Khaled Amara

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.

Identification of a novel chemokine-dependent molecular mechanism underlying rheumatoid arthritis-associated autoantibody-mediated bone loss

Objectives Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and to identify key cellular mediators in this process. Methods Polyclonal ACPA were isolated from the synovial fluid (SF) and peripheral blood of patients with RA. Monoclonal ACPAs were isolated from single SF B-cells of patients with RA. OCs were developed from blood cell precursors with or without ACPAs. We analysed expression of citrullinated targets and peptidylarginine deiminases (PAD) enzymes by immunohistochemistry and cell supernatants by cytometric bead array. The effect of an anti-interleukin (IL)-8 neutralising antibody and a pan-PAD inhibitor was tested in the OC cultures. Monoclonal ACPAs were injected into mice and bone structure was analysed by micro-CT before and after CXCR1/2 blocking with reparixin. Results Protein citrullination by PADs is essential for OC differentiation. Polyclonal ACPAs enhance OC differentiation through a PAD-dependent IL-8-mediated autocrine loop that is completely abolished by IL-8 neutralisation. Some, but not all, human monoclonal ACPAs derived from single SF B-cells of patients with RA and exhibiting distinct epitope specificities promote OC differentiation in cell cultures. Transfer of the monoclonal ACPAs into mice induced bone loss that was completely reversed by the IL-8 antagonist reparixin. Conclusions We provide novel insights into the key role of citrullination and PAD enzymes during OC differentiation and ACPA-induced OC activation. Our findings suggest that IL8-dependent OC activation may constitute an early event in the initiation of the joint specific inflammation in ACPA-positive RA.

Glycosylation of immunoglobulin G determines osteoclast differentiation and bone loss

Immunglobulin G (IgG) sialylation represents a key checkpoint that determines the engagement of pro- or anti-inflammatory Fcγ receptors (FcγR) and the direction of the immune response. Whether IgG sialylation influences osteoclast differentiation and subsequently bone architecture has not been determined yet, but may represent an important link between immune activation and bone loss. Here we demonstrate that desialylated, but not sialylated, immune complexes enhance osteoclastogenesis in vitro and in vivo. Furthermore, we find that the Fc sialylation state of random IgG and specific IgG autoantibodies determines bone architecture in patients with rheumatoid arthritis. In accordance with these findings, mice treated with the sialic acid precursor N-acetylmannosamine (ManNAc), which results in increased IgG sialylation, are less susceptible to inflammatory bone loss. Taken together, our findings provide a novel mechanism by which immune responses influence the human skeleton and an innovative treatment approach to inhibit immune-mediated bone loss.

Autoantibodies to citrullinated proteins induce joint pain independent of inflammation via a chemokine-dependent mechanism

Objective An interesting and so far unexplained feature of chronic pain in autoimmune disease is the frequent disconnect between pain and inflammation. This is illustrated well in rheumatoid arthritis (RA) where pain in joints (arthralgia) may precede joint inflammation and persist even after successful anti-inflammatory treatment. In the present study, we have addressed the possibility that autoantibodies against citrullinated proteins (ACPA), present in RA, may be directly responsible for the induction of pain, independent of inflammation. Methods Antibodies purified from human patients with RA, healthy donors and murinised monoclonal ACPA were injected into mice. Pain-like behaviour was monitored for up to 28 days, and tissues were analysed for signs of pathology. Mouse osteoclasts were cultured and stimulated with antibodies, and supernatants analysed for release of factors. Mice were treated with CXCR1/2 (interleukin (IL) 8 receptor) antagonist reparixin. Results Mice injected with either human or murinised ACPA developed long-lasting pronounced pain-like behaviour in the absence of inflammation, while non-ACPA IgG from patients with RA or control monoclonal IgG were without pronociceptive effect. This effect was coupled to ACPA-mediated activation of osteoclasts and release of the nociceptive chemokine CXCL1 (analogue to human IL-8). ACPA-induced pain-like behaviour was reversed with reparixin. Conclusions The data suggest that CXCL1/IL-8, released from osteoclasts in an autoantibody-dependent manner, produces pain by activating sensory neurons. The identification of this new pain pathway may open new avenues for pain treatment in RA and also in other painful diseases associated with autoantibody production and/or osteoclast activation.

Naturally Occurring Human Phosphorylcholine Antibodies Are Predominantly Products of Affinity-Matured B Cells in the Adult

Phosphorylcholine (PC) is a classic T-independent Ag that is exposed on apoptotic cells, oxidized phospholipids, and bacterial polysaccharides. Experimental as well as epidemiological studies have over the past decade implicated Abs against PC (anti-PC) as anti-inflammatory and a strong protective factor in cardiovascular disease. Although clinically important, little is known about the development of anti-PC in humans. This study was conceived to dissect the human anti-PC repertoire and generate human mAbs. We designed a PC-specific probe to identify, isolate, and characterize PC-reactive B cells from 10 healthy individuals. The donors had all mounted somatically mutated Abs toward PC using a broad variety of Ig genes. PC-reactive B cells were primarily found in the IgM+ memory subset, although significant numbers also were detected among naive, IgG+, and CD27+CD43+ B cells. Abs from these subsets were clonally related, suggesting a common origin. mAbs derived from the same donors exhibited equivalent or higher affinity for PC than the well-characterized murine T-15 clone. These results provide novel insights into the cellular and molecular ontogeny of atheroprotective PC Abs, thereby offering new opportunities for Ab-based therapeutic interventions.

