Objective Anti–citrullinated protein antibodies (ACPAs) are disease‐specific biomarkers in rheumatoid arthritis (RA). More than 90% of IgG ACPAs harbor N‐linked glycans in the antibody variable (V) domain. The corresponding N‐glycosylation sites in ACPA V‐region sequences result from somatic hypermutation, a T cell–dependent process. As ample evidence indicates that T cells drive the maturation of the ACPA response prior to arthritis onset, we undertook this study to investigate whether the presence of glycans in IgG ACPA V domains predicts the transition from predisease autoimmunity to overt RA. Methods We analyzed 2 independent sets of serum samples obtained from 126 ACPA‐positive first‐degree relatives (FDRs) of RA patients. Both sets originated from an Indigenous North American population and comprised cross‐sectional and longitudinal samples of individuals who did or did not develop inflammatory arthritis. Serum IgG ACPAs were affinity‐purified and subjected to ultra high‐performance liquid chromatography–based glycan analysis. Results In both data sets, FDR‐derived IgG ACPA displayed markedly lower levels of V domain glycans (<50%) compared to IgG ACPA from RA patients. Notably, FDRs who later developed RA showed extensive V‐domain glycosylation before the onset of arthritis. Moreover, IgG ACPA V‐domain glycosylation was strongly associated with future development of RA (hazard ratio 6.07 [95% confidence interval 1.46–25.2]; P = 0.013). Conclusion Extensive glycosylation of the IgG ACPA V domain is present in a subset of predisposed FDRs of Indigenous North American RA patients. The presence of this feature substantially increases the risk of RA development. Based on these findings, we propose that glycosylation of the IgG ACPA V domain represents a predictive marker for RA development in ACPA‐positive individuals and may serve to better target prevention measures.

Objective To determine the incidence of inflammatory arthritis and autoantibody prevalence in Indigenous North American people. Methods Unaffected relatives of Indigenous North Americans with rheumatoid arthritis (RA) from central Canada and Alaska were systematically monitored from 2005 to 2017. Rheumatoid factor (RF) and anti–citrullinated protein antibodies (ACPAs) were tested at every visit, and a subset was tested for ACPA fine specificity using a custom multiplex assay. Multistate models based on all available study visits were developed to determine the likelihood of transitioning between autoantibody states, or to inflammatory arthritis. Results Eighteen of 374 relatives (4.8%) developed inflammatory arthritis during follow‐up (after a mean ± SD of 4.7 ± 2.4 years), yielding a transition rate of 9.2 cases/1,000 person‐years. Thirty percent of those who developed inflammatory arthritis were seronegative at baseline, but all were seropositive at inflammatory arthritis onset. Although 30% of ACPA/RF double‐seropositive individuals developed inflammatory arthritis (after 3.2 ± 2.2 years), the majority of these individuals did not develop inflammatory arthritis. Multistate modeling indicated a 71% and 68% likelihood of ACPA and RF seropositive states, respectively, reverting to a seronegative state after 5 years, and a 39% likelihood of an ACPA/RF double‐seropositive state becoming seronegative. Fine specificity testing demonstrated an expansion of the ACPA repertoire prior to the development of inflammatory arthritis. Conclusion Despite a high incidence of inflammatory arthritis in this cohort of at‐risk relatives of Indigenous North Americans with RA, a large proportion of autoantibody‐positive individuals do not develop inflammatory arthritis and revert back to an autoantibody‐negative state.

