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.

The rheumatoid arthritis synovial fluid citrullinome reveals novel citrullinated epitopes in apolipoprotein E, myeloid nuclear differentiation antigen, and ß-actin.

OBJECTIVE: To generate a catalog of citrullinated proteins that are present in the synovia of patients with rheumatoid arthritis (RA) and to elucidate their relevance for the anti-citrullinated protein antibody response in RA. METHODS: Polypeptides isolated from the synovial fluid of patients with RA were identified by mass spectrometry. Three proteins (apolipoprotein E [Apo E], myeloid nuclear differentiation antigen [MNDA], and ß-actin) were studied in more detail, using immunoprecipitation and Western blotting. The presence of autoantibodies to synthetic peptides derived from these proteins in sera from patients with RA, sera from patients with other diseases, and sera from healthy control subjects was studied by enzyme-linked immunosorbent assay (ELISA). RESULTS: RA synovial fluid samples displayed several distinct patterns of citrullinated proteins. Using mass spectrometry, (fragments of) 192 proteins were identified, including 53 citrullinated proteins, some of which contained multiple citrullinated residues. In addition to previously reported citrullinated proteins in RA synovia (e.g., vimentin and fibrinogen), a series of novel citrullinated proteins, including Apo E, MNDA, ß-actin, and cyclophilin A, was identified. Immunoprecipitation experiments confirmed the citrullination of Apo E and MNDA. ELISAs demonstrated the presence of autoreactive citrullinated epitopes in Apo E, MNDA, and ß-actin. CONCLUSION: Synovial fluid samples from the inflamed joints of patients with RA contain many citrullinated proteins. Citrullinated Apo E, MNDA, and ß-actin are novel antigens identified in RA synovial fluid, and only a limited number of their citrullinated epitopes are targeted by the immune system in RA.

Identifying therapeutic monoclonal antibodies using target protein collision electrophoresis reflex assay to separate the wheat from the chaff.

Monoclonal gammopathies are characterized by the presence of monoclonal immunoglobulins, also known as M-proteins. Therapeutic monoclonal antibodies (t-mAbs) can interfere in laboratory assays used to monitor the state of disease, such as serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE). To establish a correct interpretation of IFE, Target protein-Collision Immunofixation Electrophoresis Reflex Assay (T-CIERA) was developed to identify t-mAbs in IFE. Here we demonstrate that T-CIERA is applicable to a wide variety of t-mAbs for which the target protein is commercially available. Moreover, the shift observed was characteristic for each t-mAb, and T-CIERA enabled the identification of multiple t-mAbs sharing a common target protein. Additionally, the lower limit of detection (LLOD) was determined objectively, and T-CIERA demonstrated an adequate LLOD for all tested t-mAbs. Furthermore, T-CIERA was also successfully applied to serum samples obtained from patients receiving daratumumab, isatuximab, elotuzumab, and durvalumab treatment. In conclusion, T-CIERA is a suitable reflex assay for identifying a wide variety of t-mAbs, including those for which no commercial assay is available to deal with their interference. Moreover, CD38-CIERA could serve as an alternative or complementary test to the commercially available Hydrashift assay kits. T-CIERA would enable laboratories without mass spectrometry equipment and expertise in this area to distinguish between drug and disease to improve clinical response monitoring and diagnosis of monoclonal gammopathies.

Anti-Sm antibodies in the classification criteria of systemic lupus erythematosus.

Systemic lupus erythematosus is characterized by autoantibodies and immune complex deposition. Several autoantibodies against mainly nuclear autoantigens have been described. One of these nuclear autoantigens is the Smith antigen. In this review, we focus on the position of autoantibodies against the Smith antigen in the classification criteria, the characteristics of the antigen, the production of anti-Smith antibodies in SLE and we discuss the different test methods available, together with their pitfalls, to detect these autoantibodies.

Treatment of Autoimmune Diseases with Therapeutic Antibodies: Lessons Learned from PID Patients Allow for Stratification of the Infection Risk.

