Johanna Steen

Monoclonal IgG antibodies generated from joint-derived B cells of RA patients have a strong bias toward citrullinated autoantigen recognition

Synovial IgG-expressing B cells from patients with rheumatoid arthritis show specificity for citrullinated autoantigens.

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 antibodies are associated with inflammatory and autoimmune 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, and 3.5% of memory B cells and 2.3% of plasma cells were found to be anti-MDA positive. Several clones were highly specific for MDA-modification with no cross-reactivity to other antigen modifications such as citrullination, carbamylation or 4-HNE-carbonylation. The mAbs recognized MDA-adducts in a variety of proteins including albumin, histone 2B, fibrinogen and vimentin. Interestingly, the most reactive clone, originated from an IgG1-bearing memory B cell, was encoded by near germline variable genes, and showed similarity to previously reported natural IgM. Other anti-MDA clones display somatic hypermutations and lower reactivity. Importantly, these anti-MDA antibodies had significant inxa0vitro 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.

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 decrease 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 through non‐antigen driven mechanisms.

Human plasma cell derived monoclonal antibodies to post-translationally modified proteins recognize amino acid motifs rather than specific proteins

Antibodies against posttranslationally modified proteins are a hallmark of rheumatoid arthritis (RA), but the emergence and pathogenicity of these autoantibodies are still incompletely understood. The aim of this study was to analyze the antigen specificities and mutation patterns of monoclonal antibodies (mAb) derived from RA synovial plasma cells and address the question of antigen cross‐reactivity.

A5.14 Homocitrulline-Reactive Antibodies can be Generated from Synovial B-Cells from ACPA-Negative RA Patients

Background and Objectives Roughly two-thirds of rheumatoid arthritis (RA) patients carry antibodies, so-called ACPA, against peptides containing citrulline, a post-translationally modified version of the amino acid (AA) arginine in their sera and/or affected joints. A recent study reported the presence of antibodies against a different kind of post-translationally modified AA, homocitrulline, also known as carbamylated proteins (CarP), where lysine residues are altered by a non-enzymatic reaction involving cyanate. The authors reported IgG antibodies recognising homocitrullinated fibrinogen in the sera of >45% of RA-patients and that 16% of ACPA-negative patients carried such anti-CarP antibodies. Furthermore, in vivo studies describe that immunisation of mice with homocitrulline-containing peptides induced erosive arthritis. Thus, homocitrulline represents an interesting immune target in the context of RA. Therefore, we aimed to assess the proportion of anti-CarP antibodies sourced from the joints of RA patients with active disease. Materials and Methods Using a single B cell-based cloning technology to isolate the immunoglobulin (Ig) genes from joint-derived IgG+ CD19+ B-cells, we then co-transfected the paired heavy and light chains into a human cell line and produced recombinant monoclonal antibodies from each individual B cell. This approach has previously allowed for the generation of citrulline-reactive monoclonal antibodies. Here, we analysed for reactivity to carbamylated (homocitrullinated) fibrinogen and compared to its counterpart unmodified fibrinogen, by ELISA. So far, we have analysed 42 monoclonal antibodies from three ACPA-positive patients and one ACPA-negative patient, for homocitrulline reactivity. Results Between six and eleven antibodies were tested for homocitrulline activity for the ACPA-positive patients and eleven were examined for the ACPA-negative patient. All clones screened for CarP-reactivity were negative for citrulline binding. Antibodies were made up to concentrations of 5 ug/ml down to 0.63 ug/ml in a 4-step dilution. So far, one antibody displayed reactivity to the carbamylated peptide and this antibody originated from the ACPA-negative patient. Conclusions Although the frequency of anti-CarP reactivity amongst the 42 antibodies examined does not match that previously reported for sera, the finding of one anti-CarP antibody from an ACPA negative patient, does support the earlier finding of significant frequencies of anti-Carp antibodies amongst ACPA-negative patients. It will be of great interest to expand the investigation for anti-Carp specificities, particularly among ACPA-negative patients and determine whether these antibodies could have pathological effects in RA patients.