B cells expressing the IgA receptor FcRL4 participate in the autoimmune response in patients with rheumatoid arthritis

The clinical efficacy of B cell targeting therapies highlights the pathogenic potential of B cells in inflammatory diseases. Expression of Fc Receptor like 4 (FcRL4) identifies a memory B cell subset, which is enriched in the joints of patients with rheumatoid arthritis (RA) and in mucosa-associated lymphoid tissue. The high level of RANKL production by this B cell subset indicates a unique pathogenic role. In addition, recent work has identified a role for FcRL4 as an IgA receptor, suggesting a potential function in mucosal immunity. Here, the contribution of FcRL4+ B cells to the specific autoimmune response in the joints of patients with RA was investigated. Single FcRL4+ and FcRL4- B cells were sorted from synovial fluid and tissue from RA patients and their immunoglobulin genes characterized. Levels of hypermutation in the variable regions in both populations were largely consistent with memory B cells selected by an antigen- and T cell-dependent process. Recombinant antibodies were generated based on the IgH and IgL variable region sequences and investigated for antigen specificity. A significantly larger proportion of the recombinant antibodies generated from individual synovial FcRL4+ B cells showed reactivity towards citrullinated autoantigens. Furthermore, both in analyses based on heavy chain sequences and flow cytometric detection, FcRL4+ B cells have significantly increased usage of the IgA isotype. Their low level of expression of immunoglobulin and plasma cell differentiation genes does not suggest current antibody secretion. We conclude that these activated B cells are a component of the local autoimmune response, and through their RANKL expression, can contribute to joint destruction. Furthermore, their expression of FcRL4 and their enrichment in the IgA isotype points towards a potential role for these cells in the link between mucosal and joint inflammation.

A2.33 Citrullinated self antigen-specific blood B cells carry cross-reactive immunoglobulins with effector potential

Background and objectives Presence of autoantibodies targeting citrullinated proteins (ACPA) and clinical response from anti-CD20 Rituximab therapy indicate a critical role for B cells in rheumatoid arthritis (RA). Due to the difficulty of reliably tracking rare B cells with a defined specificity, the composition of the autoreactive B cell repertoire contributing to ACPA+ RA has not been analysed. We previously developed a tetramer enrichment strategy for capturing autoreactive B cells. We now employ cyclic citrullinated peptide (CCP) tetramers for cloning and expressing citrulline-specific immunoglobulin (Ig) from single blood B cells in RA. Materials and methods We analysed B cells specific for two previously described CCP, which are citrullinated epitopes from human filaggrin (Cfc1) and alpha-enolase (CEP-1). Antigen-specific B cells were detected by flow cytometry – with fluorescent CCP tetramer for citrulline-specific capture and non-citrullinated peptide decoy tetramer for distinguishing B cells that bind irrelevant epitopes on the tetramer complex. Tetramers were subsequently used to sort single B cells from RA patient (n = 3) blood. Reverse transcription-PCR generated Ig cDNA from the cells. Ig was amplified, cloned, and expressed as monoclonal antibody (mAb). Results Single B cells sorted by either CCP tetramer (Cfc1 or CEP-1) from RA patients’ peripheral blood have highly mutated Ig sequences and show preferential use of the VH4 gene family. When expressed as mAb, 9 of 25 tetramer-sorted clones demonstrated high reactivity towards the targeted CCP epitope, as opposed to 0 of 21 randomly selected memory B cells isolated from the same patient samples. All of the highly citrulline-specific clones further exhibited cross-reactivity to citrullinated epitopes from other proteins and unique binding patterns to these autoantigens on peptide array. The mAb generated from tetramer-selected cells also induced osteoclastogenesis and bone erosion in vitro. Conclusions Single B cells selected by CCP tetramers have highly mutated Ig sequences and receptors with citrulline-specific reactivity. Efficient isolation of mAb clones from the autoreactive B cell repertoire by this strategy will be used to better understand the pathogenesis of autoimmunity. By re-engineering the expressed Ig Fc domain towards blocking or immunosuppressive functions, mAb with defined disease-associated specificities could also offer intriguing possibilities in targeted therapeutic design for RA and other conditions.

A2.24 Plasma cell derived monoclonal anti-citrulline antibodies from ra synovial fluid are multireactive

Background and objectives Human 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 methods Spontaneously 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. Results We 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. Conclusions Only 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.

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.

Pathogenic Citrulline-Multispecific B Cell Receptor Clades in Rheumatoid Arthritis

Anti–citrullinated protein antibodies (ACPAs) have proven highly useful as biomarkers for rheumatoid arthritis (RA). However, composition and functionality of the associated autoreactive B cell repertoire have not been directly assessed. We aimed to selectively investigate citrullinated autoantigen–specific B cell receptors (BCRs) involved in RA and initiate studies on their pathogenicity.