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A hallmark of rheumatoid arthritis (RA) is the increased levels of autoantibodies preceding the onset and contributing to the classification of the disease. These autoantibodies, mainly anti-citrullinated protein antibody (ACPA) and rheumatoid factor, have been assumed to be pathogenic and many attempts have been made to link them to the development of bone erosion, pain and arthritis. We and others have recently discovered that most cloned ACPA protect against experimental arthritis in the mouse. In addition, we have identified suppressor B cells in healthy individuals, selected in response to collagen type II, and these cells decrease in numbers in RA. These findings provide a new angle on how to explain the development of RA and maybe also other complex autoimmune diseases preceded by an increased autoimmune response., Competing Interests: Competing interests: RH is named as an inventor in an approved patent protecting the E4 antibody., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ on behalf of EULAR.)

Objectives: To investigate how individual rheumatoid arthritis (RA) autoantibodies associate with individual signs and symptoms at the time of RA diagnosis., Methods: IgA, IgG, IgM rheumatoid factor (RF), antibodies against cyclic citrullinated peptide version 2 (anti-CCP2) and 16 individual antibodies against citrullinated protein (ACPA) reactivities were analysed centrally in baseline sera from 1600 patients with RA classified according to the 1987 American College of Rheumatology (ACR) criteria. These results were related to C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), number of swollen and tender joints (SJC and TJC), 28-joint disease activity scores (DAS28 and DAS28CRP), global disease activity evaluated by the patients and Health Assessment Questionnaire, all obtained at baseline., Results: Individually, all autoantibodies except immunoglobulin G (IgG) RF associated with low SJC and TJC and with high ESR. In IgM RF-negative patients, ACPA associated strictly with low number of swollen and tender joints. This association persisted in multiple regression and stratified analyses where IgM and IgA RF instead associated with inflammation expressed as ESR. Among subjects without any ACPA peptide reactivity, there was no association between RF isotypes and ESR. The effect of RF on ESR increased with the number of ACPA reactivities, especially for IgM RF. In patients fulfilling the 1987 ACR criteria without taking RF into account, associations between IgM RF and high ESR, as well as between ACPA and low joint counts, remained., Conclusion: Whereas ACPA associate with low counts of affected joints in early RA, RF associates with elevated measures of systemic inflammation in an ACPA-dependent manner. This latter finding corroborates in vitro models of ACPA and RF in immune complex-induced inflammation. These phenotypic associations are independent of classification criteria., Competing Interests: Competing interests: EP and LM-A are employees at Thermo Fisher Scientific. JA has had or have research agreements with AbbVie, BMS, Eli Lilly, Galapagos, MSD, Pfizer, Roche, Samsung Bioepis and Sanofi, mainly for the national safety monitoring of rheumatology immunomodulators in Sweden (ARTIS). JR has been a member of the Scientific Advisory Board for Thermo Fisher Scientific and for Inova/Werfen and has received consulting fees, speaking fees and/ or honoraria by Thermo Fisher Scientific. RH has received consulting fees from Regor, Lipum AB and Cyxone AB and is the founder of Vacara AB. LK has been a co-ordinator of several IMI-funded projects which included collaborations with Janssen, Pfizer, Sanofi, UCB, GSK and Eli-Lilly. This work has been presented as a poster in a preliminary form at EULAR (June 2019) and to the European Workshop for Rheumatology Research (March 2023)., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: To identify the arthritogenic B cell epitopes of glucose-6-phosphate isomerase (GPI) and their association with rheumatoid arthritis (RA)., Methods: IgG response towards a library of GPI peptides in patients with early RA, pre-symptomatic individuals and population controls, as well as in mice, were tested by bead-based multiplex immunoassays and ELISA. Monoclonal IgG were generated, and the binding specificity and affinity were determined by ELISA, gel size exclusion chromatography, surface plasma resonance and X-ray crystallography. Arthritogenicity was investigated by passive transfer experiments. Antigen-specific B cells were identified by peptide tetramer staining., Results: Peptide GPI 293-307 was the dominant B cell epitope in K/BxN and GPI-immunised mice. We could detect B cells and low levels of IgM antibodies binding the GPI 293-307 epitopes, and high affinity anti-GPI 293-307 IgG antibodies already 7 days after GPI