BackgroundAnti-citrullinated protein antibodies (ACPA) are disease-specific biomarkers in rheumatoid arthritis (RA). Recently, we described that more than 90% of ACPA-IgGs harbour N-linked glycans in the antibody variable (V) domain. The corresponding N-glycosylation sites in the amino acid backbone of ACPA V-regions result from somatic hypermutation, a T cell-dependent process. Notably, both genetic evidence and data obtained from the analysis of serum ACPA indicate that T-cells drive the maturation of the ACPA-response prior to the onset of arthritis.ObjectivesWe investigated whether ACPA-IgG carry V-domain N-glycans prior to the development of arthritis and whether the occurrence of such glycans predicts the transition from pre-disease autoimmunity to overt RA.MethodsTwo independent sets of serum samples were obtained from RA patients and from ACPA-positive first-degree relatives (FDR) of RA-patients (n=126) of an Indigenous North American (INA) population with high incidence rates of ACPA-positive RA. These samples comprised cross-sectional and longitudinal samples of individuals who did or did not transition to inflammatory arthritis. Serum ACPA-IgG were affinity-purified and subjected to enzymatic glycan release and UHPLC-based glycan analysis.ResultsACPA-IgG V-domain glycosylation could be detected in RA patients and in FDR of RA patients. In both datasets, FDR-derived ACPA-IgG displayed markedly lower levels of V-domain glycans (<50%) compared to ACPA-IgG from RA-patients. Notably, FDRs that later developed RA showed extensive V-domain glycosylation before the onset of arthritis. Moreover, the degree of ACPA-IgG V-domain glycosylation in FDRs was strongly associated with future development of RA (HR: 6.07 [95% CI: 1.46-25.2]; p=0.013).ConclusionGlycosylation of the ACPA-IgG V-domain can be detected prior to the onset of disease. Extensive glycosylation is present in a subset of predisposed FDRs of INA RA patients. The presence of this feature substantially increases the risk of RA development. These observations fit well with a pivotal role for T cells in the selection and expansion of ACPA-expressing B cells, possibly by facilitating the introduction of N-glycosylation sites in ACPA-IgG V-domains. Moreover, glycosylation of the ACPA-IgG V-domain represents a predictive marker for RA development in ACPA-positive individuals and may serve to better time and target preventive therapeutic interventions.Disclosure of InterestsLise Hafkenscheid: None declared, Emma C. de Moel: None declared, Irene Smolik: None declared, Xiaobo Meng: None declared, Stacy Tanner: None declared, Bas C. Jansen: None declared, Albert Bondt: None declared, Manfred Wuhrer: None declared, Thomas Huizinga Consultant for: Merck, UCB, Bristol Myers Squibb, Biotest AG, Pfizer, GSK, Novartis, Roche, Sanofi-Aventis, Abbott, Crescendo Bioscience Inc., Nycomed, Boeringher, Takeda, Zydus, Epirus, Eli Lilly, Rene Toes Grant/research support from: Sanofi, Hani El-Gabalawy: None declared, Hans Ulrich Scherer Grant/research support from: Sanofi, BMS

A 33-year-old man presented with a 1-week history of nausea, vomiting, and abdominal distension. Initial laboratory tests were significant for white blood cell count (12 × 10 9 /L), creatinine (1.4 mg/dL), and lactic acid dehydrogenase (2014 U/L). Computed tomography scan showed severe thickening of the gastric body, ascites, and peritoneal induration (• " Fig. 1 a). Based on these findings the patient underwent esophagogastroduodenoscopy, which revealed diffusely ulcerated mucosa involving the majority of the gastric body (• " Fig. 1 b). Endoscopic ultrasound (EUS) demonstrated gastric wall thickening of up to 50 mm (• " Fig. 1 c). Mucosal biopsies and fine-needle aspiration (FNA) of the gastric wall were performed. Epstein-Barr virus and HIV serology were negative, whereas Helicobacter pylori serology was positive. Flow cytometry and cytologic examination of the FNA specimen demonstrated monotonous atypical CD10-positive B-cells with immunohistochemical stains positive for BCL-6 and C-MYC (• " Fig. 1 df) consistent with a diagnosis of Burkitt's lymphoma. Chemotherapy was initiated with the R-CHOP regimen as well as H. pylori eradication therapy. Burkitt's lymphoma is a rare cause of B-cell non-Hodgkin lymphoma with an aggressive clinical course. Its sporadic

BackgroundDespite immune cell dysregulation being an important event preceding the onset of rheumatoid arthritis (RA), the phenotype of T and B cells in preclinical RA is less understood. The aim of this study was to characterize T and B cell populations in RA patients and their autoantibody (aAb) negative and positive first-degree relatives (FDR).MethodsCryopreserved peripheral blood mononuclear cells (PBMCs) collected at scheduled visits from aAb-(n=25), and aAb+ FDR (n=10) and RA patients (n=13) were thawed and stained using optimized antibody cocktails as per a specific 13-color T or B cell panel. Immunophenotyping was performed using a Cytoflex LX (Beckman-Coulter) flow cytometer and FlowJo software was used for analyzing the frequency of immune cell populations.ResultsMulticolor flow cytometry experiments identified an increased TIGIT expression in circulating lymphocytes of aAb+ FDR and RA patients, relative to aAb- FDR (P&lt;0.01). These TIGIT+ T cells exhibited a memory phenotype and expressed high levels of PD-1, ICOS, HLA-DR, CXCR3 and CXCR5. Moreover, increased TIGIT+ CD4 T cell frequency correlated with the frequency of PD-1+ CD4 T cells (r = 0.4705: P = 0.0043) and circulating levels of ACPA and RF. We also identified a decreased frequency of CD27+IgD- switched memory B cells in RA patients (P &lt; 0.01), while increased frequency of TIGIT+ CD4 T cells in FDR correlated with the frequency of PD1+PTEN+ B cells (r = 0.6838, P = 0.0004) and autoantibody positivity (P = 0.01).ConclusionWe demonstrate TIGIT as a distinct CD4 T cell marker for differentiating aAb- FDR from aAb+FDR and might play a critical role in regulating T and B cell crosstalk in preclinical RA.