A major subset of patients with rheumatoid arthritis (RA) is characterized by the presence of circulating autoantibodies directed to citrullinated proteins/peptides (ACPAs). These autoantibodies, which are commonly detected by using an enzyme-linked immunosorbent assay (ELISA) based on synthetic cyclic citrullinated peptides (CCPs), predict clinical onset and a destructive disease course. In the present study, we have used plasma and synovial fluids from patients with RA, for the affinity purification and characterization of anti-CCP2 reactive antibodies, with an aim to generate molecular tools that can be used in vitro and in vivo for future investigations into the pathobiology of the ACPA response. Specifically, this study aims to demonstrate that the surrogate marker CCP2 can capture ACPAs that bind to autoantigens expressed in vivo in the major inflammatory lesions of RA (that is, in the rheumatoid joint). Plasma (n = 16) and synovial fluid (n = 26) samples were collected from RA patients with anti-CCP2 IgG levels of above 300 AU/mL. Total IgG was isolated on Protein G columns and subsequently applied to CCP2 affinity columns. Purified anti-CCP2 IgG was analyzed for reactivity and specificity by using the CCPlus® ELISA, in-house peptide ELISAs, Western blot, and immunohisto-/immunocytochemistry. Approximately 2% of the total IgG pool in both plasma and synovial fluid was CCP2-reactive. Purified anti-CCP2 reactive antibodies from different patients showed differences in binding to CCP2 and differences in binding to citrullinated peptides from α-enolase, vimentin, fibrinogen, and collagen type II, illustrating different ACPA fine-specificity profiles. Furthermore, the purified ACPA bound not only in vitro citrullinated proteins but, more importantly, in vivo-generated epitopes on synovial fluid cells and synovial tissues from patients with RA. We have isolated ACPAs from plasma and synovial fluid and demonstrated that the CCP2 peptides, frequently used in diagnostic ELISAs, de facto act as surrogate antigens for at least four different, well-characterized, largely non-cross-reactive, ACPA fine specificities. Moreover, we have determined the concentration and proportion of CCP2-reactive IgG molecules in rheumatoid plasma and synovial fluid, and we have shown that the purified ACPAs can be used to detect both in vitro- and in vivo-generated citrullinated epitopes by various techniques. We anticipate that these antibodies will provide us with new opportunities to investigate the potential pathogenic effects of human ACPAs.
Autoimmunity in rheumatoid arthritis (RA) is characterised by autoantibodies to citrullinated proteins/peptides (ACPA). These antibodies, present in 60–70% of patients, antedate clinical onset and associate with an erosive disease course, suggesting a direct pathogenic involvement in disease initiation and progression. With this study, the authors aimed to develop an efficient method for the purification of human ACPA, and to characterise their frequency and fine-specificity pattern in synovial fluid (SF) and plasma of RA patients.
Materials and methods
SF and plasma samples were collected with informed consent and ethical approval from patients (fulfilling the American college of rheumatology criteria for RA) with high anti-CCP antibody levels. SF samples (n=36) were first treated with hyaluronidase to decrease viscosity, then proteins were precipitated with ammonium sulphate, dissolved and further dialysed against PBS, before the IgG fractions were purified on Protein G columns (GE Healthcare, Uppsala, Sweden).Plasma samples (n=10) were diluted in PBS before applied to the Protein G column. ACPAs were further purified using CCP2 affinity columns, kindly provided by Euro-Diagnostica. Recovery and purity of total IgG and anti-CCP immunoglobulin G (IgG) were analysed using SDS-PAGE, Nanodrop (Thermo Scientific, Wilmington, DE, USA) and the CCP2-ELISA kit. Fine-specificity of the purified ACPAs were investigated using inhouse ELISAs, with peptides from citrullinated α-enolase (CEP-1), -vimentin (Cit-vim), -fibrinogen (Cit-fib) and -collagen type II (Cit-C1).
Results
Anti-CCP IgG could efficiently be purified from SF and plasma, using ProteinG-, followed by CCP2-, columns. No CCP IgG response could be detected in the flow-through fractions. Higher concentrations of total IgG were found in plasma (13.6 mg/ml) compared to SF (4.2 mg/ml), while a higher percentage of CCP-specific IgG was detected in SF (3%), compared to plasma (2%). The purified anti-CCP IgG fractions cross-reacted with CEP-1, Cit-vim, Cit-fib and Cit-C1, while no reactivity to these citrullinated antigens were detected in the IgG flow-through fractions. Anti-CCP IgG dilution curves (starting at 10 µg/ml of purified antibodies), demonstrated differences in affinity between patients, which may correspond to the different ACPA-fine specificity patterns seen in patients.
Conclusions
The described methodology efficiently purifies ACPAs with multiple specificities, which will allow for their use in in vivo and in vitro studies, to further elucidate their arthritogenic and pathogenic capacity. In addition, the ACPAs will be tools for future immunoprecipitation-, immunoblotting- and immunohistochemistry experiments.
Human-induced pluripotent stem cells (hiPSCs) are generated from somatic cells by ectopic expression of the 4 reprogramming factors (RFs) Oct-4, Sox2, Klf4, and c-Myc. To better define the stoichiometric requirements and dynamic expression patterns required for successful hiPSC induction, we generated 4 bicistronic lentiviral vectors encoding the 4 RFs co-expressed with discernable fluorescent proteins. Using this system, we define the optimal stoichiometry of RF expression to be highly sensitive to Oct4 dosage, and we demonstrate the impact that variations in the relative ratios of RF expression exert on the efficiency of hiPSC induction. Monitoring of expression of each individual RF in single cells during the course of reprogramming revealed that vector silencing follows acquisition of pluripotent cell markers. Pronounced lentiviral vector silencing was a characteristic of successfully reprogrammed hiPSC clones, but lack of complete silencing did not hinder hiPSC induction, maintenance, or directed differentiation. The vector system described here presents a powerful tool for mechanistic studies of reprogramming and the optimization of hiPSC generation.