immunisation, immediately before arthritis onset. Transfer of anti-GPI 293-307 IgG antibodies induced arthritis in mice. Moreover, anti-GPI 293-307 IgG antibodies were more frequent in individuals prior to RA onset (19%) than in controls (7.5%). GPI 293-307 -specific antibodies were associated with radiographic joint damage. Crystal structures of the Fab-peptide complex revealed that this epitope is not exposed in native GPI but requires conformational change of the protein in inflamed joint for effective recognition by anti-GPI 293-307 antibodies., Conclusions: We have identified the major pathogenic B cell epitope of the RA-associated autoantigen GPI, at position 293-307, exposed only on structurally modified GPI on the cartilage surface. B cells to this neo-epitope escape tolerance and could potentially play a role in the pathogenesis of RA., Competing Interests: Competing interests: RH is the founder, Outi Sareila is an employee and Erik Lönnblom is a consultant of Vacara AB., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: Rheumatoid arthritis (RA) is an autoimmune disease strongly associated with the major histocompatibility complex (MHC) class II allele DRB1*04:01, which encodes a protein that binds self-peptides for presentation to T cells. This study characterises the autoantigen-presenting function of DRB1*04:01 (HLA-DRA*01:01/HLA-DRB1*04:01) at a molecular level for prototypic T-cell determinants, focusing on a post-translationally modified collagen type II (Col2)-derived peptide., Methods: The crystal structures of DRB1*04:01 molecules in complex with the peptides HSP70 289-306 , citrullinated CILP 982-996 and galactosylated Col2 259-273 were determined on cocrystallisation. T cells specific for Col2 259-273 were investigated in peripheral blood mononuclear cells from patients with DRB1*04:01-positive RA by cytofluorometric detection of the activation marker CD154 on peptide stimulation and binding of fluorescent DRB1*0401/Col2 259-273 tetramer complexes. The cDNAs encoding the T-cell receptor (TCR) α-chains and β-chains were cloned from single-cell sorted tetramer-positive T cells and transferred via a lentiviral vector into TCR-deficient Jurkat 76 cells., Results: The crystal structures identified peptide binding to DRB1*04:01 and potential side chain exposure to T cells. The main TCR recognition sites in Col2 259-273 were lysine residues that can be galactosylated. RA T-cell responses to DRB1*04:01-presented Col2 259-273 were dependent on peptide galactosylation at lysine 264. Dynamic molecular modelling of a functionally characterised Col2 259-273 -specific TCR complexed with DRB1*04:01/Col2 259-273 provided evidence for differential allosteric T-cell recognition of glycosylated lysine 264., Conclusions: The MHC-peptide-TCR interactions elucidated in our study provide new molecular insights into recognition of a post-translationally modified RA T-cell determinant with a known dominant role in arthritogenic and tolerogenic responses in murine Col2-induced arthritis., Competing Interests: Competing interests: NS, BX, SW, RH and HB are listed as inventors on Patent EP2020072287 (https://www.onscope.com/ipowner/en/ip/ptwo/EP2020072287.html). SW, N-ND, RH and HB are lasted as inventors on Patent EP2020072280 (https://www.onscope.com/ipowner/en/ip/ptwo/EP2020072280.html). The owner of both patents is Fraunhofer-Gesellschaft zur Förderung der Angewandten Forschung E.V. (Germany). SW is listed on these patents under her maiden name of Sylvia Cienciala., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Animal models for inflammatory arthritides such as rheumatoid arthritis (RA) and psoriatic arthritis are widely accepted and frequently used to identify pathological mechanisms and validate novel therapeutic strategies. Unfortunately, many publications reporting on these animal studies lack detailed description and appropriate assessment of the distinct histopathological features of arthritis: joint inflammation, cartilage damage and bone erosion. Therefore, the European consortium BeTheCure, consisting of 38 academic and industrial partners from 15 countries, set as goal to standardise the histological evaluation of joint sections from animal models of inflammatory arthritis. The consensual approach of a task force including 16 academic and industrial scientists as well as laboratory technicians has resulted in the development of the Standardised Microscopic Arthritis Scoring of Histological sections ('SMASH') recommendations for a standardised processing and microscopic scoring of the characteristic histopathological features of arthritis, exemplified by four different rodent models for arthritis: murine collagen-induced arthritis, collagen-antibody-induced arthritis, human tumour necrosis factor transgenic Tg197 mice and rat pristane-induced arthritis, applicable to any other inflammatory arthritis model. Through