although increases in IgG4 levels cannot be wholly accounted for within the allergen-specific fraction, allergen immunotherapy might result in the production of high-affinity allergen-specific blocking IgG4.

Section snippets

Patient data

Five patients (Table I) receiving subcutaneous immunotherapy with an alum-absorbed 6-grass mix (Alutard; ALK-Abelló, Hørsholm, Denmark) were recruited at the Royal Brompton Hospital (London, United Kingdom). The study was approved by the London and City Research Ethics Committee, and blood samples were obtained after obtaining written informed consent. Levels of serum IgE and IgG4 specific for the grass pollen allergens Phl p 1, Phl p 2, Phl p 5, and Phl p 7 were determined by using ELISA (see

Molecular characteristics of IgG4

Immunoglobulin heavy- and light-chain V-region sequences were amplified from 14 individual IgG4+ memory B cells isolated from 5 patients (Table I) receiving grass pollen immunotherapy. Variable (V), diversity (D) and joining (J) IgH gene and V and J IgL gene sequences were compared with known immunoglobulin germline sequences by using IMGT/V-quest (Table II). IgH V gene use in the 14 IgG4 antibodies consisted of VH1 (n = 4), VH3 (n = 4), VH4 (n = 3), and VH5 (n = 3) families. The ratio of κ to

Discussion

We have described the use of single-cell RT-PCR for the molecular analysis and expression of IgG4 antibodies derived from 5 patients treated with grass pollen immunotherapy. Although we did not formally assess the clinical response to treatment, all participants reported an improvement in their hay fever symptoms. IgG4 is the least abundant subclass in adult human serum, ranging from 0.2 to 1 g/L compared with 5 to 12 g/L for IgG1.21 Allergen immunotherapy results in the production of high

References (41)

  • R.E. Snow et al.

    Analysis of immunoglobulin E VH transcripts in a bronchial biopsy of an asthmatic patient confirms bias towards VH5, and indicates local clonal expansion, somatic mutation and isotype switch events

    Immunology

    (1999)
  • R.E. Snow et al.

    Analysis of Ig VH region genes encoding IgE antibodies in splenic B lymphocytes of a patient with asthma

    J Immunol

    (1995)
  • N. van der Stoep et al.

    Molecular evolution of the human immunoglobulin E response: high incidence of shared mutations and clonal relatedness among epsilon VH5 transcripts from three unrelated patients with atopic dermatitis

    J Exp Med

    (1993)
  • V. Visco et al.

    Human IgG monoclonal antibodies that modulate the binding of specific IgE to birch pollen Bet v 1

    J Immunol

    (1996)
  • J. Wrammert et al.

    Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection

    J Exp Med

    (2011)
  • K. Smith et al.

    Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen

    Nat Protoc

    (2009)
  • J.F. Scheid et al.

    Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals

    Nature

    (2009)
  • J. Wrammert et al.

    Rapid cloning of high-affinity human monoclonal antibodies against influenza virus

    Nature

    (2008)
  • X. Brochet et al.

    IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis

    Nucleic Acids Res

    (2008)
  • U. Hershberg et al.

    Improved methods for detecting selection by mutation analysis of Ig V region sequences

    Int Immunol

    (2008)
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    Supported by a grant from Asthma UK (S.R.D. and H.J.G.) and a fellowship from the London Law Trust (L.K.J.). The authors acknowledge financial support from the Department of Health through a National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy's & St Thomas' NHS Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust.

    Disclosure of potential conflict of interest: A. J. Beavil has received research support from Asthma UK and the Medical Research Council UK. S. R. Durham has received lecture fees from ALK-Abelló and Merck, has received consultancy fees from Circassia, has received research support from Novartis and ALK-Abelló, has provided legal consultation/expert witness testimony for Merck, and is on the Immune Tolerance Network National Institute of Allergy and Infectious Diseases Steering Committee. The rest of the authors declare that they have no relevant conflicts of interest.

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