Clonal Evolution of Autoreactive Germinal Centers.

Germinal centers (GCs) are the primary sites of clonal B cell expansion and affinity maturation, directing the production of high-affinity antibodies. This response is a central driver of pathogenesis in autoimmune diseases, such as systemic lupus erythematosus (SLE), but the natural history of autoreactive GCs remains unclear. Here, we present a novel mouse model where the presence of a single autoreactive B cell clone drives the TLR7-dependent activation, expansion, and differentiation of other autoreactive B cells in spontaneous GCs. Once tolerance was broken for one self-antigen, autoreactive GCs generated B cells targeting other self-antigens. GCs became independent of the initial clone and evolved toward dominance of individual clonal lineages, indicating affinity maturation. This process produced serum autoantibodies to a breadth of self-antigens, leading to antibody deposition in the kidneys. Our data provide insight into the maturation of the self-reactive B cell response, contextualizing the epitope spreading observed in autoimmune disease.

Spatiotemporal dissection of the cell cycle with single-cell proteogenomics.

The cell cycle, over which cells grow and divide, is a fundamental process of life. Its dysregulation has devastating consequences, including cancer1-3. The cell cycle is driven by precise regulation of proteins in time and space, which creates variability between individual proliferating cells. To our knowledge, no systematic investigations of such cell-to-cell proteomic variability exist. Here we present a comprehensive, spatiotemporal map of human proteomic heterogeneity by integrating proteomics at subcellular resolution with single-cell transcriptomics and precise temporal measurements of individual cells in the cell cycle. We show that around one-fifth of the human proteome displays cell-to-cell variability, identify hundreds of proteins with previously unknown associations with mitosis and the cell cycle, and provide evidence that several of these proteins have oncogenic functions. Our results show that cell cycle progression explains less than half of all cell-to-cell variability, and that most cycling proteins are regulated post-translationally, rather than by transcriptomic cycling. These proteins are disproportionately phosphorylated by kinases that regulate cell fate, whereas non-cycling proteins that vary between cells are more likely to be modified by kinases that regulate metabolism. This spatially resolved proteomic map of the cell cycle is integrated into the Human Protein Atlas and will serve as a resource for accelerating molecular studies of the human cell cycle and cell proliferation.

Characterising the autoantibody repertoire in systemic sclerosis following myeloablative haematopoietic stem cell transplantation.

OBJECTIVES: Results from the SCOT (Scleroderma: Cyclophosphamide Or Transplantation) clinical trial demonstrated significant benefits of haematopoietic stem cell transplant (HSCT) versus cyclophosphamide (CTX) in patients with systemic sclerosis. The objective of this study was to test the hypothesis that transplantation stabilises the autoantibody repertoire in patients with favourable clinical outcomes. METHODS: We used a bead-based array containing 221 protein antigens to profile serum IgG autoantibodies in participants of the SCOT trial. RESULTS: Comparison of autoantibody profiles at month 26 (n=23 HSCT; n=22 CTX) revealed antibodies against two viral antigens and six self-proteins (SSB/La, CX3CL1, glycyl-tRNA synthetase (EJ), parietal cell antigen, bactericidal permeability-increasing protein and epidermal growth factor receptor (EGFR)) that were significantly different between treatment groups. Linear mixed model analysis identified temporal increases in antibody levels for hepatitis B surface antigen, CCL3 and EGFR in HSCT-treated patients. Eight of 32 HSCT-treated participants and one of 31 CTX-treated participants had temporally varying serum antibody profiles for one or more of 14 antigens. Baseline autoantibody levels against 20 unique antigens, including 9 secreted proteins (interleukins, IL-18, IL-22, IL-23 and IL-27), interferon-α2A, stem cell factor, transforming growth factor-ß, macrophage colony-stimulating factor and macrophage migration inhibitory factor were significantly higher in patients who survived event-free to month 54. CONCLUSIONS: Our results suggest that HSCT favourably alters the autoantibody repertoire, which remains virtually unchanged in CTX-treated patients. Although antibodies recognising secreted proteins are generally thought to be pathogenic, our results suggest a subset could potentially modulate HSCT in scleroderma.

Molecular mimicry between Anoctamin 2 and Epstein-Barr virus nuclear antigen 1 associates with multiple sclerosis risk.

