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Clinical research projects often use traditional methods in which data collection and signing informed consent forms rely on patients’ visits to the research institutes. However, during challenging times when the medical community is in dire need of information, such as the current COVID-19 pandemic, it becomes more urgent to use digital platforms that can rapidly collect data on large numbers of patients. In the current manuscript, we describe a novel digital rheumatology research platform, consisting of almost 5000 patients with autoimmune diseases and healthy controls, that was set up rapidly during the COVID-19 pandemic, but which is sustainable for the future. Using this platform, uniform patient data can be collected via questionnaires and stored in a single database readily available for analysis. In addition, the platform facilitates two-way communication between patients and researchers, so patients become true research partners. Furthermore, blood collection via a finger prick for routine and specific laboratory measurements has been implemented in this large cohort of patients, which may not only be applicable for research settings but also for clinical care. Finally, we discuss the challenges and potential future applications of our platform, including supplying tailored information to selected patient groups and facilitation of patient recruitment for clinical trials.

Objectives To perform a systematic literature review and meta-analysis on endothelial cell (EC) markers that are involved and dysregulated in systemic lupus erythematosus (SLE) in relation to disease activity, as EC dysregulation plays a major role in the development of premature atherosclerosis in SLE. Methods Search terms were entered into Embase, MEDLINE, Web of Science, Google Scholar and Cochrane. Inclusion criteria were 1) studies published after 2000 reporting measurements of EC markers in serum and/or plasma of SLE patients (diagnosed according to ACR/SLICC criteria), 2) English language peer reviewed articles, and 3) disease activity measurement. For meta-analysis calculations, the Meta-Essentials tool by Erasmus Research Institute and of Management (ERIM) was used. Only those EC markers, which were 1) reported in at least two articles and 2) reported a correlation coefficient (i.e. Spearman’s rank or Pearson’s) between the measured levels of the EC marker and disease activity were included. For meta-analyses, a fixed effect model was used. Results From 2133 hits, 123 eligible articles were selected. The identified SLE-related endothelial markers were involved in EC activation, EC apoptosis, disturbed angiogenesis, defective vascular tone control, immune dysregulation and coagulopathy. Meta-analyses of primarily cross-sectional studies showed significant associations between marker levels and disease activity for the following endothelial markers: Pentraxin-3, Thrombomodulin, VEGF, VCAM-1, ICAM-1, IP-10 and MCP-1. Dysregulated EC markers without associations with disease activity were: Angiopoeitin-2, vWF, P-Selectin, TWEAK and E-Selectin. Conclusions We provide a complete literature overview for dysregulated EC markers in SLE comprising a wide range of different EC functions. SLE-induced EC marker dysregulation was seen with, but also without, association with disease activity. This study provides some clarity in the eminent complex field of EC markers as biomarkers for SLE. Longitudinal data on EC markers in SLE are now needed to guide us more in unravelling the pathophysiology of premature atherosclerosis and cardiovascular events in SLE patients.