Over the years, a wide variety of therapeutic antibodies has been successfully introduced in the autoimmunology clinic and many more are on the edge to follow. Many of these treatments address either a pathogenic circulating molecule or a cell-bound molecule. Whereas the former target results in neutralization of the soluble factor, the latter target either inhibits cellular function or induces selective cell death. If this targeted molecule or cell is part of the immune system, this therapy evokes a state of immunodeficiency. Knowing the exact function of the respective components enables the risk stratification for possible infectious complications in patients treated with biologics. Much of the understanding of the function of immune cells and their associated molecules, in relation to redundancy in the immune system, is derived from studies in knockout mice. However, as mice are not men in terms of their life-expectancy, their infection exposure, or the composition of their immune system, the most useful knowledge for estimating the consequence of therapeutic intervention on immune competence comes from monitoring patients. In the current chapter, we focus on patients with a primary immunodeficiency (PID) because they provide us with a unique perspective to estimate the redundancy of a certain genetic defect for overall immune competence. These patients have inborn errors of the immune system that, in general, are due to single gene defects. Depending on the immunological pathway that is defective, patients can present with different types of (opportunistic) infectious diseases, as well as other clinical manifestations. Based on selected examples, we focus in this chapter on finding parallels in the infectious risk of autoimmune patients treated with biologics and PID patients with a defect in the immunological pathway that is affected by the respective biologic. The goal is to learn from the (dis)similarities between both patient populations in terms of safety profiles of biologic treatments.

The Role of Autoantibodies in the Diagnosis of Autoimmune Liver Disease: Lessons Learned from Clinical Practice.

BACKGROUND: Primary biliary cholangitis (PBC), autoimmune hepatitis (AIH) and primary sclerosing cholangitis (PSC) are autoimmune liver diseases associated with distinct autoantibodies. Diagnosis is based upon clinical, serological, and histopathology findings. The role of autoantibodies in the diagnosis of these autoimmune liver diseases, with the focus on PBC and AIH, will be discussed. CONTENT: When AIH or PBC is suspected, testing for multiple autoantibodies can be requested. In this mini-review, the different ways in which autoantibodies can be tested (indirect immunofluorescence and antigen-specific tests) in the context of PBC and AIH are discussed, as well as the pitfalls in interpreting the test results. SUMMARY: For appropriate interpretation of test results, an important prerequisite is that the doctor knows which test is used in the laboratory of choice and that the laboratory specialist is aware of what the doctor wants to test for. Good communication between clinician and laboratory specialist can, therefore, aid in the diagnosis of autoimmune liver diseases.

B-Cell Analysis for Monitoring Patients Undergoing B-Cell Depletion for the Treatment of Autoimmune Diseases.

B-cell depleting therapy is increasingly used in the treatment of many distinct autoimmune diseases. This not only involves remission induction therapy, but also maintenance therapy. In this respect, it is of importance to monitor composition of the B-cell compartment in the peripheral blood. This can be performed at the time of initiation of the therapy, especially in those cases in which the expected clinical effect is not achieved. If B-cells are absent, B-cell depletion may not be the best treatment option; if B-cells are present, the efficacy may be hampered by neutralizing antibodies. For monitoring B-cell recovery it is important not to just enumerate B-cells, but to also phenotype the B-cells. A phenotype of IgD-CD27++CD38++ indicates the presence of circulating plasmablasts that lack CD20 and which are therefore not sensitive for B-cell depletion with anti-CD20 biologicals. A phenotype of IgD+CD27-CD38++ on the other hand, indicates recovery from the bone marrow with transitional B-cells. This chapter will focus on B-cell analyses by flow cytometry.

Immune Monitoring upon Treatment with Biologics in Sjögren's Syndrome: The What, Where, When, and How.

Over the years, a wide variety of therapeutic antibodies has been successfully introduced in the auto-immunology clinic, and many more are on the way. Many of these treatments address either a pathogenic circulating molecule or a cell-bound molecule. Whereas addressing the former target results in neutralization of the soluble factor and binding to the latter target either inhibits cellular function or induces selective cell death. If this targeted molecule or cell is part of the immune system, this therapy evokes a state of immunodeficiency with infections as a possible consequence. Therefore, immune monitoring is needed to prevent such adverse side effects of immunotherapy. In this paper, different immunotherapies used in Sjögren's syndrome, as well as different approaches to monitoring the immune system, are discussed.

Evaluation of the diagnostic performance of an immunoblot for ANCA and anti-GBM antibody detection.