Multiple sclerosis (MS) is a chronic inflammatory, likely autoimmune disease of the central nervous system with a combination of genetic and environmental risk factors, among which Epstein-Barr virus (EBV) infection is a strong suspect. We have previously identified increased autoantibody levels toward the chloride-channel protein Anoctamin 2 (ANO2) in MS. Here, IgG antibody reactivity toward ANO2 and EBV nuclear antigen 1 (EBNA1) was measured using bead-based multiplex serology in plasma samples from 8,746 MS cases and 7,228 controls. We detected increased anti-ANO2 antibody levels in MS (P = 3.5 × 10-36) with 14.6% of cases and 7.8% of controls being ANO2 seropositive (odds ratio [OR] = 1.6; 95% confidence intervals [95%CI]: 1.5 to 1.8). The MS risk increase in ANO2-seropositive individuals was dramatic when also exposed to 3 known risk factors for MS: HLA-DRB1*15:01 carriage, absence of HLA-A*02:01, and high anti-EBNA1 antibody levels (OR = 24.9; 95%CI: 17.9 to 34.8). Reciprocal blocking experiments with ANO2 and EBNA1 peptides demonstrated antibody cross-reactivity, mapping to ANO2 [aa 140 to 149] and EBNA1 [aa 431 to 440]. HLA gene region was associated with anti-ANO2 antibody levels and HLA-DRB1*04:01 haplotype was negatively associated with ANO2 seropositivity (OR = 0.6; 95%CI: 0.5 to 0.7). Anti-ANO2 antibody levels were not increased in patients from 3 other inflammatory disease cohorts. The HLA influence and the fact that specific IgG production usually needs T cell help provides indirect evidence for a T cell ANO2 autoreactivity in MS. We propose a hypothesis where immune reactivity toward EBNA1 through molecular mimicry with ANO2 contributes to the etiopathogenesis of MS.

The autoantibody response to cyclic citrullinated collagen type II peptides in rheumatoid arthritis.

OBJECTIVES: The detection of anti-citrullinated peptide antibodies (ACPAs) is a serological hallmark of RA. Autoantibodies reactive with collagen type II (CII) are present in RA sera and synovial fluid and are potentially pathogenic. Here, we investigate the prevalence and specificity of the autoantibody responses to defined citrullinated cyclic peptides derived from CII in a China RA cohort. METHODS: Using bead-based multiplex assay, we examined the presence of autoantibodies binding to 54 cyclic 17-mer citrullinated CII peptides, encompassing all citrullinate epitopes in CII, and the corresponding unmodified peptides in 415 RA patients, in addition to 304 patients with OA. Furthermore, the autoantibody responses to a selected set of 10 cyclic citrullinated peptides were also examined in 203 healthy individuals. RESULTS: Autoantibody responses to cyclic citrullinated CII peptides were higher in RA patients as compared with OA patients or healthy individuals, whereas little or negligible antibody responses to cyclic unmodified CII peptides were observed. Interestingly, several novel citrullinated CII epitopes were identified. Antibodies to these novel citrullinated CII epitopes showed not only substantial overlapping reactivities but also had unique specificities. CONCLUSION: We found a high prevalence of autoantibodies against cyclic citrullinated CII in the sera of patients in a China RA cohort. The present study revealed heterogeneous binding patterns against novel citrullinated CII epitopes, which may help to stratify RA patients into different subgroups.

Anoctamin 2 identified as an autoimmune target in multiple sclerosis.

Multiple sclerosis (MS) is the most common chronic inflammatory disease of the central nervous system and also is regarded as an autoimmune condition. However, the antigenic targets of the autoimmune response in MS have not yet been deciphered. In an effort to mine the autoantibody repertoire within MS, we profiled 2,169 plasma samples from MS cases and population-based controls using bead arrays built with 384 human protein fragments selected from an initial screening with 11,520 antigens. Our data revealed prominently increased autoantibody reactivity against the chloride-channel protein anoctamin 2 (ANO2) in MS cases compared with controls. This finding was corroborated in independent assays with alternative protein constructs and by epitope mapping with peptides covering the identified region of ANO2. Additionally, we found a strong interaction between the presence of ANO2 autoantibodies and the HLA complex MS-associated DRB1*15 allele, reinforcing a potential role for ANO2 autoreactivity in MS etiopathogenesis. Furthermore, immunofluorescence analysis in human MS brain tissue showed ANO2 expression as small cellular aggregates near and inside MS lesions. Thus this study represents one of the largest efforts to characterize the autoantibody repertoire within MS. The findings presented here demonstrate that an ANO2 autoimmune subphenotype may exist in MS and lay the groundwork for further studies focusing on the pathogenic role of ANO2 autoantibodies in MS.