Background:Over the past 10 years, several trials have investigated prevention of rheumatoid arthritis (RA) by biological disease modifying antirheumatic drug (DMARD) therapies. Early treatment in RA-risk individuals in the preclinical phase – i.e. before the disease has been fully established – has the potential to prevent disease or delay its onset, which may have a positive impact on both patient and society. The PRAIRI study was a randomized, double-blind, controlled trial in which seropositive arthralgia patients received a single dose of rituximab (RTX) or placebo (PBO). This resulted in a significant delay of arthritis development of up to 12 months [1]. Secondary outcomes were the effects of RTX treatment on quality of life as measured by various patient reported outcomes (PROs).Objectives:To evaluate the impact of RTX treatment on the quality of life of RA-risk individuals in the PRAIRI study as measured by various patient reported outcomes (PROs).Methods:Eighty-one RA-risk individuals were included in the PRAIRI study between 2010 and 2013, and treated with one single dose of either PBO or 1000 mg RTX; all study subjects also received standard co-medication, consisting of 100 mg methylprednisolone and anti-histamines. Data on quality of life were collected at baseline and 1, 4, 6, 12, and 24 months using the following PRO-questionnaires: visual analogue scale (VAS) pain, health assessment questionnaire (HAQ) score, EuroQol five dimension (EQ-5D), and both physical component score (PCS) and mental component score (MCS) of the 36-item short form heath survey (SF-36). Changes in quality of life over time and potential impact on perceived arthritis severity at onset of clinically overt disease were analyzed for both treatment groups.Results:PRO data were available for 78 patients. No convincing effect on VAS pain, HAQ score, EQ-5D, or PCS and MCS of SF-36 was found in either RTX or PBO groups. Compared to the PBO group, the RTX group had slightly worse baseline scores for VAS pain (mean ± SEM: PBO = 23.11 ± 3.68; RTX = 30.88 ± 3.41) and HAQ score (PBO = 0.26 ± 0.06; RTX = 0.56 ± 0.09). However, baseline PRO scores were similar to the general “healthy” population and remained stable during the two-year follow up period for both RTX and PBO group (Figure 1A). At the time of arthritis development, we also did not observe a significant difference in VAS pain (mean ± SEM: PBO = 51.29 ± 5.99; RTX = 55.83 ± 6.76), HAQ score (PBO = 0.86 ± 0.20; RTX = 0.93 ± 0.15), EQ-5D (PBO = 0.64 ± 0.05; RTX = 0.63 ± 0.06), PCS (PBO = 39.63; ± 3.08; RTX = 41.13 ± 2.54) or MCS (PBO = 50.91 ± 1.83; RTX = 48.31 ± 3.93) of the SF-36 questionnaire (Figure 1B) compared to baseline.Conclusion:A single dose of RTX in RA-risk individuals delayed the onset of arthritis by a year, but did not have a positive impact on quality of life as measured by various PROs. In RA-risk individuals developing arthritis, RTX also did not significantly alter PROs and/or perceived disease severity at the time of arthritis development. Since RTX did not have a negative effect on quality of life either, these data underscore that RTX treatment is well-tolerated in the preclinical phase of RA but additional research is required to determine whether RTX based strategies can prevent RA.REFERENCES:[1] Gerlag DM et al., “Effects of B-cell directed therapy on the preclinical stage of rheumatoid arthritis: the PRAIRI study”, Ann Rheum Dis. 2019 Feb;78(2):179-185.Figure 1.PRO scores for VAS pain, HAQ, EQ-5D, PCS, and MCS of SF-36 questionnaire, stratified for treatment (PBO=orange line; RTX=blue line), reported as mean and SEM. On the left, PRO scores are shown for a two-year period (n at baseline: PBO= 37; RTX= 41) (A). On the right, PRO scores (n PBO= 14; n RTX=12)at baseline and arthritis visit are shown for patients that developed RA within study time (B). Median (IQR) time of RA onset was 12 (2-15) months in PBO group and 16.5 (9.75-28) in RTX group.Acknowledgements:NIL.Disclosure of Interests:Giulia Frazzei: None declared, Sophie Cramer: None declared, Robert Landewé: None declared, Karen Maijer: None declared, Danielle Gerlag: None declared, Paul Peter Tak: None declared, Niek de Vries: None declared, Lisa G.M. van Baarsen: None declared, Ronald F. van Vollenhoven Consultancy and/or speaker: AbbVie, AstraZeneca, Biogen, BMS, Galapagos, GSK, Janssen, Pfizer, RemeGen, UCB, Support for Research or Educational programs (institutional grants): AstraZeneca, BMS, Galapagos, MSD, Novartis, Pfizer, Roche, Sanofi, UCB, Sander W. Tas: None declared.

