Variable domain N-linked glycosylation and negative surface charge are key features of monoclonal ACPA: implications for B-cell selection

Autoreactive B cells have a central role in the pathogenesis of rheumatoid arthritis (RA), and recent findings have proposed that anti-citrullinated protein autoantibodies (ACPA) may be directly pathogenic. Herein, we demonstrate the frequency of variable-region glycosylation in single-cell cloned mAbs. A total of 14 ACPA mAbs were evaluated for predicted N-linked glycosylation motifs in silico and compared to 452 highly-mutated mAbs from RA patients and controls. Variable region N-linked motifs (N-X-S/T) were strikingly prevalent within ACPA (100%) compared to somatically hypermutated (SHM) RA bone marrow plasma cells (21%), and synovial plasma cells from seropositive (39%) and seronegative RA (7%). When normalized for SHM, ACPA still had significantly higher frequency of N-linked motifs compared to all studied mAbs including highly-mutated HIV broadly-neutralizing and malaria-associated mAbs. The Fab glycans of ACPA-mAbs were highly sialylated, contributed to altered charge, but did not influence antigen binding. The analysis revealed evidence of unusual B-cell selection pressure and SHM-mediated decreased in surface charge and isoelectric point in ACPA. It is still unknown how these distinct features of anti-citrulline immunity may have an impact on pathogenesis. However, it is evident that they offer selective advantages for ACPA+ B cells, possibly also through non-antigen driven mechanisms

Autoreactivity to malondialdehyde-modifications in rheumatoid arthritis is linked to disease activity and synovial pathogenesis

Oxidation-associated malondialdehyde (MDA) modification of proteins can generate immunogenic neo-epitopes that are recognized by autoantibodies. In health, IgM antibodies to MDA-adducts are part of the natural antibody pool, while elevated levels of IgG anti-MDA are associated with inflammatory conditions. Yet, in human autoimmune disease IgG anti-MDA responses have not been well characterized and their potential contribution to disease pathogenesis is not known. Here, we investigate MDA-modifications and anti-MDA-modified protein autoreactivity in rheumatoid arthritis (RA). While RA is primarily associated with autoreactivity to citrullinated antigens, we also observed increases in serum IgG anti-MDA in RA patients compared to controls. IgG anti-MDA levels significantly correlated with disease activity by DAS28-ESR and serum TNF-alpha, IL-6, and CRP. Mass spectrometry analysis of RA synovial tissue identified MDA-modified proteins and revealed shared peptides between MDA-modified and citrullinated actin and vimentin. Furthermore, anti-MDA autoreactivity among synovial B cells was discovered when investigating recombinant monoclonal antibodies (mAbs) cloned from single B cells. Several clones were highly specific for MDA-modification with no cross-reactivity to other antigen modifications. The mAbs recognized MDA-adducts in a variety of proteins. Interestingly, the most reactive clone, originated from an IgG1-bearing memory B cell, was encoded by germline variable genes, and showed similarity to previously reported natural IgM. Other anti-MDA clones display somatic hypermutations and lower reactivity. These anti-MDA antibodies had significant in vitro functional properties and induced enhanced osteoclastogenesis, while the natural antibody related high-reactivity clone did not. We postulate that these may represent distinctly different facets of anti-MDA autoreactive responses

Rheumatoid arthritis patients display B-cell dysregulation already in the naive repertoire consistent with defects in B-cell tolerance

B cells are postulated to be central in seropositive rheumatoid arthritis (RA). Here, we use exploratory mass cytometry (n = 23) and next-generation sequencing (n = 19) to study B-cell repertoire shifts in RA patients. Expression of several B-cell markers were significantly different in ACPA(+) RA compared to healthy controls, including an increase in HLA-DR across subsets, CD22 in clusters of IgM(+) B cells and CD11c in IgA(+) memory. Moreover, both IgA(+) and IgG(+) double negative (IgD(-) CD27(-)) CD11c(+) B cells were increased in ACPA+ RA, and there was a trend for elevation in a CXCR5/CCR6(high) transitional B-cell cluster. In the RA BCR repertoire, there were significant differences in subclass distribution and, notably, the frequency of VH with low somatic hypermutation (SHM) was strikingly higher, especially in IgG1 (p < 0.0001). Furthermore, both ACPA(+) and ACPA(-) RA patients had significantly higher total serum IgA and IgM compared to controls, based on serology of larger cohorts (n = 3494 IgA; n = 397 IgM). The observed elevated Ig-levels, distortion in IgM(+) B cells, increase in double negative B cells, change in B-cell markers, and elevation of unmutated IgG(+) B cells suggests defects in B-cell tolerance in RA. This may represent an underlying cause of increased polyreactivity and autoimmunity in RA

The human bone marrow plasma cell compartment in rheumatoid arthritis-Clonal relationships and anti-citrulline autoantibody producing cells

A majority of circulating IgG is produced by plasma cells residing in the bone marrow (BM). Long-lived BM plasma cells constitute our humoral immune memory and are essential for infection-specific immunity. They may also provide a reservoir of potentially pathogenic autoantibodies, including rheumatoid arthritis (RA)-associated anti-citrullinated protein autoantibodies (ACPA). Here we investigated paired human BM plasma cell and peripheral blood (PB) B-cell repertoires in seropositive RA, four ACPA+ RA patients and one ACPA- using two different single-cell approaches, flow cytometry sorting, and transcriptomics, followed by recombinant antibody generation. Immunoglobulin (Ig) analysis of >900 paired heavy-light chains from BM plasma cells identified by either surface CD138 expression or transcriptome profiles (including gene expression of MZB1, JCHAIN and XBP1) demonstrated differences in IgG/A repertoires and N-linked glycosylation between patients. For three patients, we identified clonotypes shared between BM plasma cells and PB memory B cells. Notably, four individuals displayed plasma cells with identical heavy chains but different light chains, which may indicate receptor revision or clonal convergence. ACPA-producing BM plasma cells were identified in two ACPA+ patients. Three of 44 recombinantly expressed monoclonal antibodies from ACPA+ RA BM plasma cells were CCP2+, specifically binding to citrullinated peptides. Out of these, two clones reacted with citrullinated histone-4 and activated neutrophils. In conclusion, single-cell investigation of B-cell repertoires in RA bone marrow provided new understanding of human plasma cells clonal relationships and demonstrated pathogenically relevant disease-associated autoantibody expression in long-lived plasma cells