The use of high-quality antigen-specific immunoassays for detecting anti-neutrophil cytoplasmic antibodies (ANCA) and anti-glomerular basement membrane (GBM) autoantibodies is recommended in patients with suspected ANCA vasculitis and/or anti-GBM disease. We analysed the diagnostic performance of a semi-quantitative and rapid immunoblot (EUROIMMUN AG, Lübeck, Germany) in two settings. Patient sera from different cohorts (ANCA vasculitis n = 187, anti-GBM disease n = 19, and disease controls n = 51) were used. The diagnostic performance of the immunoblot was assessed when used as a confirmatory test for the presence of ANCA in suspected ANCA vasculitis and when evaluating the presence of ANCA and/or anti-GBM antibodies in AAV and/or anti-GBM disease patients with a rapidly progressive glomerulonephritis (RPGN). In a confirmatory test setting, the immunoblot had an optimal sensitivity and specificity of 97.4 and 98.1% for PR3-ANCA and 98.5 and 96.4% for MPO-ANCA, respectively. With increasing test result ranges, a higher interval likelihood ratio (LR) was found for both ANCA entities. When evaluating for ANCA in patients with RPGN, the highest diagnostic accuracy (sensitivity 92.9% and specificity 100%) was obtained by using different cut-off values of positivity for PR3- (>5) and MPO-ANCA (>10). Also, the diagnostic performance for detecting anti-GBM was good (sensitivity 100% and specificity 100%). There are advantages over other assays in terms of time, costs, and interpretation of results. The immunoblot is a useful addition to current guidelines, particularly when a rapid diagnosis is necessary.

Performance analysis of automated evaluation of antinuclear antibody indirect immunofluorescent tests in a routine setting.

PURPOSE: Indirect immunofluorescence (IIF) on the human epithelial cell-line HEp-2 (or derivatives) serves as the gold standard in antinuclear antibody (ANA) screening. IIF, and its evaluation, is a labor-intensive method, making ANA testing a major challenge for present clinical laboratories. Nowadays, several automated ANA pattern recognition systems are on the market. In the current study, the EUROPattern Suite is evaluated for its use in daily practice in a routine setting. METHODS: A total of 1033 consecutive routine samples was used to screen for ANA. Results (positive/negative ANA screening, pattern identification and titer) were compared between software-generated results (EUROPattern) and visual interpretation (observer) of automatically acquired digital images. RESULTS: Considering the visual interpretation as reference, a relative sensitivity of 99.3% and a relative specificity of 88.9% were obtained for negative and positive discrimination by the software (EPa). A good agreement between visual and software-based interpretation was observed with respect to pattern recognition (mean kappa: for 7 patterns: 0.7). Interestingly, EPa software distinguished more patterns per positive sample than the observer (on average 1.5 and 1.2, respectively). Finally, a concordance of 99.3% was observed within the range of 1 titer step difference between EPa and observer. CONCLUSIONS: The ANA IIF results reported by the EPa software are in very good agreement with the results reported by the observer with respect to being negative/positive, pattern recognition and titer, making automated ANA IIF evaluation an objective and time-efficient tool for routine testing.

Peptidylarginine deiminase expression and activity in PAD2 knock-out and PAD4-low mice.

Citrullination, the conversion of peptidylarginine to peptidylcitrulline is catalyzed by peptidylarginine deiminases (PAD). The expression of PAD isoforms displays great variation among different tissues as demonstrated by PAD mRNA analyses. Here we have analyzed the differential expression of PAD2, PAD4 and PAD6 in mouse tissues at the protein level and by enzymatic activity assays using PAD2 and PAD4 knock-out strains. As expected, no PAD2 expression was detected in the PAD2-/- mice. In contrast, the PAD4 protein was observed in several tissues of the PAD4 knock-out mice, albeit at reduced levels in most tissues, and are therefore referred to as PAD4-low mice. In material from PAD2-/- mice, except for leukocyte lysates, hardly any PAD activity was found and no citrullinated proteins were detected after incubation in the presence of calcium. PAD activity in the PAD4-low mice was similar to that in wild-type mice. In both PAD knock-out strains the expression of PAD6 appeared to be up-regulated in all tissues analyzed, with the exception of spleen and testis. Our data demonstrate that the PAD2 protein is expressed in brain, spinal cord, spleen, skeletal muscle and leukocytes, but not detectably in liver, lung, kidney and testis. PAD4 was detected in each of these tissues, although the expression levels varied. In all tissues where PAD2 was detected, except for blood cells, this PAD isoform appeared to be responsible for virtually all peptidylarginine deiminase activity.

