Background and objectives We have recently identified a subset of pro-inflammatory memory B cells in RA synovial fluid and tissue, characterised by the expression of Fc receptor-like 4 (FcRL4). These cells produce RANKL, and express high levels of TNFα mRNA, indicating a destructive, pro-inflammatory role. Their immunophenoptype suggest that they are activated B cells; however, they show no signs of differentiation towards plasmablasts, suggesting that their functional role does not involve antibody production. The aim of this project was to investigate the characteristics of their immunoglobulin genes and determine whether their B cell receptors recognise autoantigens.
Methods Single FcRL4-positive and -negative B cells were sorted from synovial tissue (n = 2) and synovial fluid (n = 5) of patients with active RA. Their Ig variable region genes were sequenced and subsequently expressed to generate recombinant monoclonal antibodies. Antigen specificities of the generated monoclonal antibodies were determined by ELISA.
Results In total, we have cloned and sequenced B cell receptors from 332 individual B-cells (171 from FcRL4+ and 160 from FcRL4- cells). Ig gene sequence analysis demonstrated that the Ig repertoire was highly diverse with no major differences in the IGH and IGK or IGL gene segment usage or IGH CDR3 features between FcRL4+ and FcRL4- memory B cells. The prevalence of mutations, suggesting somatic hypermutation and selection in germinal centres, was equivalent in the FcRL4+ and FcRL4- populations. From the sequenced clones we have generated recombinant monoclonal antibodies from both FcRL4+ (n = 29) and FcRL4– B cells (n = 26) and have determined their specificity for citrullinated candidate autoantigens. A similar proportion of recombinant antibodies derived from the FcRL4+ and FcRL4- B cell populations (23% and 24% respectively) reacted with citrullinated antigens (including α-enolase, fibrinogen, vimentin and histones)
Conclusion We conclude that memory B cells from both the FcRL4+ and FcRL4- populations are post-germinal centre hypermutated B cell subsets with similar Ig gene features. Their antigen specificity suggests that these functionally distinct B subpopulations both emerge from the autoantigen driven immune response in the inflamed joint.