Our findings suggest that the high level of cross reactivity amongst the RA autoreactive B cells is the result of different antigen encounters, possibly at different sites and at different time points. This is in line with the notion that RA is initiated in one context, such as in mucosal organs, and thereafter target other sites, such as joints. This article is protected by copyright. All rights reserved.

Single B cell technologies, which avoid traditional hybridoma fusion and combinatorial display, provide a means to interrogate the naturally-selected antibody repertoire of immunized animals. Many methods enable the sampling of memory B cell subsets, but few allow for the direct interrogation of the plasma cell repertoire, i.e., the subset of B cells responsible for producing immunoglobulin in serum. Here, we describe the use of a robust and simple fluorescence-based technique, called the fluorescent foci method, for the identification and isolation of antigen-specific IgG-secreting cells, such as plasma cells, from heterogeneous bone marrow preparations. Following micromanipulation of single cells, cognate pairs of heavy and light chain variable region genes were recovered by reverse transcription (RT)-polymerase chain reaction (PCR). During the PCR, variable regions were combined with a promoter fragment and a relevant constant region fragment to produce two separate transcriptionally-active PCR (TAP) fragments that were directly co-transfected into a HEK-293F cell line for recombinant antibody expression. The technique was successfully applied to the generation of a diverse panel of high-affinity, functional recombinant antibodies to human tumor necrosis factor (TNF) receptor 2 and TNF derived from the bone marrow of immunized rabbits and rats, respectively. Progression from a bone marrow sample to a panel of functional recombinant antibodies was possible within a 2-week timeframe.

Background and objectivesHuman recombinant monoclonal antibodies have previously been generated at our laboratory from isolated patient-derived memory B cells to assess the repertoire and specificity of human anti-citrulline antibodies.The aim of the current study was to access joint-derived antibody secreting plasma cells and explore the proportion of citrulline reactivity, the immunoglobulin mutation patterns and antigen reactivity patterns. Clearly these antibodies can be utilised as tools and can help dissect the contribution antibodies make towards disease manifestations.Materials and methodsSpontaneously IgG antibody-secreting cells (referred to as plasma cells) from synovial fluid of the inflamed joints of RA patients were isolated by a fluorescent foci method. The cells were identified and selected utilising a micromanipulator-equipped microscope. Recombinant monoclonal antibodies were generated from the isolated cells by amplification of the heavy and light chain variable antibody regions and were subsequently cloned into expression vectors. The monoclonal antibodies were screened for citrulline-reactivity by in-house ELISA or by a multiplex peptide array of RA associated antigens.ResultsWe generated 375 IgG sequences and 101 recombinant antibodies from both ACPA+ (n = 3) and ACPA- (n = 1) patients. Four of the antibodies from ACPA+ synovial fluid displayed strong reactivity towards a citrullinated RA antigen peptide, but not to the cognate native peptide. The antibodies were multireactive to a diverse backbone with only the citrulline residue as common denominator, however with different reactivity patterns. The majority of the anti-citrulline clones are from the VH4 gene family. We found clonality within the plasma cells (including within the anti-citrulline antibodies), but also between different B cell populations.ConclusionsOnly a minority of the local plasma cells in the synovial fluid were autoreactive as measured by citrulline reactivity. However, the citrulline reactive plasma cells in the inflamed joint of RA patients where shown to be multireactive to several different citrulline peptides, with unique reactivity patterns. Further studies are needed to distinguish whether these plasma cells have differentiated locally in the joint. Still, it is tempting to speculate that the antibody characteristics that lead to multireactivity are driven by interaction towards several modified antigens, as has been suggested in the setting of influenza.