Background:One of the main characteristics of rheumatoid arthritis (RA) is angiogenesis, which is linked to both the disease’s activity and duration. Two key proteins, vimentin and αvβ3 integrin, are critically involved in angiogenesis in RA. As a result, both may be used as diagnostic candidates for early detection and precise RA treatment. Positron emission tomography (PET) scans using radiotracers for αvβ3 and vimentin may provide non-invasive monitoring of angiogenesis which is currently being investigated in a rat model of arthritis.Objectives:To investigate the potential of integrin and vimentin as targets for molecular imaging of angiogenesis in RA in a preclinical Antigen-Induced Arthritis (AIA) rat model of arthritis.Methods:AIA rats’ knee tissues were examined using immunohistochemical (IHC) staining to determine the expression of vimentin and integrin αvβ3. For both targets we have PET tracers available. In the AIA model, following systemic immunization by injecting a mixture of methylated bovine serum albumin (mBSA) in complete Freund’s adjuvant (CFA) and a customized Bordetella pertussis (CBP) antigen, arthritis is induced in one knee with the contralateral knee serving as control [1]. QuPath software was used to analyze stained sections from AIA and their contralateral control knees, and healthy rat knees (mean ± SEM). In addition to IHC, we investigated the inhibitory effects of microdose Fluciclatide, a small synthetic cyclic peptide that has a high affinity for integrins αvβ3/5. This was performed in a 3D spheroid angiogenesis model [2]. Statistical analysis involved one-way ANOVA for all experiments (n=3-6).Results:In arthritic knee tissues, vimentin expression was markedly and significantly increased in arthritic compared to both control and normal knees (6.5 and 7.5 fold respectively, p≤ 0.001) (Figure 1A). Additionally, αvβ3 expression was elevated in arthritic knees compared to normal rat knees (3.7-fold, p<0.05), and contralateral knees (2.6-fold), however this difference did not achieve statistical significance (Figure 1B). With demonstrated efficacy in cancer imaging research, [18F]-Fluciclatide has potential in RA imaging as well. The 3D spheroid angiogenesis model demonstrated that Fluciclatide at microdoses ≤ 0.5 μM does not exhibit inhibitory biologic effects, making it a viable agent for in vivo imaging (Figure 2).Conclusion:The current study shows that the AIA model is suitable to study novel PET tracers targeting both vimentin and integrin αvβ3 for angiogenesis imaging in arthritis. Moreover, markedly increased vimentin and integrin αvβ3 expression in arthritic knees underscores aberrant angiogenesis that provide valuable insights into arthritis pathology. Ongoing PET imaging with [18F]-Fluciclatide for αvβ3 [3] and 89Zr-anti-vimentin nanobodies [4] holds promise for precise visualization and assessment of angiogenesis in RA.REFERENCES:[1] Chandrupatla DM, et al. Biomed Res Int. 2015;2015:509295.[2] Maracle, C.X., et al. Rheumatology (Oxford). 2017;56(2):294-302.[3] Battle, M.R., et al. J Nucl Med. 2011;52(3):424-430.[4] van Beijnum, J.R., et al. Nat Commun. 2022;13(1):2842.Figure 1.IHC staining of vimentin (A) and integrin αvβ3 (B) in AIA and healthy rats knee tissues. The scale bars represent 200 μm for the reference images and 100 μm for the magnified images. Quantitative analysis of stained sections from AIA knees, their respective control knees, as well as healthy rat knees was conducted using QuPath software by training object classifier for recognizing positive cells. The data is presented as mean ± SEM for AIA rats (n=6) and normal rats (n=5). (**p≤ 0.001, *p< 0.05, ns:not significant)Figure 2.Representative confocal pictures (10X) of the 3D spheroid-based model of angiogenesis composed of endothelial cells (EC) (cyan) and normal human dermal fibroblasts (NHDF) (magenta) and impact of microdosing of Fluciclatide on sprouting. Cell tracker dyes was used to monitor cells. Significant induction of sprouting was observed following stimulation with the growth factors VEGF/bFGF. Data represents Mean ± SEM of 3 independent experiments. All the scale bars are 200 μm. (* p<0.05, ns: not significant)REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a small-vessel autoimmune vasculitis which can result in life-threatening kidney failure or pulmonary haemorrhage. The most prevalent phenotypes are granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA), mainly associated with anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO), respectively. Besides the presence of ANCA, increased BAFF levels and alterations in the