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.

Objectives Early treatment of individuals at risk of developing rheumatoid arthritis (RA-risk) in the preclinical phase has the potential to positively impact both patients and society by preventing disease onset and improving patients' quality of life. The PRAIRI study was a randomised, double-blind, placebo-controlled trial with the B-cell depleting agent rituximab (RTX), which resulted in a significant delay of arthritis development of up to 12 months in seropositive RA-risk individuals. Here, we report our findings on patient-reported outcomes (PROs) in this study population.Methods Seventy-eight RA-risk individuals were treated with one single dose of either placebo (PBO) or 1000 mg RTX plus 100 mg methylprednisolone (MP) and anti-histamines, regardless of treatment allocation, as co-medication. Data on quality of life were collected at baseline and 1, 4, 6, 12 and 24 months using established PRO questionnaires (visual analogue scale (VAS) pain, health assessment questionnaire disability index (HAQ-DI) score, EuroQol five dimension (EQ-5D) and both physical and mental component score of the 36-item short-form heath survey (SF-36)).Results No significant changes in quality of life over a 2 year follow-up were observed in at-risk individuals treated with RTX compared to PBO given the PRO scores at 24 months (mean difference +/- SEM: HAQ score=0.07 +/- 0.16; EQ-5D=-0.02 +/- 0.05; VAS pain=11.11 +/- 7.40). Furthermore, no significant effect of treatment on perceived arthritis severity at the time of clinically manifest disease (arthritis) was found.Conclusion One single dose of RTX plus MP administered to RA-risk individuals does not have a meaningful and measurable positive effect on PROs after 2 years of follow-up and/or perceived disease severity at the time of arthritis development.Trial registration number Trial registered at EU Clinical Trial Register, EudraCT Number: 2009-010955-29 (https://www.clinicaltrialsregister.eu/ctr-search/search?query=Prevention+of+RA+by+B+cell+directed+therapy).

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. 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.Figure 1PRO 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.

Background Immunological and genetic studies implicate T cells in the initiation and progression of rheumatoid arthritis (RA) [1]. Additionally, the diversity of the T cell repertoire is known to be reduced in RA, suggesting an enrichment of disease related clonal signatures [2]. Objectives To study whether changes in the T-cell receptor repertoire (TCR) in lymph node (LN) and peripheral blood (PB) precede the onset of arthritis in seropositive at-risk individuals. Methods Unique molecular identifier (UMI)-based RNA sequencing of the TCRβ repertoire in LN biopsies and PB was performed in 9 healthy controls and 26 IgM-RF and/or ACPA positive at-risk individuals. Six of these individuals developed arthritis (RA) during at least 3 years of follow-up. PB and LN biopsies were analyzed during the at-risk phase and at RA onset. Results The TCRβ repertoire in LNs of autoantibody positive at-risk individuals harbors expanded TCRβ clones which can hardly be found in LNs of healthy controls. Regardless of whether the at-risk individuals developed arthritis or not, expanded TCRβ clones in LNs could be retrieved in peripheral blood. On RA onset, the LNs showed a marked decrease in the diversity of their TCRβ repertoire and, expanded TCRβ clones present during the at-risk phase could still be found in lymph nodes on arthritis onset. Conclusion Expanded TCRβ clones are present in lymph nodes and peripheral blood of individuals at risk of developing RA which can be retrieved in peripheral blood. Expanded TCRβ clones present in LNs during the at-risk phase persist on arthritis onset. Our analyses show a clear decrease in the diversity of the TCRβ repertoire towards onset of RA, implicating a key role for T-cell adaptive responses in steering onset of seropositive RA. References [1]Cope, A.P., T cells in rheumatoid arthritis. Arthritis Research & Therapy, 2008. 10(1): p. S1. [2]Jiang, X., et al., Comprehensive TCR repertoire analysis of CD4+ T-cell subsets in rheumatoid arthritis. Journal of Autoimmunity, 2020. 109: p. 102432. Acknowledgements: NIL. Disclosure of Interests None Declared.