standardisation, the SMASH recommendations are designed to improve and maximise the information derived from in vivo arthritis experiments and to promote reproducibility and transparent reporting on such studies. In this manuscript, we will discuss and provide recommendations for analysis of histological joint sections: identification of the regions of interest, sample preparation, staining procedures and quantitative scoring methods. In conclusion, awareness of the different features of the arthritis pathology in animal models of inflammatory arthritis is of utmost importance for reliable research outcome, and the standardised histological processing and scoring methods in these SMASH recommendations will help increase uniformity and reproducibility in preclinical research on inflammatory arthritis., Competing Interests: Competing interests: MCD, CG and GK report personal fees from Biomedcode, outside the submitted work; and Biomedcode is a service provider of in vivo efficacy studies within the area of inflammatory diseases. NW is employed by Redoxis AB; and Redoxis AB is a service provider selling in vivo studies within the area of autoimmunity and inflammation., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

​OBJECTIVES: A single nucleotide polymorphism in the NCF1 gene (NCF1-339, rs201802880), encoding NADPH oxidase type II subunit NCF1/p47 phox , reducing production of reactive oxygen species (ROS) is strongly associated with the development of systemic lupus erythematosus (SLE). This study aimed at characterising NCF1-339 effects on neutrophil extracellular trap (NET) formation, type I interferon activity and antibody profile in patients with SLE. ​METHODS: Neutrophil NET-release pathways (n=31), serum interferon (n=141) and finally antibody profiles (n=305) were investigated in SLE subjects from Lund, genotyped for NCF1-339. Then, 1087 SLE subjects from the rheumatology departments of four Swedish SLE centres, genotyped for NCF1-339, were clinically characterised to validate these findings. ​RESULTS: Compared with patients with normal-ROS NCF1-339 genotypes, neutrophils from patients with SLE with low-ROS NCF1-339 genotypes displayed impaired NET formation (p<0.01) and increased dependence on mitochondrial ROS (p<0.05). Low-ROS patients also had increased frequency of high serum interferon activity (80% vs 21.4%, p<0.05) and positivity for anti-β2 glycoprotein I (p<0.01) and anticardiolipin antibodies (p<0.05) but were not associated with other antibodies. We confirmed an over-representation of having any antiphospholipid antibody, OR 1.40 (95% CI 1.01 to 1.95), anti-β2 glycoprotein I, OR 1.82 (95% CI 1.02 to 3.24) and the antiphospholipid syndrome (APS), OR 1.74 (95% CI 1.19 to 2.55) in all four cohorts (n=1087). ​CONCLUSIONS: The NCF1-339 SNP mediated decreased NADPH oxidase function, is associated with high interferon activity and impaired formation of NETs in SLE, allowing dependence on mitochondrial ROS. Unexpectedly, we revealed a striking connection between the ROS deficient NCF1-339 genotypes and the presence of phospholipid antibodies and APS., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: The second generation anticycliccitrullinated peptide (anti-CCP2) assay detects the majority but not all anticitrullinated protein/peptide antibodies (ACPA). Anti-CCP2-positive rheumatoid arthritis (RA) is associated with HLA-DRB1* shared epitope (SE) alleles and smoking. Using a multiplex assay to detect multiple specific ACPA, we have investigated the fine specificity of individual ACPA responses and the biological impact of additional ACPA reactivity among anti-CCP2-negative patients., Methods: We investigated 2825 patients with RA and 551 healthy controls with full data on anti-CCP2, HLA-DRB1* alleles and smoking history concerning reactivity against 16 citrullinated peptides and arginine control peptides with a multiplex array., Results: The prevalence of the 16 ACPA specificities ranged from 9% to 58%. When reactivity to arginine peptides was subtracted, the mean diagnostic sensitivity increased by 3.2% with maintained 98% specificity. Of the anti-CCP2-negative patients, 16% were found to be ACPA positive. All ACPA specificities associated with SE, and all but one with smoking. Correction for arginine reactivity also conveyed a stronger association with SE for 13/16 peptides. Importantly, when all ACPA specificities were analysed together, SE and smoking associated with RA in synergy among ACPA positive, but not among ACPA-negative subjects also in the anti-CCP2-negative subset., Conclusions: Multiplexing detects an enlarged group of ACPA-positive but anti-CCP2-negative patients with genetic and environmental attributes previously assigned to anti-CCP2-positive patients. The individual correction for arginine peptide reactivity confers both higher diagnostic sensitivity and stronger association