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Identification and characterisation of citrullinated antigen-specific B cells in peripheral blood of patients with rheumatoid arthritis
  1. Priscilla F Kerkman1,
  2. Emeline Fabre1,
  3. Ellen I H van der Voort1,
  4. Arnaud Zaldumbide2,
  5. Yoann Rombouts1,3,
  6. Theo Rispens4,
  7. Gertjan Wolbink4,
  8. Rob C Hoeben2,
  9. Hergen Spits5,
  10. Dominique L P Baeten6,
  11. Tom W J Huizinga1,
  12. René E M Toes1,
  13. Hans U Scherer1
  1. 1Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands
  2. 2Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands
  3. 3Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands
  4. 4Sanquin Research and Landsteiner Laboratory, Academic Medical Center, Amsterdam, The Netherlands
  5. 5Department of Cell Biology and Histology, Academic Medical Center, Amsterdam, The Netherlands
  6. 6Department of Clinical Immunology and Rheumatology, Academic Medical Center, Amsterdam, The Netherlands
  1. Correspondence to Dr Hans Ulrich Scherer, Department of Rheumatology, Leiden University Medical Centre, P.O. Box 9600, Leiden 2300RC, The Netherlands; h.u.scherer{at}lumc.nl

Abstract

Objectives Immunity to citrullinated antigens is a hallmark of rheumatoid arthritis (RA). We set out to elucidate its biology by identifying and characterising citrullinated antigen-specific B cells in peripheral blood of patients with RA.

Methods Differentially labelled streptavidin and extravidin tetramers were conjugated to biotinylated CCP2 or control antigens and used in flow cytometry to identify citrullinated antigen-specific B cells in peripheral blood. Tetramer-positive and tetramer-negative B cells were isolated by fluorescence activated cell sorting (FACS) followed by in vitro culture and analysis of culture supernatants for the presence of antibodies against citrullinated protein antigens (ACPA) by ELISA. Cells were phenotypically characterised by flow cytometry.

Results By combining differentially labelled CCP2 tetramers, we successfully separated citrullinated antigen-specific B cells from non-specific background signals. Isolated tetramer-positive B cells, but not tetramer-negative cells, produced large amounts of ACPA upon in vitro stimulation. Phenotypic analyses revealed that citrullinated antigen-specific B cells displayed markers of class-switched memory B cells and plasmablasts, whereas only few cells displayed a naïve phenotype. The frequency of tetramer-positive cells was high (up to 1/500 memory B cells with a median of 1/12 500 total B cells) and correlated with ACPA serum titres and spontaneous ACPA production in culture.

Conclusions We developed a technology to identify and isolate citrullinated antigen-specific B cells from peripheral blood of patients with RA. Most cells have a memory phenotype, express IgA or IgG and are present in relatively high frequencies. These data pave the path for a direct and detailed molecular characterisation of ACPA-expressing B cells and could lead to the identification of novel therapeutic targets.

  • Ant-CCP
  • B cells
  • Rheumatoid Arthritis
  • Autoimmunity

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