gms | German Medical Science

Background: The immune response against citrullinated protein antigens is a hallmark of patients with rheumatoid arthritis. It generates polyclonal autoantibodies of the IgG isotype that are remarkably glycosylated in the variable domain. So far, the reason for this phenomenon and its potential relevance for the development of the underlying B cell response are poorly understood. To gain further insight in the development of the ACPA-response, we here performed a detailed analysis of the B cell receptor (BCR) repertoire of ACPA-expressing B cells.

Methods: ACPA-expressing B cells were isolated from peripheral blood of 12 ACPA-positive RA patients using antigen-specific tetramer staining and fluorescence activated cell sorting. Full-length immunoglobulin (Ig) transcripts of heavy chains (HCs) and light chains (LCs) were obtained using anchoring reverse transcription of Ig sequences and amplification by nested PCR. Sequences were analysed for the degree of somatic hypermutation (SHM) and the presence of N-glycosylation sites. IgG sequences of 12 healthy donors (HD) and sequences from tetanus toxoid-specific B cells were used as controls.

Results: BCR-sequencing showed that 82% of 168 unique ACPA-IgG HC, 67% of 142 unique ACPA-Igκ LC and 47% of 102 unique ACPA-Igλ LC contained ≥1 N-glycosylation site in the variable region. This was in marked contrast to the frequency of sites observed in the healthy donor and tetanus toxoid-specific repertoire. All sites in ACPA transcripts were introduced by SHM. Despite a high degree of SHM in both HC and LC, no correlation was observed between the number of nucleotide mutations and the number of N-glycosylation sites. The distribution of sites across framework (FR) and complementarity determining regions (CDR) differed remarkably from sites observed in healthy donor sequences. Finally, the generation of sites in ACPA transcripts frequently required multiple mutations, in contrast to sites in the healthy donor dataset.

Conclusion: These findings obtained from BCR repertoire analysis of ACPA-expressing B cells suggest that classical selection mechanisms are altered in the development of this auto-reactive B cell response. In fact, ACPA-expressing B cells appear to gain selective survival advantages through the generation of N-glycosylation sites in the variable domain, thereby allowing these B cells to escape tolerance.