Antigen-specific Fab profiling achieves molecular-resolution analysis of human autoantibody repertoires in rheumatoid arthritis.

The presence of autoantibodies is a defining feature of many autoimmune diseases. The number of unique autoantibody clones is conceivably limited by immune tolerance mechanisms, but unknown due to limitations of the currently applied technologies. Here, we introduce an autoantigen-specific liquid chromatography-mass spectrometry-based IgG1 Fab profiling approach using the anti-citrullinated protein antibody (ACPA) repertoire in rheumatoid arthritis (RA) as an example. We show that each patient harbors a unique and diverse ACPA IgG1 repertoire dominated by only a few antibody clones. In contrast to the total plasma IgG1 antibody repertoire, the ACPA IgG1 sub-repertoire is characterised by an expansion of antibodies that harbor one, two or even more Fab glycans, and different glycovariants of the same clone can be detected. Together, our data indicate that the autoantibody response in a prominent human autoimmune disease is complex, unique to each patient and dominated by a relatively low number of clones.

Beyond the surface - Autoreactive B cells in human autoimmune diseases.

BACKGROUND: The generation and persistence of autoreactive B and plasma cells is crucial to the pathogenesis of many human autoimmune diseases. Secreted autoantibodies frequently serve as biomarkers in clinical practice and, in some cases, function as pathogenic effector molecules. Nonetheless, the primary break of B cell tolerance against autoantigens, the triggers that maintain autoreactive B cell memory, and the phenotype that autoreactive B cells adopt during the disease course are poorly understood. OBJECTIVES: To study phenotype and functional characteristics of human autoreactive B cells in the course of human disease using rheumatoid arthritis and the B cell response against posttranslationally modified antigens as prototype. METHODS: Combinatorial, antigen-specific identification and multiparameter phenotyping of autoreactive B cells by conventional and spectral flow cytometry in cohorts with well-defined clinical phenotypes, including patients in the phase preceding disease and in those reaching long-term, drugfree remission. RESULTS: Autoreactive B cells against post-translationally modified proteins operate as remarkably activated effector memory cells in patients with established disease and maintain this state throughout the disease course. The activation generates cytokine-secreting germinal center emigrants that resist conventional therapy, and migratory plasmablasts expressing homing markers that can direct the cells to sites of inflammation. In the pre-clinical at-risk phase, the degree of activation is lower, and migratory plasmablasts are less frequent. The cells are cross-reactive to different posttranslational modifications and express B cell receptors that are extensively glycosylated in the variable domain. CONCLUSIONS: Immune phenotyping of disease-specific, autoreactive B cells reveals heterogeneous features of human autoimmunity that reflect disease stage and course and that are only revealed upon antigen-specific cellular analysis. In rheumatoid arthritis, the picture of germinal center-derived B cell autoreactivity against post-translationally modified antigens emerges that displays extensive cross-reactivity and a likely dependence on T cell help. Such features may be different for other human autoimmune diseases with different disease kinetics, which each may require different strategies for (autoreactive) B cell targeting.

A robust nanoscale RP HPLC-MS approach for sensitive Fc proteoform profiling of IgG allotypes.

The conserved region (Fc) of IgG antibodies dictates the interactions with designated receptors thus defining the immunological effector functions of IgG. Amino acid sequence variations in the Fc, recognized as subclasses and allotypes, as well as post-translational modifications (PTMs) modulate these interactions. Yet, the high similarity of Fc sequences hinders allotype-specific PTM analysis by state-of-the-art bottom-up methods and current subunit approaches lack sensitivity and face co-elution of near-isobaric allotypes. To circumvent these shortcomings, we present a nanoscale reversed-phase (RP) HPLC-MS workflow of intact Fc subunits for comprehensive characterization of Fc proteoforms in an allotype- and subclass-specific manner. Polyclonal IgGs were purified from individuals followed by enzymatic digestion releasing single chain Fc subunits (Fc/2) that were directly subjected to analysis. Chromatographic conditions were optimized to separate Fc/2 subunits of near-isobaric allotypes and subclasses allowing allotype and proteoform identification and quantification across all four IgG subclasses. The workflow was complemented by a semi-automated data analysis pipeline based on the open-source software Skyline followed by post-processing in R. The approach revealed pronounced differences in Fc glycosylation between donors, besides inter-subclass and inter-allotype variability within donors. Notably, partial occupancy of the N-glycosylation site in the CH3 domain of IgG3 was observed that is generally neglected by established approaches. The described method was benchmarked across several hundred runs and showed good precision and robustness. This methodology represents a first mature Fc subunit profiling approach allowing truly subclass- and allotype-specific Fc proteoform characterization beyond established approaches. The comprehensive information obtained paired with the high sensitivity provided by the miniaturization of the approach guarantees applicability to a broad range of research questions including clinically relevant (auto)antibody characterization or pharmacokinetics assessment of therapeutic IgGs.

