Priscilla F Kerkman

Extensive glycosylation of ACPA-IgG variable domains modulates binding to citrullinated antigens in rheumatoid arthritis

Objectives To understand the molecular features distinguishing anti-citrullinated protein antibodies (ACPA) from ‘conventional’ antibodies in rheumatoid arthritis (RA). Methods Serum of ACPA-positive RA patients was fractionated by size exclusion chromatography and analysed for the presence of ACPA-IgG by ELISA. ACPA-IgG and non-citrulline-specific IgG were affinity purified from serum, plasma and/or synovial fluid and analysed by gel electrophoresis. Electrophoresis bands were excised, enzymatically digested and analysed by mass spectrometry. Binding affinity to citrullinated antigens was measured by ELISA and imaging surface plasmon resonance using recombinant monoclonal ACPA with molecular modifications. Results In all donor samples studied (n=24), ACPA-IgG exhibited a 10–20 kDa higher molecular weight compared with non-autoreactive IgG. This feature also distinguished ACPA-IgG from antibodies against recall antigens or other disease-specific autoantibodies. Structural analysis revealed that a high frequency of N-glycans in the (hyper)variable domains of ACPA is responsible for this observation. In line with their localisation, these N-glycans were found to modulate binding avidity of ACPA to citrullinated antigens. Conclusions The vast majority of ACPA-IgG harbour N-glycans in their variable domains. As N-linked glycosylation requires glycosylation consensus sites in the protein sequence and as these are lacking in the ‘germline-counterparts’ of identified variable domains, our data indicate that the N-glycosylation sites in ACPA variable domains have been introduced by somatic hypermutation. This finding also suggests that ACPA-hyperglycosylation confers a selective advantage to ACPA-producing B cells. This unique and completely novel feature of the citrulline-specific immune response in RA elucidates our understanding of the underlying B cell response.

Circulating plasmablasts/plasmacells as a source of anticitrullinated protein antibodies in patients with rheumatoid arthritis

Objective To study the characteristics and phenotype of anticitrullinated protein antibody (ACPA)-specific B cells in peripheral blood of patients with rheumatoid arthritis (RA). Methods Peripheral blood B cells from ACPA-positive patients with RA were cultured with or without stimulating factors. Following culture, supernatants were assessed for the presence of ACPA-IgG and non-specific total IgG by ELISA. Results Following stimulation, ACPA were detectable in up to 100% of culture wells. Of interest, ACPA were also produced spontaneously by unstimulated peripheral blood mononuclear cells. In both cases, the average ACPA titre per culture well correlated with ACPA serum titres. No ACPA production was detectable in B cell cultures from ACPA-negative patients with RA or healthy controls. Importantly, FACS-sorting experiments located spontaneous ACPA production to the CD20 negative B cell population corresponding to circulating plasmablasts/cells. Conclusions ACPA-specific peripheral blood B cells are not confined to the CD20 positive memory pool, as circulating plasmablasts/cells spontaneously producing ACPA are also readily detectable. The latter points to an ongoing B cell immune response against citrullinated proteins and contrasts conventional immune responses against, for example, vaccines, where antigen-specific plasmablasts appear in peripheral blood only shortly after vaccination. These circulating, ACPA-specific plasmablasts/cells might represent targets for novel therapeutic interventions.

Identification and characterisation of citrullinated antigen-specific B cells in peripheral blood of patients with rheumatoid arthritis

Objectives Immunity to citrullinated antigens is a hallmark of rheumatoid arthritis (RA). We set out to elucidate its biology by identifying and characterising citrullinated antigen-specific B cells in peripheral blood of patients with RA. Methods Differentially labelled streptavidin and extravidin tetramers were conjugated to biotinylated CCP2 or control antigens and used in flow cytometry to identify citrullinated antigen-specific B cells in peripheral blood. Tetramer-positive and tetramer-negative B cells were isolated by fluorescence activated cell sorting (FACS) followed by in vitro culture and analysis of culture supernatants for the presence of antibodies against citrullinated protein antigens (ACPA) by ELISA. Cells were phenotypically characterised by flow cytometry. Results By combining differentially labelled CCP2 tetramers, we successfully separated citrullinated antigen-specific B cells from non-specific background signals. Isolated tetramer-positive B cells, but not tetramer-negative cells, produced large amounts of ACPA upon in vitro stimulation. Phenotypic analyses revealed that citrullinated antigen-specific B cells displayed markers of class-switched memory B cells and plasmablasts, whereas only few cells displayed a naïve phenotype. The frequency of tetramer-positive cells was high (up to 1/500 memory B cells with a median of 1/12 500 total B cells) and correlated with ACPA serum titres and spontaneous ACPA production in culture. Conclusions We developed a technology to identify and isolate citrullinated antigen-specific B cells from peripheral blood of patients with RA. Most cells have a memory phenotype, express IgA or IgG and are present in relatively high frequencies. These data pave the path for a direct and detailed molecular characterisation of ACPA-expressing B cells and could lead to the identification of novel therapeutic targets.

