Abstract
Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting 1% of the world population. RA is associated with the presence of autoantibodies, of which anti-citrullinated protein antibodies (ACPA) are most prominent. ACPA are produced by citrullinated antigen-binding B cells that have presumably survived tolerance checkpoints. So far, it is unclear how and when such autoreactive B cells emerge. Light chain (LC) rearrangement and mutation rates can be informative with regard to selection steps during B-cell development. Therefore, we studied LC characteristics of ACPA-expressing B cells and secreted ACPA with the aim to better understand the development of this disease-specific, autoreactive B-cell response. Paired ACPA-IgG and ACPA-depleted IgG were isolated from serum (n = 87) and synovial fluid (SF, n = 21) of patients with established RA. We determined the LC composition for each fraction by ELISA using kappa(Igκ)- and lambda(Igλ) LC-specific antibodies. Cellular LC expression was determined using flow cytometry. In addition, we used a B-cell receptor (BCR)-specific PCR to obtain LC variable region sequences of citrullinated antigen- and tetanus toxoid (TT)-binding B cells. In serum, we observed an increased frequency of lambda LC in ACPA-IgG (1.64:1) compared to control IgG (2.03:1) and to the κ/λ ratio reported for healthy individuals (2:1). A similar trend towards higher frequencies of lambda LCs was observed for ACPA-IgG in SF (1.84:1). Additionally, the percentage of Igλ-expressing B cells was higher for citrullinated antigen-binding B cells (51%) compared to TT-specific (43%) and total CD19+CD20+ B cells (36%). Moreover, an increased Igλ percentage was observed in BCR-sequences derived from ACPA-expressing (49%) compared to TT-specific B cells (34%). Taken together, we report an enhanced frequency of lambda LCs in the secreted ACPA-IgG repertoire and, on the cellular level, in BCR sequences of ACPA-expressing B cells compared to control. This skewing in the autoreactive B-cell repertoire could reflect a process of active selection.
Highlights
The majority of rheumatoid arthritis (RA) patients harbor autoantibodies that recognize citrullinated proteins
Our results demonstrate an increased frequency of lambda Light chain (LC) in anti-citrullinated protein antibodies (ACPA)-IgG isolated from serum and synovial fluid, an increased percentage of citrullinated antigen-binding B cells expressing the lambda LC as measured by flow cytometry and an increase in lambda LCs in B-cell receptor (BCR) sequences derived from single cell- and pool-sorted citrullinated antigen-binding B cells
ACPA-IgG were isolated from synovial fluid (SF) of 21 patients to study if the higher frequency of lambda LC was present in this compartment
Summary
The majority of rheumatoid arthritis (RA) patients harbor autoantibodies that recognize citrullinated proteins (commonly termed anti-citrullinated protein antibodies, ACPA). ACPA-IgG were found to be highly glycosylated in the variable domain and to be highly cross-reactive with other post-translational protein modifications [9,10,11] This is intriguing, as it suggests that ACPA-expressing B cells deviate from the ‘conventional’ mechanisms of positive and negative selection and affinity maturation that are thought to govern the generation of high avidity, non-autoreactive clones, such as those observed against recall antigens [12]. Such selection processes occur at various stages of B-cell development and lead to modifications of the B-cell receptor (BCR) aimed at minimizing autoreactivity, while maintaining a broad repertoire capable of mounting a protective immune response. Understanding these processes in the context of human autoreactive B cells may be crucial to comprehend how ACPA-expressing B cells escape tolerance checkpoints and at what stage of B-cell development tolerance is breached
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