ACPA fine-specificity profiles in early rheumatoid arthritis patients do not correlate with clinical features at baseline or with disease progression.

INTRODUCTION: Autoantibodies against citrullinated peptides/proteins (ACPA) are found in approximately 75% of the sera of patients with rheumatoid arthritis (RA). The RA-specific ACPA are frequently present prior to disease onset and their presence associates with a more erosive disease course. ACPA can therefore be used to aid the diagnosis and prognosis of RA. Recently, it became clear that ACPA are very heterogeneous, both in an individual patient and among different patients. The aim of this study was to investigate whether clinically meaningful ACPA profiles exist in early RA patients. METHODS: Twenty citrullinated peptides and the corresponding non-citrullinated control peptides were immobilized on microarray sensor chips. Sera from 374 early arthritis patients were analyzed by surface plasmon resonance imaging (iSPR) of biomolecular interactions on the sensor chip. RESULTS: Cluster analysis of the reactivities with the citrullinated peptides, after subtraction of the reactivities with the corresponding control peptides confirmed the heterogeneity of the ACPA response in RA and revealed 12 distinct ACPA profiles. The association of the 5 most frequent profiles with clinical features at diagnosis and during the disease course was examined, showing no statistically significant associations. CONCLUSIONS: Compared to the detection of ACPA in RA sera by CCP-based assays, ACPA profiling in early arthritis patients did not reveal associations with disease activity and progression scores.

Anti-citrullinated fibronectin antibodies in rheumatoid arthritis are associated with human leukocyte antigen-DRB1 shared epitope alleles.

INTRODUCTION: Fibronectin is one of the most abundant proteins present in the inflamed joint. Here, we characterized the citrullination of fibronectin in the joints of rheumatoid arthritis (RA) patients and studied the prevalence, epitope specificity and human leukocyte antigen (HLA) association of autoantibodies against citrullinated fibronectin in RA. METHODS: Citrullinated residues in fibronectin isolated from RA patient synovial fluid were identified by mass spectrometry. The corresponding citrullinated and non-citrullinated peptides were synthesized and used to analyze the presence of autoantibodies to these peptides in RA sera and sera from other diseases and healthy controls by ELISA. The data were compared with risk factors like shared epitope HLA alleles and smoking, and with clinical features. RESULTS: Five citrullinated residues were identified in fibronectin from RA synovial fluid. RA sera reacted in a citrulline-dependent manner with two out of four citrullinated fibronectin peptides, one of which contains two adjacent citrulline residues, in contrast to non-RA sera, which were not reactive. The most frequently recognized peptide (FN-Cit1035,1036, LTVGLTXXGQPRQY, in which × represents citrulline) was primarily targeted by anti-CCP (cyclic citrullinated peptide) 2-positive RA patients. Anti-FN-Cit1035,1036 autoantibodies were detected in 50% of established anti-CCP2-positive RA patients and in 45% of such patients from a early arthritis clinic. These antibodies appeared to be predominantly of the immunoglobulin G (IgG) isotype and to be associated with HLA shared epitope alleles (odds ratio = 2.11). CONCLUSIONS: Fibronectin in the inflamed synovia of RA patients can be citrullinated at least at five positions. Together with the flanking amino acids, three of these citrullinated residues comprise two epitopes recognized by RA autoantibodies. Anti-citrullinated fibronectin peptide antibodies are associated with HLA shared epitope alleles.

Elevated levels of fibrinogen-derived endogenous citrullinated peptides in synovial fluid of rheumatoid arthritis patients.

