B Cell Receptor Sequencing of Anti-Citrullinated Protein Antibody Expressing B Cells Indicates a Selective Advantage for the Introduction of N-Glycosylation Sites during Somatic Hypermutation

Rochelle D. Vergroesen¹, Linda Slot¹, Lise Hafkenscheid¹, Marvyn T. Koning², Ellen I.H. van der Voort³, Christine A. Grooff¹, George Zervakis², Tom W.J. Huizinga¹, Theo Rispens⁴, Hendrik Veelken², Rene E.M. Toes¹ and Hans U. Scherer³, ¹Rheumatology, Leiden University Medical Center, Leiden, Netherlands, ²Hematology, Leiden University Medical Center, Leiden, Netherlands, ³Department of Rheumatology, Leiden University Medical Center, Leiden, Netherlands, ⁴Immunopathology, Sanquin Research, Amsterdam, Netherlands

Meeting: 2017 ACR/ARHP Annual Meeting

Date of first publication: September 18, 2017

Keywords: ACPA, B cell tolerance, B cells and rheumatoid arthritis (RA)

Session Information

Date: Sunday, November 5, 2017

Title: B Cell Biology and Targets in Autoimmune Disease

Session Type: ACR Concurrent Abstract Session

Session Time: 2:30PM-4:00PM

Background/Purpose:

The vast majority (>90%) of anti-citrullinated protein antibodies (ACPA) of the IgG isotype in serum and synovial fluid of patients with rheumatoid arthritis carry N-linked glycans in the antibody variable region. This remarkable degree of Fab-glycosylation is absent from ACPA-depleted control IgG and from autoantibodies in other diseases. N-glycosylation requires a specific amino acid consensus sequence in the protein backbone (termed an N-glycosylation site), which is very rare in germline-encoded variable region genes. Here, we analysed the B cell receptor (BCR) repertoire of ACPA-expressing B cells to understand the molecular basis for this remarkable glycosylation.

Methods:

We used anchoring reverse transcription of immunoglobulin (Ig) sequences and amplification by nested PCR (ARTISAN) to obtain full-length rearrangements of ACPA-expressing B cells. ACPA-expressing B cells and non citrulline-reactive control B cells were obtained from peripheral blood of patients with established RA by antigen-specific tetramer staining and fluorescence activated cell sorting. Cells were either sorted in pools, or sorted as single cells. Somatic mutations and N-glycosylation sites were identified in the sequence reads using IMGT (High)V-QUEST. Paired heavy and light chain sequences (HC/LC) were used to model the spatial positioning of N-glycosylation sites.

Results:

Sequence analysis of pools of cells identified 97 unique ACPA-IgG clones derived from n=8 donors. 87 unique ACPA-IgG clones were retrieved from single cell analysis (n=6 donors). In both datasets, over 80% of ACPA-IgG clones contained N-glycosylation sites in both HC and LC. All sites were created by somatic mutations. The mutation rate did not correlate with the number of N-glycosylation sites, and their distribution across the variable region and their preference differed from the pattern seen in controls. Structural modelling predicted N-glycosylation sites on the exterior of the antibody molecule.

Conclusion:

ACPA-expressing B cells generate BCRs with a high frequency of N-glycosylation sites. Frequency and localization of sites suggest that ACPA-expressing B cells gain a selective survival advantage by acquiring glycans in the variable domain, thereby escaping from putative checkpoints in B cell selection.

Disclosure: R. D. Vergroesen, None; L. Slot, None; L. Hafkenscheid, None; M. T. Koning, None; E. I. H. van der Voort, None; C. A. Grooff, None; G. Zervakis, None; T. W. J. Huizinga, None; T. Rispens, None; H. Veelken, None; R. E. M. Toes, None; H. U. Scherer, None.

To cite this abstract in AMA style:

Vergroesen RD, Slot L, Hafkenscheid L, Koning MT, van der Voort EIH, Grooff CA, Zervakis G, Huizinga TWJ, Rispens T, Veelken H, Toes REM, Scherer HU. B Cell Receptor Sequencing of Anti-Citrullinated Protein Antibody Expressing B Cells Indicates a Selective Advantage for the Introduction of N-Glycosylation Sites during Somatic Hypermutation [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/b-cell-receptor-sequencing-of-anti-citrullinated-protein-antibody-expressing-b-cells-indicates-a-selective-advantage-for-the-introduction-of-n-glycosylation-sites-during-somatic-hypermutation/. Accessed .

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