Levels of human proteins in plasma associated with acute paediatric malaria.

BACKGROUND: The intimate interaction between the pathophysiology of the human host and the biology of the Plasmodium falciparum parasite results in a wide spectrum of disease outcomes in malaria. Development of severe disease is associated with a progressively augmented imbalance in pro- and anti-inflammatory responses to high parasite loads and sequestration of parasitized erythrocytes. Although these phenomena collectively constitute common denominators for the wide variety of discrete severe malaria manifestations, the mechanistic rationales behind discrepancies in outcome are poorly understood. Exploration of the human pathophysiological response by variations in protein profiles in plasma presents an excellent opportunity to increase the understanding. This is ultimately required for better prediction, prevention and treatment of malaria, which is essential for ongoing elimination and eradication efforts. RESULTS: An affinity proteomics approach was used to analyse 541 paediatric plasma samples collected from community controls and patients with mild or severe malaria in Rwanda. Protein profiles were generated with an antibody-based suspension bead array containing 255 antibodies targetting 115 human proteins. Here, 57 proteins were identified with significantly altered levels (adjusted p-values < 0.001) in patients with malaria compared to controls. From these, the 27 most significant proteins (adjusted p-values < 10-14) were selected for a stringent analysis approach. Here, 24 proteins showed elevated levels in malaria patients and included proteins involved in acute inflammatory response as well as cell adhesion. The remaining three proteins, also implicated in immune regulation and cellular adhesivity, displayed lower abundance in malaria patients. In addition, 37 proteins (adjusted p-values < 0.05) were identified with increased levels in patients with severe compared to mild malaria. This set includes, proteins involved in tissue remodelling and erythrocyte membrane proteins. Collectively, this approach has been successfully used to identify proteins both with known and unknown association with different stages of malaria. CONCLUSION: In this study, a high-throughput affinity proteomics approach was used to find protein profiles in plasma linked to P. falciparum infection and malaria disease progression. The proteins presented herein are mainly involved in inflammatory response, cellular adhesion and as constituents of erythrocyte membrane. These findings have a great potential to provide increased conceptual understanding of host-parasite interaction and malaria pathogenesis.

Whole-Proteome Peptide Microarrays for Profiling Autoantibody Repertoires within Multiple Sclerosis and Narcolepsy.

The underlying molecular mechanisms of autoimmune diseases are poorly understood. To unravel the autoimmune processes across diseases, comprehensive and unbiased analyses of proteins targets recognized by the adaptive immune system are needed. Here we present an approach starting from high-density peptide arrays to characterize autoantibody repertoires and to identify new autoantigens. A set of ten plasma and serum samples from subjects with multiple sclerosis, narcolepsy, and without any disease diagnosis were profiled on a peptide array representing the whole proteome, hosting 2.2 million 12-mer peptides with a six amino acid lateral shift. On the basis of the IgG reactivities found on these whole-proteome peptide microarrays, a set of 23 samples was then studied on a targeted array with 174 000 12-mer peptides of single amino acid lateral shift. Finally, verification of IgG reactivities was conducted with a larger sample set (n = 448) using the bead-based peptide microarrays. The presented workflow employed three different peptide microarray formats to discover and resolve the epitopes of human autoantibodies and revealed two potentially new autoantigens: MAP3K7 in multiple sclerosis and NRXN1 in narcolepsy. The presented strategy provides insights into antibody repertoire reactivity at a peptide level and may accelerate the discovery and validation of autoantigens in human diseases.

Multiplexed protein profiling by sequential affinity capture.

Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off-target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi-automated sequential capture assay. This novel bead-based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read-out by a secondary capture bead array. We demonstrate in a proof-of-concept setting that target detection via two sequential affinity interactions reduced off-target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA-based signal amplification, and demonstrate the applicability of the dual capture bead-based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond.

Autoantibody profiling in multiple sclerosis using arrays of human protein fragments.

