Rheumatoid arthritis (RA) is characterised by autoantibodies to citrullinated proteins (ACPA). Smoking and specific HLA alleles are well-established risk factors for ACPA+RA, and more recently Porphyromonas gingivalis, a major cause of periodontitis (PD), has been linked to ACPA+RA. Our ambition with this study is to clone ACPA-specific B-cells from gingival tissue of patients suffering from both PD and RA, in order to demonstrate that citrulline-specific B-cells, previously only detected in RA joints and circulation, may also reside in gingival tissue.Materials and methods
Gingival tissue-derived single CD19+ B-cells from an ACPA+RA patient with PD were sorted by flow cytometry. Immunoglobulin (Ig) variable region genes were sequenced and expressed to generate recombinant monoclonal antibodies (mAbs). These mAbs will subsequently be screened for reactivity towards citrullinated antigens by ELISA and an antigen multiplex assay.Results
We successfully isolated 480 CD19+ B-cells from the gingival tissue, and to this date, we have analysed 110 variable heavy chain Ig genes (IGHV). Ig gene sequences analysis demonstrated that the B-cell repertoire was predominantly polyclonal, although two clonally related B-cell populations (approximately 2%) were detected. Compared to healthy controls, RA/PD B-cells showed decrease in VH3 and JH5 genes, and increase in VH4, JH4 and JH6. By individual VH gene segments, IGHV4–31 was overrepresented compared to controls (p<0.0001). Conversely, IGHV1-2, IGHV3-23, and IGHV4-34 were under-represented (p<0.0001). Interestingly, antibodies with positively charged IGHV CDR3 regions, a feature associated with auto-reactivity, were enriched in gingival tissue.Results
Alignment of VH sequences to their closest germline counterparts revealed that RA/PD B-cells exhibited extensive mutations in the IGH CDR regions and higher levels of somatic mutations in the V gene segments compared to controls (p<0001). These observations suggest that the B-cell response in the gingival tissue was antigen-driven. We have so far expressed 47 mAbs, and we are currently screening these for citrulline-reactivity.Conclusions
We have been able to successfully isolate and clone a number of gingival-tissue-derived B-cells. Based on the hypothesis that the ACPA-response may be initiated at mucosal surfaces such as gingival tissue, we now have the tools available to more directly address this etiological question.