Progression from suspected to definite systemic sclerosis and the role of anti-topoisomerase I antibodies.

INTRODUCTION: Early diagnosis of systemic sclerosis (SSc) is important to start therapeutic interventions timely. Important risk factors for progression to SSc are the SSc-specific autoantibodies, of whom anti-centromere antibodies (ACA) and anti-topoisomerase I antibodies (ATA) are the most frequent. ATA is associated with a severe disease course. A more detailed characterisation of the ATA-response in SSc might increase insights in preclinical disease stages and improve prognostication. To address this we identified all patients with suspected very early ATA-positive SSc, defined as all patients who are ATA-positive not fulfilling American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR) 2013 criteria, in the Leiden Combined Care in Systemic Sclerosis (CCISS)-cohort and found very low numbers. METHODS: This triggered us to search the literature on the ATA prevalence in patients with suspected very early SSc and contribution of the SSc-specific autoantibodies to progression from suspected very early to definite SSc. To increase insights on the ATA-response in suspected very early SSc, we then evaluated the association between the ATA-response and time between onset of Raynaud's phenomenon (RP) and first non-RP symptom, as a proxy for progressing to definite SSc, in all patients with ATA-positive SSc from the Leiden CCISS-cohort. RESULTS: In short, included studies show that prevalence of ATA is much lower in suspected very early SSc than in populations fulfilling ACR/EULAR 2013 criteria. After 1-15 years of follow-up, only 52% of the patients with suspected very early SSc progress to definite SSc. ATA-IgG levels tend to be higher in patients with ATA-positive SSc with more rapid disease progression. CONCLUSION: Although a role of ATA in disease progression is suggested, more studies on the ATA response in suspected very early SSc are warranted.

A subset of antibodies targeting citrullinated proteins confers protection from rheumatoid arthritis.

Although elevated levels of anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA), the in vivo functions of these antibodies remain unclear. Here, we have expressed monoclonal ACPAs derived from patients with RA, and analyzed their functions in mice, as well as their specificities. None of the ACPAs showed arthritogenicity nor induced pain-associated behavior in mice. However, one of the antibodies, clone E4, protected mice from antibody-induced arthritis. E4 showed a binding pattern restricted to skin, macrophages and dendritic cells in lymphoid tissue, and cartilage derived from mouse and human arthritic joints. Proteomic analysis confirmed that E4 strongly binds to macrophages and certain RA synovial fluid proteins such as α-enolase. The protective effect of E4 was epitope-specific and dependent on the interaction between E4-citrullinated α-enolase immune complexes with FCGR2B on macrophages, resulting in increased IL-10 secretion and reduced osteoclastogenesis. These findings suggest that a subset of ACPAs have therapeutic potential in RA.

Randomized phase I trial of antigen-specific tolerizing immunotherapy with peptide/calcitriol liposomes in ACPA+ rheumatoid arthritis.