Synovial fluid mononuclear cells provide an environment for long-term survival of antibody-secreting cells and promote the spontaneous production of anti-citrullinated protein antibodies.

Objectives In rheumatoid arthritis (RA), observations point to a crucial role for (autoreactive) B cells in disease pathogenesis. Here, we studied whether cells from the synovial environment impact on the longevity of autoreactive B cell responses against citrullinated antigens. Methods Synovial fluid mononuclear cells and peripheral blood mononuclear cells (SFMC/PBMC) were obtained from patients with established RA and assessed for the presence of B cell subpopulations. Cells spontaneously secreting anti-citrullinated protein antibodies (ACPA-IgG) directly ex vivo were detected by antigen-specific Enzyme-Linked ImmunoSpot (ELISpot) assay. SFMC and PBMC were cultured to assess the degree of spontaneous ACPA-IgG secretion. Cells surviving for several weeks were characterised by carboxyfluorescein succinimidyl ester (CFSE) labelling and Ki-67 staining. Results Cells spontaneously secreting ACPA-IgG were readily detectable in peripheral blood and synovial fluid (SF) of patients with ACPA-positive RA. SFMC showed an up to 200-fold increase in ex vivo ACPA-IgG secretion compared with PBMC despite lower numbers of B cells in SFMC. ELISpot confirmed the presence of spontaneously ACPA-IgG-secreting cells, accounting for up to 50% (median 12%) of all IgG-secreting cells in SF. ACPA-IgG secretion was remarkably stable in SFMC cultures, maintained upon depletion of the CD20+ B cell compartment and detectable for several months. CFSE labelling and Ki-67 staining confirmed the long-term survival of non-dividing plasma cells (PCs). Conclusions This study demonstrates a high frequency of differentiated, spontaneously ACPA-IgG-secreting cells in SF. These cells are supported by SFMC for prolonged survival and autoantibody secretion, demonstrating that the synovial compartment is equipped to function as inflammatory niche for PC survival.

A1.25 Visualisation and characterisation of citrullinated antigen-specific B cells from peripheral blood of patients with rheumatoid arthritis

Background and objectives In Rheumatoid arthritis (RA), anti-citrullinated protein antibodies (ACPA) represent highly disease-specific biomarkers found in the majority of patients. As ACPA have been implicated in disease pathogenesis, it is of crucial relevance to understand the underlying B cell response. In this context, visualisation of citrullinated antigen-specific B cells would allow for a detailed characterisation of the immune response to better understand its development and maintenance. Unfortunately, visualisation of autoreactive B cells in humans has proven extremely difficult. Materials and methods Using the CCP2-antigen and its arginine control variant, we developed a multicolour tetramer-based staining method to visualise citrullinated antigen-specific B cells in peripheral blood of RA patients by flow-cytometry. Specificity of the staining was verified by culturing tetramer-positive and -negative B cells isolated by FACS. Citrullinated antigen-reactive B cells were further phenotyped using cell surface markers associated with developmental and functional B cell characteristics. Finally, the frequency of citrullinated antigen-reactive B cells was correlated to ACPA serum levels and in vitro ACPA production. Results The staining procedure successfully separated citrullinated antigen-reactive B cells from non-specific background signals. Already fourteen FACS-sorted tetramer-positive B cells produced detectable amounts of ACPA, whereas no ACPA production was observed in cultures of up to 5000 tetramer-negative B cells. The majority of citrullinated antigen-reactive B cells had a post-germinal centre memory or plasmablast phenotype. Up to 1 in 200 memory B cells were directed against citrullinated antigens, and their frequency correlated with spontaneous ACPA production in culture and ACPA serum titres in vivo. Conclusions We show, for the first time, the specific and reliable identification of citrullinated antigen-specific B cells in high frequencies in peripheral blood of RA patients. The majority of this population has a memory phenotype and closely reflects the dynamics of the in vivo ACPA response. These data provide the basis for a detailed characterisation of this disease-specific immune response on a single cell level and could lead to the identification of novel therapeutic targets.