peripheral B cell compartment, the beneficial clinical effects of peripheral B cell depletion using rituximab (anti-CD20) therapy ultimately confirmed the importance of B cells in AAV. However, this treatment results in impaired humoral immune responses and permits the survival of (autoreactive) long-lived plasma cells. Hitherto, characterization of the B lineage in AAV relies mainly on the peripheral blood, but it is imperative to also study the difficult-to-access plasma cell niches in other compartments of the immune system, such as lymph nodes and bone marrow, as well as in the target tissues (i.e. lungs/kidneys), to fully determine the role of B lineage cells in the pathogenesis of AAV.Objectives:To profile immune cells in kidney biopsies from AAV patients with active disease and a nephrectomy control using a single-cell RNA sequencing (scRNAseq) approach, and characterize the different clusters of B lineage cells in more detail.Methods:Needle-core kidney biopsies were obtained from 4 patients with AAV glomerulonephritis and one nephrectomy control. Live CD45+ kidney immune cells were sorted from the kidney single-cell suspensions and processed for single-cell data generation and analysis, performing the adequate filtering and pre-processing steps. Mapping the dataset to the mature immune kidney cell atlas was used for annotation.Results:We identified 24 clusters based on the reference at resolution 1, from which 2 B cell enriched clusters (based on CD19/CD20 expression; 5.07% of total) and one plasmablast/plasma cell cluster (based on CD38/CD138 expression; 0.45% of total) were selected for further analysis. These 3 clusters were re-clustered at low and high resolutions, rendering 4 to 9 different subpopulations. The 4 clusters obtained at low resolution suggest the presence of memory B cells (MS4A1, CD19, CD27, PAX5), naïve B cells (MS4A1, CD19, IGHD, PAX5), a plasmablast-like population (MS4A1, CD19, CD27, TNFRSF13C, XBP1) and plasma cells (CD38, IGHG1, SDC1, IRF4, XBP1, PRDM1). Generally, the relative mean frequencies of the B lineage populations were 48.4% memory B cells, 41.7% naïve B cells, 4.1% plasmablasts, and 5.8% plasma cells. With higher resolution analysis, 4 different subtypes of memory B cells were observed, in addition to 3 naïve/germinal center-like B cells, and the 2 previously well-defined plasmablast-like and plasma cell clusters, which need to be characterized in more detail.Conclusion:These preliminary data characterise for the first time cells of the B and plasma cell lineage in kidney biopsies from AAV patients with glomerulonephritis using scRNAseq. Subsequent in-depth study of the resulting clusters may allow identification of subsets potentially specific for AAV (or even ANCA subtype) that could be actively involved in the immune response in the affected kidneys. Finally, gene set enrichment analysis of the data may reveal differential gene expression patterns and intracellular signalling pathways involved, which may provide novel targets for more directed therapies.REFERENCES: NIL. Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:Many rheumatoid arthritis (RA) patients experience an asymptomatic phase preceding clinical onset of disease, in which antibodies against citrullinated proteins (aCCP or ACPA) develop. These antibodies can be detected years before onset of arthritis, and along with rheumatoid factor (RF) are a risk factor for the development of RA. Moreover, in this preclinical phase of RA nonspecific signs and symptoms, such as join pain (arthralgia) and morning stiffness, may occur. However, not all RA-risk individuals will develop arthritis. Therefore, the identification of those individuals who would benefit the most from preventive treatment remains a key challenge. We analyzed data available from the expanded Reade cohort of seropositive arthralgia individuals. Part of this dataset was previously used to derive a prediction rule for the stratification of individuals at risk of developing RA (1); two hundred and thirty-four additional subjects have been included in the Reade cohort and the extended dataset was used in the analysis.Objectives:We aimed to identify predictors of RA development in a cohort of at risk-individuals with arthralgia and systemic autoimmunity associated with RA (ACPA and/or RF positive) followed for up to five years.Methods:Six hundred and seventeen ACPA- and/or RF-positive individuals with arthralgia were included in the study cohort between 2004 and 2019 and were followed up for up to five years, or until arthritis development. We performed univariable logistic regression on