Background Follicular T helper (Tfh) cells provide key help for B cell differentiation into plasma and memory B cells and are essential in germinal center formation and (auto) antibody generation (1). Objectives To gain more insights into the role of Tfh cells in RA development, we assessed whether Tfh cells have an altered frequency, phenotype and cytokine profile in peripheral blood, lymphoid and synovial tissues during the earliest stages of RA development. Methods Using flow cytometry, we analyzed the phenotpe, frequencies and cytokine profile of Tfh cells and B cells in peripheral blood and lymph node biopsies of healthy controls (HCs), autoantibody positive individuals at risk for developing RA (RA-risk individuals), and early RA patients. Using immunofluorescene we confirmed the presence of Tfh in B cell follicles of lymph nodes and synovial tissue biopsies of RA patients. Results In blood, the frequency of Tfh cells did not differ between study groups. In lymphoid and synovial tissue, Tfh cells were localized in B-cell areas, and their frequencies correlated strongly with the frequency of CD19 + B cells. Compared to lymphoid tissue of healthy controls, that of RA patients and RA-risk individuals, showed more CD19 + B cells and more CD4 + CXCR5 + and CD8 + CXCR5 + follicular T cells. Of note, compared to healthy controls, specifically lymph node Tfh cells of RA-risk and early RA patients produced less IL-21 upon ex-vivo stimulation. Conclusion Tfh cells are localised in B-cell rich areas in lymphoid and synovial tissues of early RA patients. The analysis of lymph node tissue in early RA patients showed increased frequencies of Tfh cells, where they clearly associate with B cells. Interestingly, IL-21 production is already aberrant in the very early at risk phase of the disease. Our data suggest that Tfh cells may present a novel rationale for therapeutic targeting during the preclinical stage of RA to prevent further disease progression. References [1]Ise W, Fujii K, Shiroguchi K, Ito A, Kometani K, Takeda K, et al. T Follicular Helper Cell-Germinal Center B Cell Interaction Strength Regulates Entry into Plasma Cell or Recycling Germinal Center Cell Fate. Immunity. 2018;48(4):702-15.e4. Acknowledgements We thank the participants in the study, the radiology department at the Academic Medical Center (AMC) for lymph node sampling; the flow cytometry facility at the Hematology department at AMC, especially J.A. Dobber; and the core facility Cellular Imaging of the Amsterdam UMC, location AMC. Disclosure of Interests Dornatien Anang: None declared, Tamara H.Ramwadhdoebe: None declared, Janine Hahnlein: None declared, Bo van Kuijk: None declared, Smits Noortje: None declared, Krijn P. van Lienden: None declared, Mario Maas: None declared, Danielle Gerlag Employee of: Employee of UCB Pharma. UCB pharma was not involved in this study, Paul-Peter Tak Employee of: Employee of Candel therapeutics. Candel therapeutics was not involved in the study, Niek de Vries: None declared, Lisa van Baarsen: None declared.

Interleukin (IL)-17 and tumor necrosis factor-alpha (TNF)-α are key players in psoriatic arthritis (PsA) pathogenesis. While both cytokines can be therapeutically targeted with beneficial clinical outcome, it is unclear whether inhibiting one cytokine will affect the other at sites of inflammation. If both act independently, this might provide a rationale for dual or combined inhibition of both cytokines. Here, we evaluated the effect of TNF blockade in PsA patients on IL-17 levels in both skin and synovial tissue biopsies. PsA patients with mild psoriatic skin lesions were randomized to receive either adalimumab or placebo for four weeks. Synovial and skin biopsies were obtained at weeks zero and four. Skin from healthy donors (HDs) was used for comparison. Expression of IL-17A, IL-17F, IL-17RA and IL-17RC was assessed by immunohistochemistry and analyzed with digital image analysis. We found relatively low levels of IL-17 and its receptors in the skin of PsA patients compared to HD, and only IL-17F in the dermis of lesional psoriatic skin was significantly higher compared to HD skin (p = 0.0002). Histologically IL-17A, IL-17F, IL-17RA and IL-17RC in skin and synovial tissue were not downregulated by adalimumab treatment. Thus, in this cohort of PsA patients with mild psoriasis, TNF blockade did not affect the protein levels of IL-17 cytokines and its receptors in skin and synovium, despite reduced cellular inflammation and improved clinical outcome for joint involvement.