to SE than gross ACPA measurement., Competing Interests: Competing interests: The project is part of the Innovative Medicines Initiative (IMI) project Be The Cure where Karolinska Institutet is a scientific partner and Thermo Fisher Scientific is a commercial partner. The project follows the rules for IMI projects. JR has obtained reagents from Thermo Fisher Scientific for the investigation on other rheumatology cohorts. LM-A is employed by Thermo Fisher Scientific. LK and RH were cofounders of a company, Curara AB, which has previously collaborated with Thermo Fisher Scientific concerning certain technical aspects of the multiplex assay. This development is done with partial support from an ERC Proof of Concept Grant (pRActice) to LK. RH is a coinventor of a patent (US Patent 7148020) protecting the use of the CitC1 and C1 peptides. KS is coinventor of the patents US 13/141,960 and EP 09799354.7 describing the diagnostic use of the hnRNP-A3 peptide epitopes. GS is coinventor of several international patents about ACPA antigens held by BioMérieux Cy and licenced to Eurodiagnostica Cy and Axis-Shield Cy for commercialisation of the CCP2 assays; according to French laws, he receives a part of the royalties paid to the Toulouse III University and the University Hospital of Toulouse. KL is coinventor of patent US12/524,465, describing the diagnostic use of the CEP-1 epitope. The other authors declare that they have no competing interests., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Objectives: Ncf1 polymorphisms leading to low production of reactive oxygen species (ROS) are strongly associated with autoimmune diseases in animal models. The human NCF1 gene is very complex with both functional and non-functional gene copies and genotyping requires assays specific for functional NCF1 genes. We aimed at investigating association and function of the missense single nucleotide polymorphism (SNP), rs201802880 (here denoted NCF1-339) in NCF1 with systemic lupus erythematosus (SLE)., Methods: We genotyped the NCF1-339 SNP in 973 Swedish patients with SLE and 1301 controls, using nested PCR and pyrosequencing. ROS production and gene expression of type 1 interferon-regulated genes were measured in isolated cells from subjects with different NCF1-339 genotypes., Results: We found an increased frequency of the NCF1-339 T allele in patients with SLE, 11% compared with 4% in controls, OR 3.0, 95% CI 2.4 to 3.9, p=7.0×10 -20 . The NCF1-339 T allele reduced extracellular ROS production in neutrophils (p=0.004) and led to an increase expression of type 1 interferon-regulated genes. In addition, the NCF1-339 T allele was associated with a younger age at diagnosis of SLE; mean age 30.3 compared with 35.9, p=2.0×1 -6 ., Conclusions: These results clearly demonstrate that a genetically controlled reduced production of ROS increases the risk of developing SLE and confirm the hypothesis that ROS regulate chronic autoimmune inflammatory diseases., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Objective: Anti-citrullinated protein antibodies (ACPA) are highly specific for rheumatoid arthritis (RA), but the diagnostic accuracy of ACPA in the general population has not been thoroughly assessed. We aimed to assess the diagnostic accuracy of ACPA for RA in the general population and to further characterise the citrullinated peptide recognition pattern., Methods: Serum samples from a large population-representative twin cohort consisting of 12 590 individuals were analysed for the presence of ACPA using anti-CCP2 ELISA. All ACPA-positive samples were further tested on ELISAs for four peptide-specific ACPA. RA cases were identified by linkage to the Swedish National Patient Register at inclusion and after a median follow-up of 37 months (IQR 31-49)., Results: 350 out of 12 590 individuals had a positive anti-CCP2 test, measuring ACPA. Of these, 103 had an RA diagnosis at the time of blood donation and inclusion. During a median follow-up of 3 years, an additional 21 of the remaining 247 ACPA-positive individuals developed RA. Overall, a positive anti-CCP2 test had a positive predictive value of 29% for prevalent RA at inclusion (negative predictive value of 99.6%). High titres (>3× cut-off) of anti-CCP2 increased the positive predictive value to 48% (negative predictive value of 99.5%). ACPA-positive individuals without RA had lower anti-CCP2 titres and fewer peptide-specific ACPA than ACPA-positive patients with RA and higher C reactive protein levels than ACPA-negative individuals without RA., Conclusion: Presence of ACPA and especially high titres of anti-CCP2 have a high diagnostic accuracy for an RA diagnosis in a population setting., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Objective: An interesting and so far unexplained feature of chronic pain in autoimmune disease is the frequent disconnect between pain and inflammation. This