clinically and biologically relevant parameters, such as age, gender, smoking habit and alcohol consumption, RA family history, morning stiffness, joint tenderness and stiffness, visual analogue scale (VAS) pain, length and type of symptoms experienced, presence of shared epitope (SE), high levels of C reactive protein, and auto-antibody status. Moreover, we assessed the ability of previously mentioned baseline characteristics to predict RA development via Cox proportional hazard analysis. Alcohol consumption, morning stiffness, and SE positivity were excluded from survival analysis due to the number of missing data.Results:Univariate logistic regression showed that individuals with first degree relatives (FDR) with RA, who had intermittent symptoms < 12 months at inclusion, morning stiffness > 1 h, or were positive for shared epitope were more likely to develop RA. Moreover, antibody status was also associated with RA development: individuals with high ACPA titers or being double positive for both ACPA and RF had increased risk for developing RA compared to those with only RF or low ACPA titers. Consistent with this, survival analysis via Cox hazard analysis found that having FDR with RA, intermittent symptoms, VAS pain score ≥ 50 and antibody status were significantly associated with the risk of developing RA (Table 1). Out of the 24 subjects who had a FDR with RA, intermittent symptoms AND either high ACPA titers or were double positive for ACPA and RF, 20 individuals (83%) developed RA.Conclusion:We found that, in arthralgia individuals with systemic autoimmunity associated with RA, having a FDR with RA, intermittent symptoms, a VAS pain score ≥ 50, and high ACPA or testing positive for both RF and ACPA are predictors of future RA development. These results will aid in the identification of individuals at highest risk of developing RA, and who could benefit the most from preventive treatment.REFERENCES:[1] van de Stadt LA, Witte BI, Bos WH, van Schaardenburg D. A prediction rule for the development of arthritis in seropositive arthralgia patients. Ann Rheum Dis. 2013;72(12):1920-6Acknowledgements:NIL.Disclosure of Interests:Giulia Frazzei: None declared, Robert Landewé: None declared, Carlijn Wagenaar: None declared, Lotte A. van de Stadt: None declared, D. van Schaardenburg: None declared, Sander W. Tas: None declared, Ronald F. van Vollenhoven Consultancy and/or speaker: AbbVie, AstraZeneca, Biogen, BMS, Galapagos, GSK, Janssen, Pfizer, RemeGen, UCB, Support for Research or Educational programs (institutional grants): AstraZeneca, BMS, Galapagos, MSD, Novartis, Pfizer, Roche, Sanofi, UCB.

Background:Rheumatoid Arthritis (RA) is a progressive and systemic autoimmune disorder associated with chronic and destructive inflammation of the joints. In vitro approaches simulating RA synovial tissue are crucial in preclinical and translational research to expand our knowledge on RA human pathophysiology and to test new diagnostic and therapeutic applications. Recently, we developed a novel RA synovial tissue model by incorporating RA fibroblast-like-synoviocytes (RAFLS), endothelial cells (ECs), and macrophages, in which both angiogenesis and inflammatory processes can be studied.Objectives:In the current study, we set out to examine the potential of the spheroid-based RA synovial tissue model in the presence of either “M1”-like or “M2”-like macrophages to modulate key RA inflammatory processes using the established TNF inhibitor etanercept, as well as novel small molecule inhibitors of the NF-κB intracellular signaling pathways.Methods:Spheroids of RAFLS, ECs and either pro-inflammatory “M1”-like macrophages or anti-inflammatory “M2”-like macrophages derived from monocytes were formed, followed by growth in 3 dimensions in a collagen-based matrix. The spheroids were left unstimulated, or cultured in the presence of the pro-angiogenic factors VEGF/bFGF (GF), the pro-inflammatory cytokine Tumor-Necrosis Factor α (TNF) or RA synovial fluid (SF). Next, the 3D model was compared to investigate the impact of macrophage phenotype on different readouts. Finally, the 3D model containing the “M1”-like macrophages was used to test therapeutic compounds in the presence of GF, SF or TNF. Morphological changes were quantified using confocal imaging and digital image analysis by machine learning, while concentrations of pro-inflammatory soluble factors were measured in culture supernatants.Results:The presence of SF significantly enhanced containment of the ECs and pro-inflammatory “M1”-like macrophages within the spheroid core compared to the unstimulated condition. Overall, the