Follicular T helper cells (Tfh cells) provide key B-cell help and are essential in germinal center formation and (auto) antibody generation. To gain more insight into their role during the earliest phase of rheumatoid arthritis (RA), we analyzed their frequencies, phenotypes, and cytokine profiles in peripheral blood and lymph node biopsies of healthy controls (HCs), autoantibody-positive individuals at risk for developing RA (RA-risk individuals), and early RA patients. Subsequently, we confirmed their presence in lymph nodes and synovial tissue of RA patients using immunofluorescence microscopy. In the blood, the frequency of Tfh cells did not differ between study groups. In lymphoid and synovial tissues, Tfh cells were localized in B-cell areas, and their frequency correlated with the frequency of CD19(+) B cells. Compared to lymphoid tissues of healthy controls, those of RA patients and RA-risk individuals showed more CD19(+) B cells, CD4(+)CXCR5(+) follicular helper T cells, and CD8(+)CXCR5(+) follicular T cells. These Tfh cells produced less IL-21 upon ex vivo stimulation. These findings suggest that Tfh cells may present a novel rationale for therapeutic targeting during the preclinical stage of RA to prevent further disease progression.

Background Previous work has shown subtle infiltration of synovial T cells in the absence of overt synovial inflammation in individuals at risk of developing rheumatoid arthritis (RA). Objective To study the molecular changes in synovium preceding arthritis development in at risk individuals. Materials and methods We included sixty-seven individuals with arthralgia who were IgM rheumatoid factor (RF) and/or anti-citrullinated protein antibody (ACPA) positive and without any evidence of arthritis. All individuals underwent mini-arthroscopic synovial tissue sampling of a knee joint at baseline and were followed prospectively. An explorative genome-wide transcriptional profiling study was performed on synovial tissue using Agilent arrays (discovery cohort). Survival analysis was used to identify transcripts associated with arthritis after follow up. Expression levels of differentially expressed genes were validated using quantitative real-time PCR (qPCR). Immunohistochemistry was used to study gene candidates at the protein level in situ. Results In the discovery cohort, 6 of the 13 at risk individuals developed RA after a median follow-up time of 20 months (IQR 2 – 44; pre-RA). The 7 individuals who did not develop RA had a median follow-up time of 85 months (IQR 69 – 86). Using a False Discovery Rate of <5% we found increased expression of 3,151 transcripts correlating with a higher risk of arthritis development, whereas increased expression of 2,437 transcripts correlated with a lower risk. Gene set enrichment analysis revealed that synovial biopsies of pre-RA individuals display higher expression of genes involved in several immune response-related pathways compared with biopsies of individuals who did not develop RA. In contrast, lower expression was observed for genes involved in extracellular matrix receptor interaction, Wnt-mediated signal transduction and lipid metabolism. Two-way hierarchical cluster analysis of 27 genes measured by qPCR classified the synovial biopsies of 61 individuals into two groups, where pre-RA individuals (n=16) showed a preference to cluster together. Synovial tissue from pre-RA individuals were more likely to show podoplanin positive cells and lower lipid staining compared with synovial tissue from individuals who did not develop RA. Conclusion Molecular changes can be detected in synovial tissues before clinical onset of arthritis. Alterations in the immune response genes and lipid metabolism are associated with development of arthritis.

In this study, we sought to characterize synovial tissue obtained from individuals with arthralgia and disease-specific auto-antibodies and patients with established rheumatoid arthritis (RA), by applying an integrative multi-omics approach where we investigated differences at the level of DNA methylation and gene expression in relation to disease pathogenesis. We performed concurrent whole-genome bisulphite sequencing and RNA-Sequencing on synovial tissue obtained from the knee and ankle from 4 auto-antibody positive arthralgia patients and thirteen RA patients. Through multi-omics factor analysis we observed that the latent factor explaining the variance in gene expression and DNA methylation was associated with Swollen Joint Count 66 (SJC66), with patients with SJC66 of 9 or more displaying separation from the rest. Interrogating these observed differences revealed activation of the immune response as well as dysregulation of cell adhesion pathways at the level of both DNA methylation and gene expression. We observed differences for 59 genes in particular at the level of both transcript expression and DNA methylation. Our results highlight the utility of genome-wide multi-omics profiling of synovial samples for improved understanding of changes associated with disease spread in arthralgia and RA patients, and point to novel candidate targets for the treatment of the disease.

