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

B cell autoimmunity and bone damage in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic immune-inflammatory disease associated with significant bone damage. Pathological bone remodeling in RA is primarily driven by persistent inflammation. Indeed, pro-inflammatory cytokines stimulate the differentiation of bone-resorbing osteoclasts and, in parallel, suppress osteoblast function, resulting in net loss of bone. Abating disease activity thus remains the major goal of any treatment strategy in patients with RA. Autoantibody-positive patients, however, often show a rapidly progressive destructive course of the disease, disproportionate to the level of inflammation. The epidemiological association between RA-specific autoantibodies, in particular anti-citrullinated protein autoantibodies, and poor structural outcomes has recently found mechanistic explanation in the multiple roles that B cells play in bone remodeling. In this review, we will summarize the substantial progress that has been made in deciphering how B cells and autoantibodies negatively impact on bone in the course of RA, through both inflammation-dependent and independent mechanisms

Past infections and low ACPA in RA

Background : Rheumatoid arthritis (RA), an autoimmune disease of unknown etiology, is believed to occur as the result of actions of genetic and environmental factors. In this study, we examined the relation of past histories about infectious diseases with the levels anti-citrullinated protein autoantibodies (ACPA) in RA. Methods : Results of a questionnaire about histories of infectious diseases were obtained from 85 patients with RA, and were analyzed. Results : Significantly lower level of ACPA was detected in patients with the history of tonsillitis, otitis media or urinary cystitis than in those without it. There was no difference in the level of ACPA in RA patients between with and without cold / influenza, rubella, chickenpox, herpes labialis or herpes zoster. When RA patients were divided into two groups, high-level and low-level ACPA, multiple logistic regression analysis revealed that the history of otitis media was a significantly independent factor for the low level of ACPA. There was no significant relation between the level of rheumatoid factor and histories of infectious diseases. Conclusion : This study clarified that the past history of otitis media is associated with the low level of ACPA in RA

Improved RA classification among early arthritis patients with the concordant presence of three RA autoantibodies: Analysis in two early arthritis clinics

Background: The patients with RA benefit from early identification soon after the first clinical symptoms appear. The 2010 ACR/EULAR classification criteria were developed to fulfill this need and their application has been demonstrated to be effective. However, there is still room for improvement. Therefore, we aimed to evaluate the potential of the concordant presence of RF, anti-CCP and anti-carbamylated protein antibodies to improve current RA classification among early arthritis (EA) patients. Methods: Data from the first visit of 1057 patients in two EA prospective cohorts were used. The serological scores from the 2010 ACR/EULAR criteria and the concordant presence of the three RA autoantibodies were assessed relative to a gold standard consisting of the RA classification with the 1987 ACR criteria at the 2 years of follow-up. Results: The concordant presence of three antibodies showed predictive characteristics allowing for direct classification as RA (positive predictive value = 96.1% and OR = 80.9). They were significantly better than the corresponding to the high antibody titers defined as in the 2010 classification criteria (PPV = 88.8%, OR = 26.1). In addition, the concordant presence of two antibodies was also very informative (PPV = 82.3%, OR = 15.1). These results allowed devising a scoring system based only on antibody concordance that displayed similar overall performance as the serological scoring system of the 2010 criteria. However, the best classification was obtained combining the concordance and 2010 serological systems, a combination with a significant contribution from each of the two systems. Discussion: The concordant presence of RA autoantibodies showed an independent contribution to the classification of EA patients that permitted increased discrimination and precision.This work was supported by the Instituto de Salud Carlos III (Spain) through grants [PI17/01606 and RD16/0012/0014 to AG; RD16/0012/0011 to IG-A and RD16/0012/0012 to AB]. These grants are partially financed by the European Regional Development Fund of the EU (FEDER). CR was supported by Ministerio de Educacion Cultura y Deporte (Spain) through a FPU pre-doctoral fellowship [FPU15/03434]. LR-M was supported by a Xunta de Galicia predoctoral contract

Diversity and function of anti-modified protein autoantibodies in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease particularly affecting synovial joints. Anti-citrullinated protein autoantibodies (ACPA) are detected in the serum of about 2/3 of RA patients and are being used to classify the disease. These autoantibodies may occur years before any signs of arthritis which implies that they are a cause rather than a consequence of disease. By using modified autoantigens to detect ACPAs in large patient cohorts and by purifying polyclonal autoantibodies from patients, the hunt for disease promoting autoantigens has been going on for decades. However, the relationship between ACPA specificity and any functional effects remains unclear. In order to understand the evolution, specificity, and function of autoreactive B cells in RA, the focus of this thesis is the generation of monoclonal antibodies (mAbs) from paired variable heavy- and light-chain immunoglobulin (Ig) sequences from identified single B cells from various RA tissue compartments. We have identified citrulline-reactive autoantibody producing plasma cells in the synovium of RA patients with established disease. By generating single plasma cell derived mAbs, we learned that such autoantibodies may be directly involved in the pathogenesis of RA by promoting bone degrading osteoclasts. The mAbs were all multireactive to citrulline-peptides and citrullinated proteins, but with unique distinct binding patterns. We recognized glycine in +1 position to the citrulline and a fraction of the citrulline-reactive mAbs cross-reacted with carbamyl-peptides. In addition, the identified plasma cells displayed features of high somatic mutations and fragment antigen binding (Fab) variable N-glycosylation sites introduced by affinity maturation. By analyzing a selection of RA patient B cell derived mAbs for reactivity against apoptotic cells and activated neutrophils, we learned that a subset of the citrulline reactive mAbs bound nuclear antigens. Interestingly, a fraction of these mAbs could target nuclear histones independently of the citrullinating enzyme PAD by binding to acetylated histones. We explored the extent of the ACPA mAb multireactivity and cross-reactivity by acknowledging the importance of neighboring amino acids in addition to glycine in +1 to the citrulline. By analyzing the bone marrow plasma cell repertoire of RA patients, we observed differences in Ig-frequencies and variable Fab N-linked glycosylation sites between ACPA+ and ACPA- patients. We also found RA patient bone marrow plasma cell clonotypes. In addition, we identified citrulline-reactive bone marrow plasma cells that could bind activated neutrophils which strengthen previous reports of citrullinated histones as ACPA targets. Lastly, we identified autoantibodies against the oxidation-induced post translational protein modification adducts malondialdehyde (MDA) and malondialdehyde-acetaldehyde (MAA) in the bone marrow and lung of RA patients and individuals that also harbor ACPAs. A majority of these autoantibodies, that can promote osteoclastogenesis, need cross-linked MAA-protein for recognition independent of protein backbone. Taken together, the generation of RA patient single B cell derived mAbs, have revealed remarkable features of the autoreactivity that increases the understanding of B cell involvement in the pathogenesis of RA