anti-inflammatory “M2”-like macrophages did not sustain within the core and migrated away to a larger extent than its “M1”-like counterpart. No significant changes were observed upon the different stimulation strategies. In the 3D model containing the “M1”-like macrophages, the addition of etanercept prior to GF stimulation decreased spheroid outgrowth by 30%, while a small molecule NF-κB-inducing kinase (NIK) inhibitor fully prevented spheroid outgrowth. Moreover, the concentrations of interleukin (IL)-6 in culture supernatants upon GF stimulation were diminished by around 30% for both compounds. Secondly, all compounds tested strongly inhibited macrophage containment within the core upon SF stimulation compared to the conditions with SF only. Lastly, stimulation with TNF triggered the spheroid core to collapse across all conditions and significantly increased IL-6 production, which could be inhibited by etanercept.Conclusion:We demonstrated the capacity of the 3D model of synovial inflammation to replicate alterations in cellular interactions depending on macrophage phenotype. Moreover, we showed the inhibitory effects of therapeutic compounds on spheroid outgrowth, cell migration and soluble mediator production, highlighting the potential of this novel 3D model to test the effect of various therapeutic applications on RA synovial inflammation.REFERENCES:[1] Philippon EML, van Rooijen LJE, Khodadust F, van Hamburg JP, van der Laken CJ and Tas SW (2023) A novel 3D spheroid model of rheumatoid arthritis synovial tissue incorporating fibroblasts, endothelial cells, and macrophages. Front. Immunol. 14:1188835. doi: 10.3389/fimmu.2023.1188835.Acknowledgements:NIL.Disclosure of Interests:Eva Philippon: None declared, Jan Piet Van Hamburg: None declared, Lisanne van Rooijen: None declared, Gary P Sims AstraZeneca, Conny J. van der Laken: None declared, Sander W. Tas: None declared

Idiopathic inflammatory myopathies (IIM) or myositis are characterized as inflammatory, autoimmune disorders with a wide spectrum of symptoms and affected tissues. The presence of circulating autoantibodies and B-lineage cells in affected tissues, point towards an essential role of B cells in the pathogenesis of IIM. This sparked interest in the B cell depleting anti-CD20 antibody rituximab as a new treatment opportunity in IIM. However, despite its beneficial effects, rituximab results in long-term B cell depletion and does not target CD20-negative, long-lived plasma cells, which may maintain the autoimmune response. Hence, further research aimed at more specific and reversible therapies targeting B and plasma cells is warranted. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signalling pathway is essential for several aspects of B cell biology, making it an attractive novel target for patients with IIM. To study whether targeting the JAK-STAT signalling pathway using tofacitinib, a small molecule inhibitor specific for JAK1/JAK3, impairs essential functions of B cells isolated from patients with IIM. B lineage cell phenotyping was performed by flow cytometry, using PBMCs obtained from 3 IIM phenotypes (dermatomyositis, overlap-myositis, anti-synthetase syndrome) and healthy donors (HD). Functional assays were performed culturing PBMCs in the presence of tofacitinib, and the stimuli IL-21/anti-CD40 or IL-2/CpG to activate JAK/STAT signalling in a T cell-independent or T cell-dependent manner, respectively. After a 6-day culture, the effects of tofacitinib on B cell proliferation, differentiation, and antibody production were assessed by flow cytometry and ELISA. Peripheral blood B cell composition from patients with IIM showed reduced fractions of memory B cells, and an increase in transitional and naive B cells in comparison to HD reference values. Targeting JAK/STAT signalling using tofacitinib significantly reduced IL-21/anti-CD40 induced B cell proliferation, plasmablast differentiation and antibody production in B cells of patients with IIM and HD. However, this effect was observed to a lesser extent upon stimulation with IL-2/CpG. Interestingly, tofacitinib exerted its effects irrespective of the IIM phenotype. Our data demonstrate that the composition of the peripheral B cell compartment in IIM patients is altered. Furthermore, tofacitinib treatment in vitro results in the inhibition of functional B cell responses, including proliferation, plasmablast differentiation, and antibody production, which was irrespective of the clinical phenotype. Consequently, targeting of JAK/STAT signalling may represent a novel treatment modality in myositis. NIL. NIL. None Declared.