A1.45 Hyperglycosylation of ACPA-IGG variable domains modulates reactivity to citrullinated antigens

Background and Objectives Autoantibodies specific for citrullinated antigens are highly relevant diagnostic and prognostic biomarkers in rheumatoid arthritis and have been considered to be involved in disease pathogenesis. Previous studies have indicated that the ACPA-specific immune response differs from conventional B cell responses by generating polyclonal, cross-reactive antibodies of mostly low-avidity. In addition, ACPA were found to carry aberrant glycosylation patterns at the IgG-Fc tail. The present study was undertaken to further characterise the molecular make-up of ACPA and its potential functional consequences in the context of RA. Materials and Methods Serum components of RA patients were fractionated by size exclusion chromatography and analysed for the presence of ACPA-IgG by ELISA. In addition, ACPA-IgG and non-citrulline-specific IgG were affinity purified from RA patient serum and synovial fluid and analysed by gel electrophoresis. Electrophoresis bands were excised and subsequently analysed by HPLC and mass-spectrometry. Recombinant monoclonal ACPA with variations in ACPA molecular structure were used to study binding affinity by surface plasmon resonance. Results We discovered that ACPA-IgG from RA patients have a higher apparent molecular weight as compared to other IgG molecules including antibodies against recall antigens and other autoantibodies. This higher molecular weight was explained by the overrepresentation of N-linked glycans in the variable domain (Fab region) of ACPA-IgG. Detailed structural analysis of these glycans demonstrated that ACPA-IgG linked Fab glycans are complex-type biantennary N-glycans that differ from the conventional Fc-linked N-glycans by a high degree of sialylation, galactosylation, and fucosylation together with the presence of bisecting N-acetylglucosamine. Using recombinant ACPA-IgG monoclonal antibodies with and without Fab-glycans, we found that Fab-glycans modulate binding affinity of ACPA-IgG for citrullinated antigens. Finally, lectin-immunoblotting showed that ACPA Fab-glycans can bind to sialic acid-binding immunoglobulin-type lectins. Conclusions This study describes an unusual and novel molecular feature of the citrulline-specific immune response in RA. ACPA-IgG, in contrast to non-citrulline-specific IgG, are highly glycosylated in the variable region, which modulates recognition of citrullinated antigens. Moreover, ACPA-IgG linked Fab glycans can be the target of specific lectins, suggesting additional functional features potentially involved in ACPA-mediated pathogenetic effects. This finding points to aberrations in the development of ACPA-specific B cells and further elucidates our understanding of basic disease mechanisms in RA.

Generation and characterization of anti-citrullinated protein antibody-producing B-cell clones from rheumatoid arthritis patients

Anti–citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA). Aside from autoantibody production, the function of autoantigen‐specific B cells remains poorly understood in the context of this disease. This study set out to elucidate autoantigen‐specific B cell functions through the isolation and immortalization of unique citrullinated protein/peptide (CP)–reactive B cell clones from RA patients.

OP0200 Circulating, Auto-Reactive B Cells Specific for Citrullinated Antigens in Patients with Rheumatoid Arthritis Display An Activated, Proliferative Phenotype

Background Rheumatoid arthritis (RA) is characterized by the formation of autoantibodies against citrullinated antigens (CA). Intriguingly, depletion of CD20+ B cells leads to significant clinical improvement in the majority of patients, despite the persistence of high titer autoantibodies. This indicates that the CD20+ B cell compartment, potentially more than the autoantibodies themselves, contributes to the pathogenesis and/or chronicity of RA. Auto-reactive B cells within this compartment could be the main drivers of this effect, but evidence for this hypothesis is lacking. We recently developed the technology to visualize and characterize CA-specific B cells in patients and demonstrated that a large proportion has a CD20+ memory phenotype1. Objectives To study the phenotype of CA-specific B cells in comparison to tetanus-toxoid (TT)-specific B cells in peripheral blood of individual patients. Methods Differentially labeled CA and their arginine control variants were used as tetramers to identify CA-specific B cells in peripheral blood of patients with RA. TT-specific B cells were identified in the same samples using differentially labeled TT. Both antigen-specific cell populations were enumerated and phenotypically characterized by flow cytometry. Results CA-specific B cells and TT-specific B cells were detected in peripheral blood in comparable frequency (median 0.012% vs. 0.014%). Both CA- and TT-specific B cells displayed mainly markers of class-switched memory B cells. Intriguingly, CA-specific memory B cells showed remarkable overexpression of co-stimulatory molecules and markers of active proliferation. Conclusions This is the first phenotypic analysis of auto-reactive, CA-specific memory B cells in comparison to recall antigen-specific memory B cells in individual patients with RA. CA-specific memory B cells overexpress co-stimulatory molecules and proliferation markers, indicative of an active immune response against CA in these patients. These data support the hypothesis that CA-specific memory B cells contribute to RA pathogenesis and fuel efforts for their antigen-specific depletion. References Kerkman PF et al. Identification and characterisation of citrullinated antigen-specific B cells in peripheral blood of patients with rheumatoid arthritis. Ann Rheum Dis. 2015 Jun 1. doi: 10.1136/annrheumdis-2014-207182. Acknowledgement Supported by grants from the Dutch Arthritis Foundation, The Netherlands Organization for Scientific Research and the Innovative Medicines Initiative funded project BeTheCure. Disclosure of Interest H. Kristyanto: None declared, P. Kerkman: None declared, E. van der Voort: None declared, H. Spits Employee of: AIMM Therapeutics, D. Baeten: None declared, T. Huizinga: None declared, R. Toes: None declared, H. Scherer: None declared