Arthritis autoantibodies in individuals without rheumatoid arthritis:follow-up data from a Dutch population-based cohort (Lifelines)

Objectives. To assess whether the presence of arthritis autoantibodies alongside IgG ACPA predicts clinically suspect arthralgia in ACPA-positive subjects without RA. Methods. In the population-based Lifelines cohort (n=40 136), 308 IgG ACPA-positive individuals without RA were present. Serum levels of IgA ACPA, IgA and IgM RF, and IgG anti-carbamylated antibodies were measured at baseline. Individuals were divided based on the Connective tissue disease Screening Questionnaire after 2 years follow-up. Antibodies to Porphyromonas gingivalis were determined at baseline and related to presence of periodontitis and joint complaints at 2 years follow-up. Results. Of 308 subjects 53.6% were also seropositive for IgA ACPA, 42.2% for IgM RF, 23.7% for IgA RF and 13.6% for anti-carbamylated antibodies. We defined 75 persons with clinically suspect arthralgia at risk for RA based on CTD Screening Questionnaire at follow-up. Significantly more seropositivity for IgM RF and higher levels of IgG ACPA, IgA ACPA and IgM RF were found in clinically suspect arthralgia compared with no-clinically suspect arthralgia. In multivariate logistic regression correcting for age, gender and never smoking, positivity for three or more extra autoantibodies was significantly associated with clinically suspect arthralgia. Although levels of anti-P. gingivalis were not different between groups, they were significantly correlated to levels of both RFs, and both ACPAs in clinically suspect arthralgia. Conclusions. ACPA-positive individuals without RA who develop clinically suspect arthralgia have more and higher levels of other arthritis autoantibodies at baseline. Levels of anti-P. gingivalis are not related to self-reported periodontitis or clinically suspect arthralgia, but are correlated to arthritis autoantibodies in clinically suspect arthralgia

Autoantibodies in the Pathogenesis, Diagnosis, and Prognosis of Juvenile Idiopathic Arthritis

Autoantibody production occurs in juvenile idiopathic arthritis (JIA) and numerous other autoimmune diseases. In some conditions, the autoantibodies are clearly pathogenic, whereas in others the roles are less defined. Here we review various autoantibodies associated with JIA, with a particular focus on antinuclear antibodies and antibodies recognizing citrullinated self-antigens. We explore potential mechanisms that lead to the development of autoantibodies and the use of autoantibody testing in diagnosis and prognosis. Finally, we compare and contrast JIA-associated autoantibodies with those found in adults with rheumatoid arthritis (RA)

Efficacy and safety of NI-0101, an anti-toll-like receptor 4 monoclonal antibody, in patients with rheumatoid arthritis after inadequate response to methotrexate: a phase II study

Objectives Anti-citrullinated protein antibodies (ACPAs) form immune complexes with citrullinated proteins binding toll-like receptor (TLR) 4, which has been proposed as a mediator of rheumatoid arthritis (RA). NI-0101 is a first-in-class humanised monoclonal antibody blocking TLR4, as confirmed by inhibition of in vivo lipopolysaccharide-induced cytokine release in healthy volunteers. This study was design to confirm preclinical investigations supporting a biomarker-driven approach for treatment of patients with RA who present positive for these immune complexes. Methods Placebo-controlled, double-blind, randomised (2:1) trial of the tolerability and efficacy of NI-0101 (5 mg/kg, every 2 weeks for 12 weeks) versus placebo in ACPA-positive RA patients with inadequate response to methotrexate. Efficacy measures included Disease Activity Score (28-joint count) with C reactive protein (DAS28-CRP), European League Against Rheumatism (EULAR) good and moderate responses, and American College of Rheumatology (ACR) 20, ACR50 and ACR70 responses. Subgroup analyses defined on biomarkers were conducted. Pharmacokinetics, pharmacodynamics and safety were reported. Results 90 patients were randomised (NI-0101 (61) and placebo (29)); 86 completed the study. No significant between-group difference was observed for any of the efficacy endpoints. Subgroup analyses using baseline parameters as covariants did not reveal any population responding to NI-0101. Treatment-emergent adverse events occurred in 51.7% of patients who received placebo versus 52.5% for NI-0101. Conclusions We demonstrate for the first time that in RA, a human immune-mediated inflammatory disease, blocking the TLR4 pathway alone does not improve disease parameters. Successful targeting of innate immune pathways in RA may require broader and/or earlier inhibitory approaches