is illustrated well in rheumatoid arthritis (RA) where pain in joints (arthralgia) may precede joint inflammation and persist even after successful anti-inflammatory treatment. In the present study, we have addressed the possibility that autoantibodies against citrullinated proteins (ACPA), present in RA, may be directly responsible for the induction of pain, independent of inflammation., Methods: Antibodies purified from human patients with RA, healthy donors and murinised monoclonal ACPA were injected into mice. Pain-like behaviour was monitored for up to 28 days, and tissues were analysed for signs of pathology. Mouse osteoclasts were cultured and stimulated with antibodies, and supernatants analysed for release of factors. Mice were treated with CXCR1/2 (interleukin (IL) 8 receptor) antagonist reparixin., Results: Mice injected with either human or murinised ACPA developed long-lasting pronounced pain-like behaviour in the absence of inflammation, while non-ACPA IgG from patients with RA or control monoclonal IgG were without pronociceptive effect. This effect was coupled to ACPA-mediated activation of osteoclasts and release of the nociceptive chemokine CXCL1 (analogue to human IL-8). ACPA-induced pain-like behaviour was reversed with reparixin., Conclusions: The data suggest that CXCL1/IL-8, released from osteoclasts in an autoantibody-dependent manner, produces pain by activating sensory neurons. The identification of this new pain pathway may open new avenues for pain treatment in RA and also in other painful diseases associated with autoantibody production and/or osteoclast activation., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Objective: To investigate the role of genetic and environmental factors in the development of anticitrullinated protein antibodies (ACPA) and ACPA-positive rheumatoid arthritis (RA) in a twin cohort., Methods: A total of 12 590 twins were analysed for the presence of ACPAs (CCP2 ELISA), HLA-DRB1 shared epitope (SE) gene alleles, and exposure to smoking. Twins with established RA were identified in national public care registers. Antibody reactivities against citrullinated and native forms of α-enolase, vimentin, fibrinogen and type II collagen peptides were tested by ELISA in anti-CCP2-positive subjects and their cotwins. Structural equation models and ORs for the development of ACPA and ACPA-positive RA were computed for smokers and SE carriers., Results: A total of 2.8% (350/12 590) of the twins were ACPA positive, and 1.0% (124/12 590) had ACPA-positive RA. Most of the variability in the ACPA status was accounted for by non-shared environmental or stochastic factors (78%, 95% CI 55% to 100%) rather than shared environmental and genetic factors. Analysis of specific risk factors revealed an association between smoking and SE and the presence of ACPAs. Twins with ACPA-positive RA were more frequently SE positive than twins with ACPAs without RA. Reactivities against multiple citrullinated peptides were present in most twins with ACPA-positive RA but in fewer twins with ACPAs without RA., Conclusions: Environment, lifestyle and stochastic factors may be more important than genetics in determining which individuals develop ACPAs. Genetic factors (particularly SE) may have a relatively larger role in determining which ACPA-positive individuals will ultimately develop arthritis., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Introduction: Collagen-induced arthritis (CIA) has traditionally been performed in MHC class II A(q)-expressing mice, whereas most genetically modified mice are on the C57BL/6 background (expressing the b haplotype of the major histocompatibility complex (MHC) class II region). However, C57BL/6 mice develop arthritis after immunisation with chicken-derived collagen type II (CII), but arthritis susceptibility has been variable, and the immune specificity has not been clarified., Objective: To establish a CIA model on the C57BL/6 background with a more predictable and defined immune response to CII., Results: Both chicken and rat CII were arthritogenic in C57BL/6 mice provided they were introduced with high doses of Mycobacterium tuberculosis adjuvant. However, contaminating pepsin was strongly immunogenic and was essential for arthritis development. H-2(b)-restricted T cell epitopes on chicken or rat CII could not be identified, but expression of A(q) on the C57BL/6 background induced T cell response to the CII260-270 epitope, and also prolonged the arthritis to be more chronic., Conclusions: The putative (auto)antigen and its arthritogenic determinants in C57BL/6 mice remains undisclosed, questioning the value of the model for addressing T cell-driven pathological pathways in arthritis. To circumvent this impediment, we recommend MHC class II congenic C57BL/6N.Q mice, expressing A(q), with which T cell determinants have been thoroughly characterised.