BACKGROUNDAntigen-specific regulation of autoimmune disease is a major goal. In seropositive rheumatoid arthritis (RA), T cell help to autoreactive B cells matures the citrullinated (Cit) antigen-specific immune response, generating RA-specific V domain glycosylated anti-Cit protein antibodies (ACPA VDG) before arthritis onset. Low or escalating antigen administration under "sub-immunogenic" conditions favors tolerance. We explored safety, pharmacokinetics, and immunological and clinical effects of s.c. DEN-181, comprising liposomes encapsulating self-peptide collagen II259-273 (CII) and NF-κB inhibitor 1,25-dihydroxycholecalciferol.METHODSA double-blind, placebo-controlled, exploratory, single-ascending-dose, phase I trial assessed the impact of low, medium, and high DEN-181 doses on peripheral blood CII-specific and bystander Cit64vimentin59-71-specific (Cit-Vim-specific) autoreactive T cell responses, cytokines, and ACPA in 17 HLA-DRB1*04:01+ or *01:01+ ACPA+ RA patients on methotrexate.RESULTSDEN-181 was well tolerated. Relative to placebo and normalized to baseline values, Cit-Vim-specific T cells decreased in patients administered medium and high doses of DEN-181. Relative to placebo, percentage of CII-specific programmed cell death 1+ T cells increased within 28 days of DEN-181. Exploratory analysis in DEN-181-treated patients suggested improved RA disease activity was associated with expansion of CII-specific and Cit-Vim-specific T cells; reduction in ACPA VDG, memory B cells, and inflammatory myeloid populations; and enrichment in CCR7+ and naive T cells. Single-cell sequencing identified T cell transcripts associated with tolerogenic TCR signaling and exhaustion after low or medium doses of DEN-181.CONCLUSIONThe safety and immunomodulatory activity of low/medium DEN-181 doses provide rationale to further assess antigen-specific immunomodulatory therapy in ACPA+ RA.TRIAL REGISTRATIONAnzctr.org.au identifier ACTRN12617001482358, updated September 8, 2022.FUNDINGInnovative Medicines Initiative 2 Joint Undertaking (grant agreement 777357), supported by European Union's Horizon 2020 research and innovation programme and European Federation of Pharmaceutical Industries and Associations; Arthritis Queensland; National Health and Medical Research Council (NHMRC) Senior Research Fellowship; and NHMRC grant 2008287.

From risk to chronicity: evolution of autoreactive B cell and antibody responses in rheumatoid arthritis.

The presence of disease-specific autoantibody responses and the efficacy of B cell-targeting therapies in rheumatoid arthritis (RA) indicate a pivotal role for B cells in disease pathogenesis. Important advances have shaped our understanding of the involvement of autoantibodies and autoreactive B cells in the disease process. In RA, autoantibodies target antigens with a variety of post-translational modifications such as carbamylation, acetylation and citrullination. B cell responses against citrullinated antigens generate anti-citrullinated protein antibodies (ACPAs), which are themselves modified in the variable domains by abundant N-linked glycans. Insights into the induction of autoreactive B cells against antigens with post-translational modifications and the development of autoantibody features such as isotype usage, epitope recognition, avidity and glycosylation reveal their relationship to particular RA risk factors and clinical phenotypes. Glycosylation of the ACPA variable domain, for example, seems to predict RA onset in ACPA+ healthy individuals, possibly because it affects B cell receptor signalling. Moreover, ACPA-expressing B cells show dynamic phenotypic changes and develop a continuously proliferative and activated phenotype that can persist in patients who are in drug-induced clinical remission. Together, these findings can be integrated into a conceptual framework of immunological autoreactivity in RA, delineating how it develops and persists and why disease activity recurs when therapy is tapered or stopped.

Multifunctional, Multivalent PIC Polymer Scaffolds for Targeting Antigen-Specific, Autoreactive B Cells.

