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A2.33 Citrullinated self antigen-specific blood B cells carry cross-reactive immunoglobulins with effector potential
  1. PJ Titcombe1,2,
  2. K Amara1,
  3. LO Barsness2,
  4. N Zhang2,
  5. A Krishnamurthy1,
  6. A Shmagel2,
  7. M Hansson1,
  8. L Israelsson1,
  9. P Sahlström1,
  10. L Giacobbe2,
  11. AI Catrina1,
  12. EC Gillespie2,
  13. L Klareskog1,
  14. EJ Peterson2,
  15. V Malmström1,
  16. DL Mueller2
  1. 1Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden
  2. 2University of Minnesota Medical School, Minneapolis, Minnesota, USA

Abstract

Background and objectives Presence of autoantibodies targeting citrullinated proteins (ACPA) and clinical response from anti-CD20 Rituximab therapy indicate a critical role for B cells in rheumatoid arthritis (RA). Due to the difficulty of reliably tracking rare B cells with a defined specificity, the composition of the autoreactive B cell repertoire contributing to ACPA+ RA has not been analysed. We previously developed a tetramer enrichment strategy for capturing autoreactive B cells. We now employ cyclic citrullinated peptide (CCP) tetramers for cloning and expressing citrulline-specific immunoglobulin (Ig) from single blood B cells in RA.

Materials and methods We analysed B cells specific for two previously described CCP, which are citrullinated epitopes from human filaggrin (Cfc1) and alpha-enolase (CEP-1). Antigen-specific B cells were detected by flow cytometry – with fluorescent CCP tetramer for citrulline-specific capture and non-citrullinated peptide decoy tetramer for distinguishing B cells that bind irrelevant epitopes on the tetramer complex. Tetramers were subsequently used to sort single B cells from RA patient (n = 3) blood. Reverse transcription-PCR generated Ig cDNA from the cells. Ig was amplified, cloned, and expressed as monoclonal antibody (mAb).

Results Single B cells sorted by either CCP tetramer (Cfc1 or CEP-1) from RA patients’ peripheral blood have highly mutated Ig sequences and show preferential use of the VH4 gene family. When expressed as mAb, 9 of 25 tetramer-sorted clones demonstrated high reactivity towards the targeted CCP epitope, as opposed to 0 of 21 randomly selected memory B cells isolated from the same patient samples. All of the highly citrulline-specific clones further exhibited cross-reactivity to citrullinated epitopes from other proteins and unique binding patterns to these autoantigens on peptide array. The mAb generated from tetramer-selected cells also induced osteoclastogenesis and bone erosion in vitro.

Conclusions Single B cells selected by CCP tetramers have highly mutated Ig sequences and receptors with citrulline-specific reactivity. Efficient isolation of mAb clones from the autoreactive B cell repertoire by this strategy will be used to better understand the pathogenesis of autoimmunity. By re-engineering the expressed Ig Fc domain towards blocking or immunosuppressive functions, mAb with defined disease-associated specificities could also offer intriguing possibilities in targeted therapeutic design for RA and other conditions.

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