The specificity of monoclonal anti citrulline protein antibodies - reply to Dr Holmdahl's letter

Rikard Holmdahl has commented on our recently published paper on specificity and function of human monoclonal antibodies (mAbs) from synovial plasma cells that react with citrullinated proteins and peptides (1). He also comments on our previous study from 2013 (2) that was based on mAbs generated from synovial B cells. Dr Holmdahls letter provides us with an opportunity to discuss and clarify some methodological issues concerning RA monoclonal autoantibody expression, specificity, and functionality. In our eyes, cloning and characterization of human monoclonal autoantibodies from patients represents an important approach to the understanding of origins and functions of autoantibodies in the pathogenesis of immune-mediated diseases. In fact, the RA field has been especially active on making use of mAbs derived from the immunoglobulin sequences from patient-derived B cells and plasma cells. This article is protected by copyright. All rights reserved.

08.41 cloning of gingival tissue b cells from an acpa+ ra patient with periodontitis

Background 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 480u2009CD19+ 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. 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.

FRI0006 Protein citrullinations by pad enzymes promote dendritic cell transdifferentiation into osteoclast and generate targets for ra-specific antibodies

Background Immature dendritic cells (DCs) are able to trans-differentiate into osteoclasts (OCs) although the mechanisms regulating this process are little understood. We have recently described an important role for protein citrulliantion and peptidylarginine deiminase (PAD) enzyme activity in the regulation of OC development (1). Objectives We studied the molecular bases of DC-OC trans-differentiation and aimed at understanding the role of protein citrullination in this process. Methods Monocyte-derived DCs and peripheral blood CD1c+ DCs were cultured in the presence of osteoclastogenic cytokines. Polyclonal ACPAs were isolated from the serum of RA patients and applied in OC cultures. DC and OC differentiation was analyzed in vitro using gene expression analyses, flow cytometry-based methods, DC-T cell co-culture experiments, tartrate-resistant acid phosphatase stainings and osteolysis assays. Protein citrullination was monitored with the help of mass spectrometry. PAD activity was measured by ELISA. Results Different DC types showed different capacities to develop into OCs. OC-prone DCs were characterized by little immunogenicity and their development was potentiated by the increase of lactic acid, a side product of glycolytic metabolism. The more immunogenic DC types, characterized by prominent ability to migrate towards secondary lymphoid tissues and trigger T cell activation, showed a limited capacity to develop into OCs (2). The differentiation switch towards the OC lineage was associated with increased activity of the Protein Arginine Deiminase (PAD) enzymes and with higher level of protein citrullination in DCs. The PAD inhibitor Cl-Amidine efficiently interfered with OC development form DC precursors. In addition, the deposition of citrullinated proteins on the cell surface made the cells sensitive for anti-citrullinated protein autoantibodies, which could further stimulate DC-OC trans-differentiation through inducing the cytokine IL-8. Conclusions Our results indicated that DCs are heterogenic in their ability to form OCs and lineages for immunostimulatory and OC-prone DCs might separate early during DC differentiation. Plasticity towards OC differentiation might be influenced by the metabolic environment and the upregulation of PAD activity in DCs. References Krishnamurthy A et al. Ann Rheum Dis 2016. Nasi A et al. J Immunol 2013. Disclosure of Interest None declared

