Background/Purpose:    Our recent findings identified a subset of pro-inflammatory memory B cells in the RA synovium characterized by the expression of the surface protein Fc receptor like 4 (FcRL4)¹. FcRL4+ve B cells produce RANKL, and express high levels of TNF mRNA, indicating a destructive, pro-inflammatory role for this B cell subpopulation. It is however unclear how synovial FcRL4+ve B cells develop, whether they have undergone hypermutation in germinal centers and the nature of their relationship with FcRL4-ve B cells at the same site. The aim of this project was to investigate whether autoreactive features would accumulate in the FcRL4+ve subset by investigating the antigen-specificity and Ig gene characteristics of FcRL4 +ve versus -ve B cells from both synovial tissue and fluid of RA patients.  

Methods: Single FcRL4+ve and -ve memory 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). The Ig gene sequence analyses demonstrated that the Ig repertoire was highly diverse with no major differences in the IGH and IGK or IGL gene segment usage or IGHCDR3 features between FcRL4+ and FcRL4- memory B cells. From the sequenced clones we have so far generated 38 recombinant monoclonal antibodies (from both FcRL4+ve and FcRL4–ve B cells) and determined their specificity for citrullinated candidate autoantigens. We found no difference in the frequency of autoreactive Ig in the FcRL4 +ve versus -ve cells. 13 antibodies reacted to citrullinated antigens (including α-enolase, fibrinogen, and vimentin) without recognizing the unmodified arginine control peptides.

Conclusion: We conclude that the FcRL4+ve and FcRL4-ve memory B cells, while functionally and phenotypically distinct, are both post-germinal center hypermutated B cell subsets with similar Ig gene features. We have not identified a clonal relationship, but overall both B cell receptor characteristics and antigen specificities suggest that the two B cell subpopulations may originate from similar immune responses differentiating into functionally distinct subsets.

¹ Yeo L, Lom H, Juarez M, et al. Ann Rheum Dis (2014) doi:10.1136/annrheumdis-2013-204116

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/pro-inflammatory-fcrl4-memory-b-cells-in-joints-of-ra-patients-immunoglobulin-gene-characteristics-and-antigen-specificity/