OP0177 A High Frequency of N-Glycans in the Acpa-Igg Variable Domain Modulates Reactivity to Citrullinated Antigens

Background Antibodies against citrullinated proteins antigens (ACPA) are the most relevant prognostic and diagnostic biomarker for rheumatoid arthritis (RA). ACPA positive patients are characterized by progressive disease, a high rate of joint destruction, and a low chance to achieve remission. Despite these strong and well-defined associations with clinical phenotype, however, molecular mechanisms underlying the citrulline-specific immune response are incompletely understood. Of note, this immune response is highly polyclonal with considerable cross-reactivity, develops and matures prior to disease onset, and is of remarkably low avidity. These features indicate that the underlying B cell response is different from “conventional” antibody responses, but also different from other autoantibody responses such as the high affinity double-stranded DNA antibody response in SLE. Objectives To study molecular characteristics of ACPA in order to obtain insight into the properties of the underlying B cell response. Methods Serum of ACPA positive RA patients was fractionated by size exclusion chromatography and analysed for the presence of ACPA-IgG by ELISA. In addition, ACPA-IgG and non-citrulline-specific IgG were affinity purified from serum and synovial fluid and analysed by gel electrophoresis. Electrophoresis bands were excised, enzymatically digested and subsequently analysed by HPLC and mass-spectrometry. Recombinant monoclonal ACPA were used to study binding affinity for multiple antigens by surface plasmon resonance. Results In almost all donors studied (23 out of 24), ACPA-IgG molecules were found to have a higher molecular weight than non auto-reactive IgG. Structural analysis pinpointed this observation to a high frequency of N-glycans in the ACPA variable regions, which was found on up to 90% of all ACPA. These Fab-glycans were linked to N-glycosylation sites generated by somatic hypermutation, were absent from other disease-specific autoantibodies, were characterized by a high degree of sialylation, and clearly differentiated ACPA from “conventional” IgG molecules. Moreover, ACPA Fab glycans modulated reactivity to citrullinated antigens. Conclusions These data describe a unique and completely novel feature of the citrulline-specific immune response in RA, with important implications for ACPA pathogenicity and disease prognostication. The high frequency of variable domain Fab glycans points to specific developmental abnormalities of the underlying B cell response. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4717

A5.29 Spontaneous Production of Anti-Citrullinated Protein Antibodies in Cultures of Peripheral Blood Mononuclear Cells and Synovial Fluid Mononuclear Cells Isolated from Patients with Rheumatoid Arthritis

Background and Objectives Anti-citrullinated protein antibodies (ACPA) are among the most important molecular candidates that could drive the inflammatory immune response in a subset of patients with rheumatoid arthritis (RA). So far, however, little is known on the phenotype and functional characteristics of ACPA producing B cells. Therefore, we studied ACPA producing B cells using ex-vivo cultures of peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC). Materials and Methods PBMC as well as SFMC from patients with ACPA-positive RA were cultured in 96 well plates without the addition of exogenous stimuli. Cultures were maintained for several weeks, with weekly complete replacement of the culture medium. Every week, supernatants were assessed for the presence of ACPA-IgG and total IgG by ELISA. B cell subsets within the culture populations were determined by flow cytometry at several time points. Results Circulating, spontaneously ACPA producing B cells were readily detectable in peripheral blood of ACPA positive RA patients, but not in ACPA negative RA patients or healthy donors. FACS sorting experiments comparing isolated B cell subsets located spontaneous ACPA production to the plasmablast compartment. Memory B cells were capable of ACPA production upon stimulation. In some culture wells, ACPA production was stable and detectable for up to 3 months. In a similar manner, we observed spontaneous, long-lasting ACPA production in (paired) SFMC cultures. The latter showed an up to 200 fold increase in ex vivo ACPA production compared to PBMC, but only a minor increase in the secretion of non-specific IgG. B cell numbers in PBMC and SFMC were comparable in the starting population. Conclusions ACPA specific plasmablasts circulate in the peripheral blood of patients with ACPA positive RA. Upon isolation, peripheral blood B cells can secrete ACPA spontaneously for several months. This observation suggests that ACPA specific B cells are either continuously recruited from the memory compartment, or that a subset of ACPA specific plasmacells might have the capacity to survive for extended periods of time. In SFMC, the frequency of ACPA specific B cells is strongly increased. These observations point to a continuously active, ACPA specific immune response in RA.