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.B cell clones from either the blood or synovial fluid of cyclic citrullinated peptide 2 (CCP2) antibody-positive RA patients were immortalized by genetic reprogramming with Bcl-6 and Bcl-xL. Enzyme-linked immunosorbent assay and flow cytometry were used to identify CCP2-reactive clones and to further characterize surface marker and cytokine expression as well as B cell receptor signaling competence. Global gene expression profiles were interrogated by RNA sequencing.Three unique CP-reactive memory B cell clones were generated from the blood or synovial fluid of 2 RA patients. CP-reactive memory B cells did not appear to be broadly cross-reactive, but rather had a fairly restricted epitope recognition profile. These clones were able to secrete both pro- and antiinflammatory cytokines and had a unique surface profile of costimulatory molecules and receptors, including CD40 and C5a receptor type 1, when compared to non-CP-reactive clones from the same patient. In addition, CP-reactive clones bound citrullinated protein, but not native protein, and could mobilize calcium in response to antigen binding.CP-reactive memory B cells comprise a rare, seemingly oligoclonal population with restricted epitope specificity and distinct phenotypic and molecular characteristics suggestive of antigen-presenting cells. Cloning by genetic reprogramming opens new avenues to study the function of autoreactive memory B cells, especially in terms of antigen processing, presentation, and subsequent T cell polarization.
In addition to anti-citrullinated protein antibodies (ACPAs), antibodies targeting carbamylated (i.e., homocitrullinated) proteins (anti-CarP antibodies) have been described in rheumatoid arthritis (RA). However, the extent to which anti-CarP antibodies are truly distinct from ACPA remains unclear, and few studies have focused on specific autoantigens. Here, we examine cross-reactivity between ACPA and anti-CarP antibodies, in the context of the candidate autoantigen α-enolase. Cross-reactivity was examined by immunoblotting of citrullinated and carbamylated proteins using purified ACPA; and by peptide absorption experiments, using the citrullinated α-enolase peptide CEP-1 and a homocitrulline-containing version (carb-CEP-1) in ELISA. The population-based case-control cohort EIRA (n = 2836 RA; 373 controls) was screened for reactivity with CEP-1 and carb-CEP-1, using the ISAC multiplex array. Associations between anti-CarP antibodies, smoking and genetic risk factors were analysed using unconditional logistic regression models. Differences in antibody levels were investigated using the Mann-Whitney U test. Affinity-purified ACPA was found to bind carbamylated proteins and homocitrulline-containing peptides, demonstrating definitive cross-reactivity between ACPA and anti-CarP antibodies. Anti-carb-CEP-1 reactivity in EIRA was almost exclusively confined to the CEP-1-positive subset, and this group of RA patients (21 %) displayed a particularly strong ACPA response with marked epitope spreading. The small RA subset (3 %) with homocitrulline reactivity in the absence of citrulline reactivity did not associate with smoking or risk genes, and importantly had significantly lower anti-carb-CEP-1 antibody levels. Our data presented herein cast doubt on the specificity of anti-CarP antibodies in RA, which we posit may be a subset of cross-reactive ACPA.
Abstract Background Rheumatoid arthritis (RA) is classified as seropositive or seronegative, depending on the presence/absence of rheumatoid factor (RF), primarily IgM RF, and/or anti-citrullinated protein antibodies (ACPA), commonly detected using anti-cyclic citrullinated peptide (CCP) assays. Known risk factors associate with the more severe seropositive form of RA; less is known about seronegative RA. Here, we examine risk factors and clinical phenotypes in relation to presence of autoantibodies in the RA subset that is traditionally defined as seronegative. Methods Anti-CCP2 IgG, 19 ACPA fine-specificities, IgM/IgG/IgA RF, anti-carbamylated-protein (CarP) antibodies, and 17 other autoantibodies, were analysed in 2755 RA patients and 370 controls. Antibody prevalence, levels, and co-occurrence were examined, and associations with risk factors and disease activity during 5 years were investigated for different antibody-defined RA subsets. Results Autoantibodies were detected in a substantial proportion of the traditionally defined seronegative RA subset, with ACPA fine-specificities found in 30%, IgA/IgG RF in 9.4%, and anti-CarP antibodies in 16%, with a 9.6% co-occurrence of at least two types of RA-associated autoantibodies. HLA-DRB1 shared epitope (SE) associated with the presence of ACPA in anti-CCP2-negative RA; in anti-CCP2-positive RA, the SE association was defined by six ACPA fine-specificities with high co-occurrence. Smoking associated with RF, but not with ACPA, in anti-CCP2-negative RA. Presence of ACPA and RF, but not anti-CarP antibodies, in conventionally defined “seronegative” RA, associated with worse clinical outcome. Conclusions “Seronegative” RA is not truly a seronegative disease subset. Additional screening for ACPA fine-specificities and IgA/IgG RF defines a group of patients that resembles seropositive patients with respect to risk factors and clinical picture and may contribute to earlier diagnosis for a subset of anti-CCP2−/IgM RF− patients with a high need for active treatment.