Background:In individuals with RA-associated antibodies (RA-risk individuals), B-cell depleting therapy delays the onset of arthritis, pointing to a key role for B-cell clones in the onset of arthritis[1]. Little is known about the dynamics and phenotype of the dominant B-cell clones in this phase of the disease, and the effect of Rituximab on these clones.Objectives:To study the dynamics and phenotype of dominant B-cell clones in peripheral blood of RA-risk patients, and to evaluate the effect of rituximab on these clones.Methods:Every 6 months we performed adaptive immune receptor sequencing (AIRR-seq) of the BCR-heavy (BCRH) chain on freshly isolated FACS-sorted phenotyped peripheral blood cells in a cohort of untreated RA-risk individuals (DOMINO study). In addition, we performed AIRR-sequencing on peripheral blood samples obtained from RA-risk individuals at screening, baseline, 6 and 12 months after treatment with rituximab (RTX; anti-CD20) or placebo (PRAIRI study).Results:In the DOMINO study we showed that dominant BCRH signatures are encoded by plasmablasts and/or plasma cells. Interestingly, after 6 and 12 months these dominant signatures in peripheral blood are present in low frequences and encoded by memory B cells. RTX induces clonal BCRH depletion in sequential peripheral blood samples, followed by a gradual re-establishment to the pre-treatment BCRH repertoire from 6 months onwards up to 12 months after treatment. Some of the pre-treatment dominant clones do come back in low frequencies, but none resurfaced as dominant clones after 12 months.Conclusion:In RA-risk individuals the BCRH repertoire in peripheral blood continuously changes over time. The most dominant BCRH signatures are encoded by plasmablasts and plasma cells, and intriguingly, over time these clonal BCRH signatures disappear from the plasmablast/plasma cell compartment and reappear as low frequency memory B cell clones. During recovery from RTX-induced B-cell depletion, after approximately 6 months, some of the dominant clones resurfaced, albeit at low frequencies.REFERENCES:[1] Gerlag, D.M., et al., Effects of B-cell directed therapy on the preclinical stage of rheumatoid arthritis: the PRAIRI study. Ann Rheum Dis, 2019. 78(2): p. 179-185.Acknowledgements:NIL.Disclosure of Interests:Anne Musters: None declared, Aram Al-soudi: None declared, Dornatien Anang: None declared, Ilse Niewold: None declared, Lisa G.M. van Baarsen: None declared, Barbera van Schaik: None declared, Antoine van Kampen: None declared, Danielle Gerlag Current employee of UCB pharma. UCB did not have any role in this project/study, Paul Peter Tak Current employee of Candel therapeutics. Candel therapeutics did not have any role in this study, Sander W. Tas: None declared, Niek de Vries: None declared.

Interleukin 10 (IL-10) is an antiinflammatory cytokine, but also promotes B cell responses and plays a pathogenic role in systemic lupus erythematosus (SLE). CD4⁺CCR6⁺IL-7R⁺T cells from human tonsils produced IL-10 following stimulation by naïve B cells, which promoted B cell immunoglobulin G (IgG) production. These tonsillar CCR6⁺B helper T cells were phenotypically distinct from follicular helper T (TFH) cells and lacked BCL6 expression. In peripheral blood, a CCR6⁺T cell population with similar characteristics was identified, which lacked Th17- and TFH-associated gene signatures and differentiation-associated surface markers. CD4⁺CCR6⁺T cells expressing IL-10, but not IL-17, were also detectable in the spleens of cytokine reporter mice. They provided help for IgG production in vivo, and expanded systemically in pristane-induced lupus-like disease. In SLE patients, CD4⁺CCR6⁺IL-7R⁺T cells were associated with the presence of pathogenic anti-dsDNA (double-stranded DNA) antibodies, and provided spontaneous help for autoantibody production ex vivo. Strikingly, IL-10–producing CCR6⁺T cells were highly abundant in lymph nodes of SLE patients, and colocalized with B cells at the margins of follicles. In conclusion, we identified a previously uncharacterized population of extrafollicular B helper T cells, which produced IL-10 and could play a prominent pathogenic role in SLE.