INTRODUCTION: Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of the joints and the presence of autoantibodies directed against proteins containing the non-standard arginine-derived amino acid citrulline. The protein fibrinogen, which has an essential role in blood clotting, is one of the most prominent citrullinated autoantigens in RA, particularly because it can be found in the inflamed tissue of affected joints. Here, we set out to analyze the presence of citrullinated endogenous peptides in the synovial fluid of RA and arthritic control patients. METHODS: Endogenous peptides were isolated from the synovial fluid of RA patients and controls by filtration and solid phase extraction. The peptides were identified and quantified using high-resolution liquid chromatography-mass spectrometry. RESULTS: Our data reveal that the synovial fluid of RA patients contains soluble endogenous peptides, derived from fibrinogen, containing significant amounts of citrulline residues and, in some cases, also phosphorylated serine. Several citrullinated peptides are found to be more abundantly present in the synovial fluid of RA patients compared to patients suffering from other inflammatory diseases affecting the joints. CONCLUSIONS: The increased presence of citrullinated peptides in RA patients points toward a possible specific role of these peptides in the immune response at the basis of the recognition of citrullinated peptides and proteins by RA patient autoantibodies.

Anti-CCP antibodies: the past, the present and the future.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by autoantibodies against citrullinated antigens. The importance of citrulline for the epitopes bound by these autoantibodies, referred to as ACPA (anti-citrullinated peptide/protein antibodies), was first described in 1998. In addition to citrullinated proteins, cyclic citrullinated peptides (CCP) can also be used as test substrates for detecting ACPA. The standard test for these antibodies is the second-generation CCP (CCP2) test, which is one of the best in terms of sensitivity and specificity. The generation of ACPA is an early event in the disease course, and is dependent on the presence of certain MHC class II alleles. ACPA in the inflamed synovium have been shown to associate with citrullinated antigens to form immune complexes, resulting in progression of the inflammatory process. The involvement of ACPA in the chronicity of RA is probably the reason why ACPA-positive patients have a more erosive disease course than ACPA-negative patients. The presence of ACPA has been included in the 2010 RA classification criteria. Thus, it is important to further standardize ACPA testing, for example by including an internal serum standard, which may lead to a better distinction between low and high ACPA levels.

Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance.

INTRODUCTION: Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology. METHODS: Human fibrinogen was citrullinated in vitro by peptidylarginine deiminases (PAD), subjected to proteolysis and the resulting peptides were fractionated by ion exchange chromatography. The peptide composition of the citrullinated peptide-containing fractions was determined by high resolution tandem mass spectrometry. The recognition of these fractions by patient sera was subsequently analyzed by imaging surface plasmon resonance on microarrays. RESULTS: In total about two-thirds of the 81 arginines of human fibrinogen were found to be susceptible to citrullination by the human PAD2, the human PAD4 or the rabbit PAD2 enzymes. Citrullination sites were found in all three polypeptide chains of fibrinogen, although the α-chain appeared to contain most of them. The analysis of 98 anti-citrullinated protein antibody-positive RA sera using the new methodology allowed the identification of three major citrullinated epitope regions in human fibrinogen, two in the α- and one in the ß-chain. CONCLUSIONS: A comprehensive overview of citrullination sites in human fibrinogen was generated. The multiplex analysis of peptide fractions derived from a post-translationally modified protein, characterized by mass spectrometry, with patient sera provides a versatile system for mapping modified amino acid-containing epitopes. The citrullinated epitopes of human fibrinogen most efficiently recognized by RA autoantibodies are confined to three regions of its polypeptides.

Anti-CCP Antibody, a Marker for the Early Detection of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a common autoimmune disease characterized by chronic inflammation of synovial joints. In most cases this will lead to the formation of pannus tissue, ultimately leading to joint destruction. Early diagnosis, coupled with aggressive use of disease-modifying antirheumatic drugs, has been shown to have a favorable effect on the course of the disease. Therefore, early and accurate diagnosis has become increasingly important. Several sets of criteria have been published to achieve such an early diagnosis, and all of them include measurement of antibodies directed to citrullinated peptides or proteins. This review summarizes our present knowledge about the most well-known and established test to measure these antibodies, the anti-CCP test, which measures antibodies directed to cyclic citrullinated peptides. We describe the current views on how these antibodies are generated and how genetic and environmental parameters are important in this process. The anti-CCP test is more specific than the commonly used RF test (95% versus less than 90%) and has a comparable sensitivity (more than 70%). These antibodies are detectable very early in the disease and are reported to predict the development of erosive RA. Increasing evidence supports a role for these antibodies in the pathology of the disease. In conclusion, testing for anti-CCP autoantibodies is widely accepted as an indispensable tool for diagnosis and early treatment in the management of rheumatoid arthritis patients.