Multivalent scaffolds that carry multiple molecules with immunophenotyping or immunomodulatory properties are invaluable tools for studying and modulating specific functions of human immune responses. So far, streptavidin-biotin-based tetramers have been widely used for B-cell immunophenotyping purposes. However, the utility of these tetramers is limited by their tetravalency, the inherent immunogenicity of streptavidin (a bacterial protein that can potentially be recognized by B cells), and the limited feasibility to functionalize these reagents. This has rendered tetramers suboptimal for studying rare, in particular, antigen-specific B-cell populations in the context of clinical applications. Here, we used polyisocyanopeptides (PICs), multivalent polymeric scaffolds functionalized with around 50 peptide antigens, to detect autoreactive B cells in the peripheral blood of patients with rheumatoid arthritis. To explore the potential immunomodulatory functionalities, we functionalized PICs with autoantigenic peptides and a trisaccharide CD22 ligand to inhibit autoreactive B-cell activation through interference with the B-cell receptor activation pathway, as evidenced by reduced phospho-Syk expression upon PIC binding. Given the possibilities to functionalize PICs, our data demonstrate that the modular and versatile character of PIC scaffolds makes them promising candidates for future clinical applications in B-cell-mediated diseases.

Surface Ig variable domain glycosylation affects autoantigen binding and acts as threshold for human autoreactive B cell activation.

The hallmark autoantibodies in rheumatoid arthritis are characterized by variable domain glycans (VDGs). Their abundant occurrence results from the selective introduction of N-linked glycosylation sites during somatic hypermutation, and their presence is predictive for disease development. However, the functional consequences of VDGs on autoreactive B cells remain elusive. Combining crystallography, glycobiology, and functional B cell assays allowed us to dissect key characteristics of VDGs on human B cell biology. Crystal structures showed that VDGs are positioned in the vicinity of the antigen-binding pocket, and dynamic modeling combined with binding assays elucidated their impact on binding. We found that VDG-expressing B cell receptors stay longer on the B cell surface and that VDGs enhance B cell activation. These results provide a rationale on how the acquisition of VDGs might contribute to the breach of tolerance of autoreactive B cells in a major human autoimmune disease.

Autologous stem cell transplantation for progressive systemic sclerosis: a prospective non-interventional study from the European Society for Blood and Marrow Transplantation Autoimmune Disease Working Party.

Three randomized controlled trials in early severe systemic sclerosis demonstrated that autologous hematopoietic stem cell transplantation was superior to standard cyclophosphamide therapy. This European Society for Blood and Marrow Transplantation multi-center prospective non-interventional study was designed to further decipher efficacy and safety of this procedure for severe systemic sclerosis patients in real-life practice and to search for prognostic factors. All consecutive adult systemic sclerosis patients undergoing a first autologous hematopoietic stem cell transplantation between December 2012 and February 2016 were prospectively included in the study. Primary endpoint was progression free survival. Secondary endpoints were overall survival, non-relapse mortality, response and incidence of progression. Eighty systemic sclerosis patients were included. Median follow-up duration was 24 (6-57) months after stem cell transplantation using cyclophosphamide plus antithymocyte globulins conditioning for all, with CD34+ selection in 35 patients. At 2 years, progression free survival was 81.8%, overall survival was 90%, response was 88.7% and incidence of progression was 11.9%. The 100 days non-relapse mortality was 6.25% (n=5) with four deaths from cardiac event, including three due to cyclophosphamide toxicity. Modified Rodnan skin score and forced vital capacity improved with time (p< 0.001). By multivariate analysis, baseline skin score >24 and older age at transplant were associated with lower progression free survival (Hazard ration 3.32) and 1.77, respectively). CD34+-selection was associated with better response (Hazard ration: 0.46). This study confirms the efficacy of autologous stem cell transplantation in real-life practice for severe systemic sclerosis using non myeloablative conditioning. Careful cardio-pulmonary assessment to identify organ involvement at patient referral, reduced cyclophosphamide doses and CD34+ selection may improve outcomes. The study was registered at ClinicalTrials.gov: NCT02516124.

Association of Anti-Topoisomerase I Antibodies of the IgM Isotype With Disease Progression in Anti-Topoisomerase I-Positive Systemic Sclerosis.