Background: In rheumatoid arthritis (RA), neutrophil granulocytes fuel inflammation and damage tissue in the joint by releasing cytotoxic agents, antimicrobial peptides, proteases and other inflammatory mediators. The human cathelicidin LL-37 has recently been implicated in the development of systemic lupus erythematosus and psoriasis., Objective: To elucidate if antimicrobial peptides (AMPs) contribute to the pathogenesis of arthritis., Methods: Expression of LL-37 was determined in synovial membranes from patients with arthritis and control subjects. Expression of the rat cathelicidin rCRAMP and defensins was characterised in joints, blood and secondary lymphoid organs during pristane-induced arthritis (PIA) in rats and in a transfer model of PIA induced by CD4 T cells. Serum samples of rats with arthritis were tested for IgG and IgM autoantibodies against rCRAMP by immunoblot and for interferon (IFNα) by ELISA., Results: Cathelicidins are strongly upregulated in RA synovial membranes and in joints from rats with arthritis as compared with healthy joints. Expression was most prominent in neutrophil granulocytes and macrophages/osteoclasts. Cathelicidin expression is also upregulated in the blood and spleen of pristane-injected rats, with strongest expression detected in activated CD62L- cells coexpressing granulocyte and monocyte markers. Pristane injection caused accumulation of low-density granulocytes in the blood. After pristane injection, the increased expression of rCRAMP coincided with higher levels of cell death, raised levels of interferon (IFN)α and development of autoantibodies., Conclusions: Our results show strong upregulation of cathelicidins and β-defensins coinciding with pathological events of arthritis. Higher expression and release of AMPs might contribute to development and/or maintenance of disease by systemic or local mechanisms.

Objectives: To increase understanding of the aetiology and pathogenesis of rheumatoid arthritis (RA), genetic and environmental risk factors for RA subsets, defined by the presence or absence of different anticitrullinated protein/peptide antibodies (ACPAs) targeting citrullinated peptides from α-enolase, vimentin, fibrinogen and collagen type II, were investigated., Methods: 1985 patients with RA and 2252 matched controls from the EIRA case-control cohort were used in the study. Serum samples were assayed by ELISA for the presence of anticyclic citrullinated peptides (anti-CCP) antibodies and four different ACPA fine specificities. Cross-reactivity between ACPAs was examined by peptide absorption experiments. Genotyping was performed for HLA-DRB1 shared epitope (SE) alleles and the PTPN22 gene, while information regarding smoking was obtained by questionnaire. The association of genetic and environmental risk factors with different subsets of RA was calculated by logistic regression analysis., Results: Limited cross-reactivity was observed between different ACPA fine specificities. In total, 17 RA subsets could be identified based on their different ACPA fine specificity profiles. Large differences in association with genetic and environmental determinants were observed between subsets. The strongest association of HLA-DRB1 SE, PTPN22 and smoking was identified for the RA subset which was defined by the presence of antibodies to citrullinated α-enolase and vimentin., Conclusion: This study provides the most comprehensive picture to date of how HLA-DRB1 SE, PTPN22 and smoking are associated with the presence of specific ACPA reactivities rather than anti-CCP levels. The new data will form a basis for molecular studies aimed at understanding disease development in serologically distinct subsets of RA.