Background: In rheumatoid arthritis (RA) the cause for loss of tolerance and anti-citrullinated protein antibody (ACPA) production remains unidentified. Mouse studies showed that lymph node stromal cells (LNSCs) maintain peripheral tolerance through presentation of peripheral tissue antigens (PTAs). We hypothesize that dysregulation of peripheral tolerance mechanisms in human LNSCs might underlie pathogenesis of RA. Method: Lymph node (LN) needle biopsies were obtained from 24 RA patients, 23 individuals positive for RA-associated autoantibodies but without clinical disease (RA-risk individuals), and 14 seronegative healthy individuals. Ex vivo human LNs from non-RA individuals were used to directly analyze stromal cells. Molecules involved in antigen presentation and immune modulation were measured in LNSCs upon interferon γ (IFNγ) stimulation (n = 15). Results: Citrullinated targets of ACPAs were detected in human LN tissue and in cultured LNSCs. Human LNSCs express several PTAs, transcription factors autoimmune regulator (AIRE) and deformed epidermal autoregulatory factor 1 (DEAF1), and molecules involved in citrullination, antigen presentation, and immunomodulation. Overall, no clear differences between donor groups were observed with exception of a slightly lower induction of human leukocyte antigen-DR (HLA-DR) and programmed cell death 1 ligand (PD-L1) molecules in LNSCs from RA patients. Conclusion: Human LNSCs have the machinery to regulate peripheral tolerance making them an attractive target to exploit in tolerance induction and maintenance.

Plain English summary Patient and public involvement (PPI) improves the quality of health research and ensures that research is relevant to patients’ needs. Though PPI is increasingly evident in clinical and health services research, there are few examples in the research literature of effective PPI in translational and laboratory-based research. In this paper, we describe the development and evaluation of PPI in a multi-centre European project (EuroTEAM – Towards Early biomarkers in Arthritis Management) that included both translational and laboratory-based and psychosocial research. We found that although most PPI in EuroTEAM was centred around the psychosocial research, there were examples of PPI in the laboratory studies. As the project evolved, researchers became better at accommodating PPI and identifying PPI opportunities. It was generally agreed that PPI had a positive impact on the project overall, particularly on public engagement with the research. We concluded that the inclusion of both psychosocial and laboratory-based research in the same project facilitated PPI across all aspects of the research. In future projects, we would try to specify individual PPI activities in more detail at the project-planning stage, and better accommodate patient partners who are not native speakers of English. Abstract Background Patient and public involvement (PPI) enhances research quality and relevance and is central to contemporary health policy. The value of PPI has been recognised in rheumatology research, though there are limited examples of PPI in basic and translational science. The EU FP7 funded ‘EuroTEAM’ (Towards Early biomarkers in Arthritis Management) project was established to develop biomarker-based approaches to predict the future development of rheumatoid arthritis and incorporated psychosocial research to investigate the perceptions of ‘at risk’ individuals about predictive testing, and to develop informational resources about rheumatoid arthritis (RA) risk. Patient involvement was central to EuroTEAM from the inception of the project. The objective of this paper is to describe the development of PPI in EuroTEAM, formatively assess the impact of PPI from the perspectives of researchers and patient research partners (PRPs), reflect on successes and lessons learned, and formulate recommendations to guide future projects. Methods Two mixed-methods surveys (for PRPs and researchers) and a teleconference were undertaken to assess the impact of PPI on individual work packages and on EuroTEAM overall. Results There was consensus about the positive impact of PPI on the research and on the experiences of those involved. In particular, the positive impact of PPI on the personal development of researchers, and on effective public engagement with EuroTEAM research were highlighted. Researchers described adapting their practice in future projects to facilitate PPI. Spin-off projects and ongoing collaborations between PRPs and researchers reflected the value of PPI to participants. PPI was more frequently integrated in psychosocial research, though examples of PPI in laboratory/translational science were also described. PRPs asked for more opportunities to contribute meaningfully to basic scientific research and for more extensive feedback on their contributions. Conclusions The findings were used to formulate recommendations to guide effective involvement of patients in future similar projects, including identifying specific training requirements for PRPs and researchers, the identification of PRP focused tasks/deliverables at the project planning stage, and supporting access to involvement for all PRPs. Importantly, the distinctive multidisciplinary approach of EuroTEAM, incorporating both basic science and psychosocial research, facilitated patient involvement in the project overall.