OBJECTIVE: Anti-topoisomerase I (anti-topo I) autoantibodies in systemic sclerosis (SSc) are associated with diffuse skin involvement and interstitial lung fibrosis. Thus far, however, the relationship between anti-topo I antibody response and disease course has not yet been fully evaluated. This study was undertaken to gain insight into the association between characteristics of the anti-topo I antibody response and clinical disease course in SSc patients positive for anti-topo I antibodies. METHODS: Levels of anti-topo I IgG, anti-topo I IgM, and anti-topo I IgA were assessed in consecutive serum samples obtained from patients at baseline who were positive for anti-topo I IgG in the Leiden Combined Care In Systemic Sclerosis (CCISS) cohort. One-year disease progression was defined by a relevant increase in modified Rodnan skin thickness score (MRSS), decline in pulmonary function, development of digital ulcers, renal crisis, and pulmonary hypertension, and/or mortality. Validation was performed in SSc patients who were positive for anti-topo I from the Oslo University Hospital and University Hospital Zurich. RESULTS: Of the 103 patients with anti-topo I IgG in the CCISS cohort, clinical data were available to assess 1-year disease progression in 81 patients. Of these 81 patients, 23 (28%) had disease progression. At baseline, patients with disease progression were significantly more often anti-topo I IgM-positive than those who did not experience disease progression (21 [91%] of 23 versus 33 [57%] of 58; P < 0.01). This finding was confirmed in the independent validation samples. CONCLUSION: In SSc patients who were anti-topo I IgG-positive, presence of anti-topo I IgM, which might be considered as a surrogate for an ongoing autoreactive B cell immune response, is associated with disease progression.

A Comparison of Immunoglobulin Variable Region N-Linked Glycosylation in Healthy Donors, Autoimmune Disease and Lymphoma.

N-linked glycans play an important role in immunity. Although the role of N-linked glycans in the Fragment crystallizable (Fc) region of immunoglobulins has been thoroughly described, the function of N-linked glycans present in Ig-variable domains is only just being appreciated. Most of the N-linked glycans harbored by immunoglobulin variable domain are of the complex biantennary type and are found as a result of the presence of N-linked glycosylation that most often have been introduced by somatic hypermutation. Furthermore, these glycans are ubiquitously present on autoantibodies observed in some autoimmune diseases as well as certain B-cell lymphomas. For example, variable domain glycans are abundantly found by anti-citrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) as well as by the B-cell receptors of follicular lymphoma (FL). In FL, variable domain glycans are postulated to convey a selective advantage through interaction with lectins and/or microbiota, whereas the contribution of variable domain glycans on autoantibodies is not known. To aid the understanding how these seemingly comparable phenomena contribute to a variety of deranged B-responses in such different diseases this study summarizes the characteristics of ACPA and other auto-antibodies with FL and healthy donor immunoglobulins, to identify the commonalities and differences between variable domain glycans in autoimmune and malignant settings. Our finding indicate intriguing differences in variable domain glycan distribution, frequency and glycan composition in different conditions. These findings underline that variable domain glycosylation is a heterogeneous process that may lead to a number of pathogenic outcomes. Based on the current body of knowledge, we postulate three disease groups with distinct variable domain glycosylation patterns, which might correspond with distinct underlying pathogenic processes.

The etiology of rheumatoid arthritis.

Rheumatoid arthritis is a heterogeneous disease, which can be, based on data combining genetic risk factors and autoantibodies, sub-classified into ACPA-positive and -negative RA. Presence of ACPA and RF as well as rising CRP-levels in some patients years before onset of clinical symptoms indicate that relevant immune responses for RA development are initiated very early. ACPA are highly specific for RA, whereas RF can also be found among healthy (elderly) individuals and patients with other autoimmune diseases or infection. The most important genetic risk factor for RA development, the shared epitope alleles, resides in the MHC class II region. Shared epitope alleles, however, only predispose to the development of ACPA-positive RA. Smoking is thus far the most important environmental risk factor associated with the development of RA. Studies on synovitis have shown the importance not only of adaptive but also of innate immune responses. In summary of the various results from immunological changes in blood and synovial tissue, the extension of the immune response from a diffuse myeloid to a lympho-myeloid inflammation appears to be associated with a more successful therapeutic response to biologics. With respect to advances in synovitis research, new targets for treatment against pathological subsets of immune cells or fibroblasts are already on the horizon. However, alternative strategies involving the microbiome may play an important role as well and research in this field is growing rapidly.