Profiling the autoantibody repertoire with large antigen collections is emerging as a powerful tool for the identification of biomarkers for autoimmune diseases. Here, a systematic and undirected approach was taken to screen for profiles of IgG in human plasma from 90 individuals with multiple sclerosis related diagnoses. Reactivity pattern of 11,520 protein fragments (representing ∼38% of all human protein encoding genes) were generated on planar protein microarrays built within the Human Protein Atlas. For more than 2,000 antigens IgG reactivity was observed, among which 64% were found only in single individuals. We used reactivity distributions among multiple sclerosis subgroups to select 384 antigens, which were then re-evaluated on planar microarrays, corroborated with suspension bead arrays in a larger cohort (n = 376) and confirmed for specificity in inhibition assays. Among the heterogeneous pattern within and across multiple sclerosis subtypes, differences in recognition frequencies were found for 51 antigens, which were enriched for proteins of transcriptional regulation. In conclusion, using protein fragments and complementary high-throughput protein array platforms facilitated an alternative route to discovery and verification of potentially disease-associated autoimmunity signatures, that are now proposed as additional antigens for large-scale validation studies across multiple sclerosis biobanks.

Affinity proteomic profiling of plasma, cerebrospinal fluid, and brain tissue within multiple sclerosis.

The brain is a vital organ and because it is well shielded from the outside environment, possibilities for noninvasive analysis are often limited. Instead, fluids taken from the spinal cord or circulatory system are preferred sources for the discovery of candidate markers within neurological diseases. In the context of multiple sclerosis (MS), we applied an affinity proteomic strategy and screened 22 plasma samples with 4595 antibodies (3450 genes) on bead arrays, then defined 375 antibodies (334 genes) for targeted analysis in a set of 172 samples and finally used 101 antibodies (43 genes) on 443 plasma as well as 573 cerebrospinal spinal fluid (CSF) samples. This revealed alteration of protein profiles in relation to MS subtypes for IRF8, IL7, METTL14, SLC30A7, and GAP43. Respective antibodies were subsequently used for immunofluorescence on human post-mortem brain tissue with MS pathology for expression and association analysis. There, antibodies for IRF8, IL7, and METTL14 stained neurons in proximity of lesions, which highlighted these candidate protein targets for further studies within MS and brain tissue. The affinity proteomic translation of profiles discovered by profiling human body fluids and tissue provides a powerful strategy to suggest additional candidates to studies of neurological disorders.

Development of a coordinated allo T cell and auto B cell response against autosomal PTK2B after allogeneic hematopoietic stem cell transplantation.

It is well known that allo-reactive T cells play a crucial role in graft-versus-leukemia and graft-versus-host disease after allogeneic hematopoietic stem cell transplantation (alloSCT). Allo-reactive CD4(+) T cells can mediate direct cytolysis, but may also stimulate production of IgG antibodies as helper cells. Immune complexes may subsequently be processed and presented by professional antigen presenting cells and stimulate induction of specific CD8(+) T cells. As such, proteins targeted in coordinated T- and B-cell responses may represent a class of immunodominant antigens in clinical responses after alloSCT. We previously identified LB-PTK2B-1T as HLA class II restricted polymorphic antigen in a patient treated with donor lymphocyte infusion for relapsed chronic myeloid leukemia after HLA-matched alloSCT. Since PTK2B has also been described as antibody target, we here investigated whether a coordinated T- and B-cell response against PTK2B was induced. Patient serum before and after alloSCT and donor lymphocyte infusion (DLI) was screened for antibodies, and we indeed observed development of a humoral immune response against PTK2B. Antibodies against PTK2B were only found after DLI and, in contrast to the CD4(+) T cells, recognized a monomorphic region of the protein. To our knowledge, this is the first description of a coordinated allo-reactive CD4(+) T-cell and auto-reactive antibody response against an autosomal antigen.

Antibody-based profiling of cerebrospinal fluid within multiple sclerosis.

Antibody suspension bead arrays have proven to enable multiplexed and high-throughput protein profiling in unfractionated plasma and serum samples through a direct labeling approach. We here describe the development and application of an assay for protein profiling of cerebrospinal fluid (CSF). While setting up the assay, systematic intensity differences between sample groups were observed that reflected inherent sample specific total protein amounts. Supplementing the labeling reaction with BSA and IgG diminished these differences without impairing the apparent sensitivity of the assay. We also assessed the effects of heat treatment on the analysis of CSF proteins and applied the assay to profile 43 selected proteins by 101 antibodies in 339 CSF samples from a multiple sclerosis (MS) cohort. Two proteins, GAP43 and SERPINA3 were found to have a discriminating potential with altered intensity levels between sample groups. GAP43 was detected at significantly lower levels in secondary progressive MS compared to early stages of MS and the control group of other neurological diseases. SERPINA3 instead was detected at higher levels in all MS patients compared to controls. The developed assay procedure now offers new possibilities for broad-scale protein profiling of CSF within neurological disorders.