08.19 Variable domain n-linked glycosylation is a key feature of monoclonal acpa-igg

Background Fab-glycosylation is found in ~15%–25%u2009serum IgG and while its exact consequence remains unknown, it may alter IgG functionality. Recent data revealed extensive Fab-glycosylation in polyclonal anti-citrullinated protein autoantibodies (ACPA) from rheumatoid arthritis (RA) patients. Herein, we characterise the Fab-glycan profile of monoclonal ACPA. Materials and methods Among 200 single-cell isolated and recombinantly expressed monoclonal antibodies (mAbs) from RA patients, we selected 12 identified ACPA mAbs, four derived from synovial antibody secreting cells (ASC), and eight from peripheral antigen-tetramer sorted memory B cells. The ACPA had high somatic hypermutation (SHM) levels with an average of 49 VH and 35 VL mutations. For comparison, we selected 14 ASC synovial antibodies with mutation rate >20 in VH or VL, and we also searched the literature for 19 highly-mutated broadly-neutralising HIV-derived antibodies (bnAbs). N-linked glycan motifs were identified using the NetNGlyc server. Glycosylation was verified in five ACPA IgG by enzymatic digestion with PNGase-F or Endo-S followed by SDS-PAGE. Antigen binding was investigated by CCP3 ELISA. VH-VL structure models were generated using the online tool PIGS and visualised by Jmol, and the GlyProt server was utilised for in silico glycosylation. Results The majority of ACPA exhibited variable region N-linked motifs (83.8%), compared to 14.3% of non-ACPA RA mAbs and 63.2% of bnAbs, featured in both framework and CDRs. When adjusted for SHM, N-linked motifs were significantly increased in ACPA compared to non-ACPA RA mAbs (p=0.001) or bnAbs (p=0.002). VH region motif rates were increased in ACPA compared to non-ACPA mAbs (p=0.002) and bnAbs (p=0.0004), while VL region motifs were only higher compared to non-ACPA (p=0.002). Deglycosylation revealed that N-linked motifs were indeed glycosylated, although preliminary data suggests glycan removal had no striking effect on antigen-binding. Homology-based structures predicted glycans to be primarily positioned outside of the potential antigen-binding site. Conclusions The results support that variable region glycosylation is a key feature of ACPA. Significant increases in N-linked motifs in ACPA compared to other highly-mutated antibodies signifies that this is not solely linked to hypermutation. Future studies are merited to further investigate the selection mechanisms and functional role of Fab-glycosylated autoantibodies.

08.24 Monoclonal acpa antibodies recognising a common citrulline motif are mainly dependent on light chain hypermutations for antigen recognition

Background Anti-citrullinated protein antibodies (ACPA) can be detected many years before disease onset, implicating that not all ACPAs are pathogenic and that the autoantibodies undergo critical maturation steps closer to disease onset. We have studied the ACPA profile of synovial fluid and of monoclonal antibodies generated from plasma cells of the same synovial fluid to address these questions. Materials and methods IgG-secreting cells from RA synovial fluid were isolated, the variable Ig regions were amplified, and recombinant monoclonal antibodies were expressed. The antibody reactivity to citrullinated peptides were addressed both by a planar multi array of RA associated peptides, as well as on a 1u200975u2009000 peptide micro-array of arginine/citrulline peptides from extracellular matrix proteins. The matching synovial fluid was also analysed on the peptide arrays. Chimeric monoclonal antibodies were produced based on IMGT predicted germline sequences of the ACPAs, to address the importance of hypermutations in the heavy versus the light chain. The induction of osteoclastogenesis by the ACPAs were tested in an in vitro assay. Results Sera from RA-patients display wide cross-reactivity between many citrullinated proteins and peptides. Synovial fluid replicates this pattern, and a glycine-rich consensus sequence could be deduced. The monoclonal ACPAs recognised different consensus sequences, of which some replicated the dominant polyclonal pattern. To dissect the contribution of the heavy and light chain to antigen recognition, chimeric variants were expressed where either the light or the heavy chain was exchanged with the respective germline sequence, or to an unrelated chain. Surprisingly, the ACPAs with the common consensus epitope were highly dependent on the light chain mutations for citrulline reactivity, while the heavy chain appeared primarily important for stability and conformation, independent on the acquired mutations. Even if these ACPA were highly cross-reactive, they did not cause osteoclastogenesis. The pathogenic ACPA was instead dependent on hypermutations in both heavy and light chains for citrulline reactivity, and had a more restricted citrulline peptide reactivity. Conclusions Our data lend support to the observed high cross-reactivity of ACPA between citrullinated peptides. We also find differences in citrulline-reactivity patterns and light chain contribution between pathogenic and non-pathogenic ACPA.