On the presence of HLA-SE alleles and ACPA-IgG variable domain glycosylation in the phase preceding the development of rheumatoid arthritis.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) patients display a unique feature defined by the abundant presence of N-linked glycans within the variable domains (V-domains). Recently, we showed that N-glycosylation sites, which are required for the incorporation of V-domain glycans, are introduced following somatic hypermutation. However, it is currently unclear when V-domain glycosylation occurs. Further, it is unknown which factors might trigger the generation of V-domain glycans and whether such glycans are relevant for the transition towards RA. Here, we determined the presence of ACPA-IgG V-domain glycans in paired samples of pre-symptomatic individuals and RA patients. METHODS: ACPA-IgG V-domain glycosylation was analysed using ultra-high performance liquid chromatography (UHPLC) in paired samples of pre-symptomatic individuals (median interquartile range (IQR) pre-dating time: 5.8 (5.9) years; n=201; 139 ACPA-positive and 62 ACPA-negative) and RA patients (n=99; 94 ACPA-positive and 5 ACPA-negative). RESULTS: V-domain glycans on ACPA-IgG were already present up to 15 years before disease in pre-symptomatic individuals and their abundance increased closer to symptom onset. Noteworthy, human leucocyte antigen class II shared epitope (HLA-SE) alleles associated with the presence of V-domain glycans on ACPA-IgG. CONCLUSION: Our observations indicate that somatic hypermutation of ACPA, which results in the incorporation of N-linked glycosylation sites and consequently V-domain glycans, occurs already years before symptom onset in individuals that will develop RA later in life. Moreover, our findings provide first evidence that HLA-SE alleles associate with ACPA-IgG V-domain glycosylation in the pre-disease phase and thereby further refine the connection between HLA-SE and the development of ACPA-positive RA.

Fra1 Controls Rheumatoid Factor Autoantibody Production by Bone Marrow Plasma Cells and the Development of Autoimmune Bone Loss.

Next to proinflammatory cytokines, autoimmunity has been identified as a key trigger for osteoclast activation and bone loss. IgG-rheumatoid factor (IgG-RF) immune complexes, which are present in patients with rheumatoid arthritis, were shown to boost osteoclast differentiation. To date, the regulation of IgG-RF production in the absence of inflammatory triggers is unknown. Herein, we describe Fra1 as a key checkpoint that controls IgG-RF production by plasma cells and regulates autoimmune-mediated bone loss. Fra1 deficiency in B cells (Fra1ΔBcell ) led to increased IgG1-producing bone marrow plasma cells, enhanced IgG-RF production, and increased bone loss associated with elevated osteoclast numbers after immunization. The effect of IgG-RF on osteoclasts in vitro and on osteoclasts associated with bone loss in vivo was dependent on FcγR, especially FcγR3. Furthermore, immunization of WT mice with T-cell-dependent antigens induced a significant and robust decrease in Fra1 expression in bone marrow B cells, which was followed by increased IgG1 production and the induction of osteoclast-mediated bone loss. Overall, these data identify Fra1 as a key mediator of IgG-RF production and autoimmune-mediated bone loss. © 2019 American Society for Bone and Mineral Research.

Sequential Prodrug Strategy To Target and Eliminate ACPA-Selective Autoreactive B Cells.