A topographic atlas defines developmental origins of cell heterogeneity in the human embryonic lung.

The lung contains numerous specialized cell types with distinct roles in tissue function and integrity. To clarify the origins and mechanisms generating cell heterogeneity, we created a comprehensive topographic atlas of early human lung development. Here we report 83 cell states and several spatially resolved developmental trajectories and predict cell interactions within defined tissue niches. We integrated single-cell RNA sequencing and spatially resolved transcriptomics into a web-based, open platform for interactive exploration. We show distinct gene expression programmes, accompanying sequential events of cell differentiation and maturation of the secretory and neuroendocrine cell types in proximal epithelium. We define the origin of airway fibroblasts associated with airway smooth muscle in bronchovascular bundles and describe a trajectory of Schwann cell progenitors to intrinsic parasympathetic neurons controlling bronchoconstriction. Our atlas provides a rich resource for further research and a reference for defining deviations from homeostatic and repair mechanisms leading to pulmonary diseases.

Longitudinal serum biomarker screening identifies malate dehydrogenase 2 as candidate prognostic biomarker for Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal disease for which no cure is available. Clinical trials have shown to be largely underpowered due to inter-individual variability and noisy outcome measures. The availability of biomarkers able to anticipate clinical benefit is highly needed to improve clinical trial design and facilitate drug development. METHODS: In this study, we aimed to appraise the value of protein biomarkers to predict prognosis and monitor disease progression or treatment outcome in patients affected by DMD. We collected clinical data and 303 blood samples from 157 DMD patients in three clinical centres; 78 patients contributed multiple blood samples over time, with a median follow-up time of 2 years. We employed linear mixed models to identify biomarkers that are associated with disease progression, wheelchair dependency, and treatment with corticosteroids and performed survival analysis to find biomarkers whose levels are associated with time to loss of ambulation. RESULTS: Our analysis led to the identification of 21 proteins whose levels significantly decrease with age and nine proteins whose levels significantly increase. Seven of these proteins are also differentially expressed in non-ambulant patients, and three proteins are differentially expressed in patients treated with glucocorticosteroids. Treatment with corticosteroids was found to partly counteract the effect of disease progression on two biomarkers, namely, malate dehydrogenase 2 (MDH2, P = 0.0003) and ankyrin repeat domain 2 (P = 0.0005); however, patients treated with corticosteroids experienced a further reduction on collagen 1 serum levels (P = 0.0003), especially following administration of deflazacort. A time to event analysis allowed to further support the use of MDH2 as a prognostic biomarker as it was associated with an increased risk of wheelchair dependence (P = 0.0003). The obtained data support the prospective evaluation of the identified biomarkers in natural history and clinical trials as exploratory biomarkers. CONCLUSIONS: We identified a number of serum biomarkers associated with disease progression, loss of ambulation, and treatment with corticosteroids. The identified biomarkers are promising candidate prognostic and surrogate biomarkers, which may support drug developers if confirmed in prospective studies. The serum levels of MDH2 are of particular interest, as they correlate with disease stage and response to treatment with corticosteroids, and are also associated with the risk of wheelchair dependency and pulmonary function.

Blood-derived biomarkers correlate with clinical progression in Duchenne muscular dystrophy.