Autoreactive B cells are thought to play a pivotal role in many autoimmune diseases. Rheumatoid arthritis (RA) is an autoimmune disease affecting ∼1% of the Western population and is hallmarked by the presence of anticitrullinated proteins antibodies (ACPA) produced by autoreactive B cells. We intend to develop a method to target and selectively eliminate these autoreactive B cells using a sequential antigen prodrug targeting strategy. As ACPA-expressing B cells are thought to play essential roles in RA-disease pathogenesis, we used this B cell response as a prototype to analyze the feasibility to generate a construct consisting of a biologically silenced, that is, blocked, antigen connected to a cytotoxic prodrug. Blocking of the antigen is considered relevant as it is anticipated that circulating autoantibodies will otherwise clear the antigen-prodrug before it can reach the target cell. The antigen-prodrug can only bind to the autoantigen-specific B cell receptor (BCR) upon enzymatic removal of the blocking group in close proximity of the B cell surface. BCR binding ultimately induces antigen-specific cytotoxicity after internalization of the antigen. We have synthesized a cyclic citrullinated peptide (CCP) antigen suitable for BCR binding and demonstrated that binding by ACPA was impaired upon introduction of a carboxy- p-nitrobenzyl (CNBz) blocking group at the side chain of the citrulline residue. Enzymatic removal of the CNBz moiety by nitroreductase fully restored citrulline-specific recognition by both ACPA and ACPA-expressing B cells and showed targeted cell death of CCP-recognizing B cells only. These results mark an important step toward antigen-specific B cell targeting in general and more specifically in RA, as successful blocking and activation of citrullinated antigens forms the basis for subsequent use of such construct as a prodrug in the context of autoimmune diseases.

To what extent do autoantibodies help to identify high-risk patients in systemic sclerosis?

OBJECTIVES: To evaluate the additive value of autoantibodies in identifying systemic sclerosis (SSc) patients with high complication risk. METHODS: Patients entering the Combined Care In SSc cohort, Leiden University Medical Centre between April 2009 and May 2016 were included. Subgroups of patients were determined using hierarchical clustering, performed on Principal Component Analysis scores, 1) using baseline data of demographic and clinical variables only and 2) with additional use of antibody status. Disease-risk within subgroups was assessed by evaluating 5-year mortality rates. Clinical and autoantibody characteristics of obtained subgroups were compared. RESULTS: In total 407 SSc patients were included, of which 91% (n=371) fulfilled ACR/EULAR 2013 criteria for SSc. Prevalences of autoantibodies were: anti-centromere 37%, anti-topoisomerase (ATA) 24%, anti-RNA polymerase III 5%, anti-fibrillarin 4% and anti-Pm/Scl 5%. Clinical cluster analysis identified 4 subgroups, with two subgroups showing higher than average mortality (resp. 17% and 7% vs. total group mortality of 4%). ATA-positivity ranged from 10 to 21% in low-risk groups and from 30 to 49% among high-risk groups. Adding autoantibody status to the cluster process resulted in 5 subgroups with 3 showing higher than average mortality. Still, 22% of ATA- positive patients were clustered into a low-risk subgroup, while the total number of patients stratified to a high-risk subgroup increased. CONCLUSIONS: Autoantibodies only partially contribute to risk-stratification and clinical subsetting in SSc. The current findings confirm that not all ATA-positive patients have worse prognosis and as such, additional biomarkers are needed to guide clinical follow-up in SSc.

The B cell response to citrullinated antigens in the development of rheumatoid arthritis.

The immune response to citrullinated antigens is found almost exclusively in patients with rheumatoid arthritis (RA). It is a dynamic response that expands before the onset of disease and generates antibodies (anti-citrullinated protein antibodies (ACPAs)) that are extensively glycosylated in the variable domain. This feature of ACPAs is remarkable and warrants detailed investigation, as it can offer insights into the earliest immunologic mechanisms that lead up to the development of RA. The acquisition of variable domain glycans, in fact, could enable ACPA-expressing B cells to breach tolerance. Although the underlying mechanisms are still elusive, data to support this concept are emerging, owing to the reliable identification and isolation of citrullinated antigen-directed B cells from patients with RA. This technological proficiency also allows for the generation of an increasing number of well-defined monoclonal ACPAs, and provides the opportunity to test and define the mechanisms by which the citrullinated antigen-directed B cell response contributes to the onset and persistence of inflammation. Together with a revised perception of the HLA-risk effect and novel insights into how T cells can govern antibody effector functions, these developments shape an increasingly clear picture of the B cell response to citrullinated antigens in the development of RA.