BACKGROUND: Duchenne Muscular Dystrophy is a severe, incurable disorder caused by mutations in the dystrophin gene. The disease is characterized by decreased muscle function, impaired muscle regeneration and increased inflammation. In a clinical context, muscle deterioration, is evaluated using physical tests and analysis of muscle biopsies, which fail to accurately monitor the disease progression. OBJECTIVES: This study aims to confirm and asses the value of blood protein biomarkers as disease progression markers using one of the largest longitudinal collection of samples. METHODS: A total of 560 samples, both serum and plasma, collected at three clinical sites are analyzed using a suspension bead array platform to assess 118 proteins targeted by 250 antibodies in microliter amount of samples. RESULTS: Nine proteins are confirmed as disease progression biomarkers in both plasma and serum. Abundance of these biomarkers decreases as the disease progresses but follows different trajectories. While carbonic anhydrase 3, microtubule associated protein 4 and collagen type I alpha 1 chain decline rather constantly over time, myosin light chain 3, electron transfer flavoprotein A, troponin T, malate dehydrogenase 2, lactate dehydrogenase B and nestin plateaus in early teens. Electron transfer flavoprotein A, correlates with the outcome of 6-minutes-walking-test whereas malate dehydrogenase 2 together with myosin light chain 3, carbonic anhydrase 3 and nestin correlate with respiratory capacity. CONCLUSIONS: Nine biomarkers have been identified that correlate with disease milestones, functional tests and respiratory capacity. Together these biomarkers recapitulate different stages of the disorder that, if validated can improve disease progression monitoring.

AIRE expression controls the peripheral selection of autoreactive B cells.

Autoimmune regulator (AIRE) mutations result in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome characterized by defective central T cell tolerance and the production of many autoantibodies targeting tissue-specific antigens and cytokines. By studying CD3- and AIRE-deficient patients, we found that lack of either T cells or AIRE function resulted in the peripheral accumulation of autoreactive mature naïve B cells. Proteomic arrays and Biacore affinity measurements revealed that unmutated antibodies expressed by these autoreactive naïve B cells recognized soluble molecules and cytokines including insulin, IL-17A, and IL-17F, which are AIRE-dependent thymic peripheral tissue antigens targeted by autoimmune responses in APECED. AIRE-deficient patients also displayed decreased frequencies of regulatory T cells (Tregs) that lacked common TCRß clones found instead in their conventional T cell compartment, thereby suggesting holes in the Treg TCR repertoire of these patients. Hence, AIRE-mediated T cell/Treg selection normally prevents the expansion of autoreactive naïve B cells recognizing peripheral self-antigens.

Plasma protein profiling reveals candidate biomarkers for multiple sclerosis treatment.

Multiple sclerosis (MS) treatment options have improved significantly over the past decades, but the consequences of MS can still be devastating and the needs for monitoring treatment surveillance are considerable. In the current study we used affinity proteomics technology to identify potential biomarkers which could ultimately be used to as facilitate treatment decisions. We profiled the intra-individual changes in the levels of 59 target proteins using an antibody suspension bead array in serial plasma samples from 44 MS patients during treatment with natalizumab followed by fingolimod. Nine proteins showed decreasing plasma levels during natalizumab treatment, with PEBP1 and RTN3 displaying the most significant changes. Protein levels remained stable during fingolimod treatment for both proteins. The decreasing PEBP1 levels during natalizumab treatment could be validated using ELISA and replicated in an independent cohort. These results support the use of this technology as a high throughput method of identifying potentially useful biomarkers of MS treatment.

Structural Basis of Cross-Reactivity of Anti-Citrullinated Protein Antibodies.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) develop many years before the clinical onset of rheumatoid arthritis (RA). This study was undertaken to address the molecular basis of the specificity and cross-reactivity of ACPAs from patients with RA. METHODS: Antibodies isolated from RA patients were expressed as monoclonal chimeric antibodies with mouse Fc. These antibodies were characterized for glycosylation using mass spectrometry, and their cross-reactivity was assessed using Biacore and Luminex immunoassays. The crystal structures of the antigen-binding fragment (Fab) of the monoclonal ACPA E4 in complex with 3 different citrullinated peptides were determined using x-ray crystallography. The prevalence of autoantibodies reactive against 3 of the citrullinated peptides that also interacted with E4 was investigated by Luminex immunoassay in 2 Swedish cohorts of RA patients. RESULTS: Analysis of the crystal structures of a monoclonal ACPA from human RA serum in complex with citrullinated peptides revealed key residues of several complementarity-determining regions that recognized the citrulline as well as the neighboring peptide backbone, but with limited contact with the side chains of the peptides. The same citrullinated peptides were recognized by high titers of serum autoantibodies in 2 large cohorts of RA patients. CONCLUSION: These data show, for the first time, how ACPAs derived from human RA serum recognize citrulline. The specific citrulline recognition and backbone-mediated interactions provide a structural explanation for the promiscuous recognition of citrullinated peptides by RA-specific ACPAs.