Background: Rheumatoid arthritis (RA) is a disease with significant remaining unmet medical needs for better treatments. Vagus nerve stimulation (VNS) to activate the inflammatory reflex (cholinergic anti-inflammatory pathway) represents a novel experimental therapy for RA. 1 Previously, we reported that inflammatory reflex activation by VNS reduced pro-inflammatory cytokine production and improved disease activity in a 17-patient rheumatoid arthritis (RA) proof-of-concept study using a reprogrammed epilepsy stimulator2; clinical improvement was sustained for 24 months without untoward safety signals. 3 Here we report the 48 months results from this long-term observational study. Objectives: Determine the long-term safety and efficacy of VNS for the treatment of RA Methods: In the primary study, a VNS device was implanted into 17 RA patients, mostly with insufficient response to multiple conventional and biologic DMARDs, on stable background of methotrexate (≤25 mg weekly) therapy 2 . The device electrically stimulated the vagus nerve, 1-4 min/day, over a 12-week open label period. On completion, subjects were offered to enroll into a follow-up study, where the study physicians were given flexibility to alter VNS dosing parameters and/or to add a biologic disease-modifying antirheumatic drug (DMARD) to the treatment regimen to induce disease remission. Clinical disease activity measures and safety were accessed over 4 years. Results: All patients electively continued VNS treatment in the long-term follow-up study, 4 subjects withdrew prior to month 48. Reasons for discontinuation were withdrawal of consent (N=3) and adverse event due to device discomfort (N=1). At the start of the follow-up study, the mean DAS28-CRP, CDAI and HAQ-DI were significantly reduced compared to the pre-implant baseline (mean difference± SD: DAS28-CRP=-1.60± 1.13, p<0.001; CDAI=-21.19± 13.5, p<0.001; HAQ-DI=-0.44± 0.49, p<0.01), and this effect was retained through 48 months. Patients using VNS monotherapy and those using a combination of VNS with biologic DMARDs exhibited stable improvements in DAS28-CRP, CDAI and HAQ-DI at month 48 (Table 1). Improvements were observed for patients who both previously had an insufficient response to targeted biological therapies as well those who had an insufficient response to standard DMARDs. No association was seen between DAS28-CRP and stimulation frequency (Range= 1X-8X/day). There was no difference in the adverse events profile between the two groups. Table 1. Efficacy of VNS treatment. Treatment Reinitiated N=9 VNS Monotherapy N=8 Total N=17 Mo. 24 Mo. 36 Mo.48 Mo. 24 Mo. 36 Mo. 48 Mo. 24 Mo. 36 Mo. 48 Mean change from baseline (SD) DAS28-CRP -2.58 (1.0)*** -2.40 (1.0)** -2.28 (1.3)** -2.61 (1.3)* -1.77 (1.8) -2.0 (1.7) -2.59 (1.1)*** -2.19 (1.2)** -2.17 (1.4)** CDAI -24.06 (8.3)*** -18.02 (13.3) * -16.2 (19.6) -33.5 (11.1)*** -27.8 (16.0)* -27.9 (12.7)* -28.20 (10.5)*** -21.93 (14.5)* -20.83 (17.5)* HAQ-DI -.60 (0.64)* -.63 (0.45)* -.31 (0.60) -.89 (0.69)* -.88 (0.92) -.88 (0.61) -0.73 (0.66)*** -0.73 (0.64)** -0.54 (0.64)* *P<0.05, **P<0.01, ***P<0.001 versus primary study baseline (month -3.5) Conclusion: VNS was safe, well-tolerated, and resulted in significant and clinically important improvements in disease activity measures that were maintained over 48 months. These results support development of VNS devices as a new therapeutic option for RA treatment. References: [1]van Maanen MA, et al. Nat Rev Rheum 2009 [2]Koopman FA, et al. PNAS 2016 [3]Koopman FA, et al. Arthritis Rheum 2018 Disclosure of Interests: Frieda Koopman: None declared, Anne Musters: None declared, Marieke Backer: None declared, Danielle Gerlag Shareholder of: GlaxoSmithKline, Employee of: GlaxoSmithKline, Sanda Miljko: None declared, Simeon Grazio Speakers bureau: Abbvie., Roche, MSD, Eli Lilly, Pfizer, Mylan, Amgen, Fresenius Kabi, Stada, Berlin-Chemie, Sekib Sokolovic: None declared, Yaakov Levine Shareholder of: SetPoint Medical, Employee of: SetPoint Medical, Emmett Glass Employee of: SetPoint Medical, David Chernoff Shareholder of: SetPoint Medical, Adamas Pharmaceuticals, Olly Nutrition, NAIA Pharma, Aquinox Pharma, Consultant of: Adamas Pharmaceuticals, Olly Nutrition, NAIA Pharma, Aquinox Pharma, Crescendo Bioscience, Employee of: SetPoint Medical, Niek de Vries Grant/research support from: AbbVie, Janssen, Ergomed Clinical Research, GlaxoSmithKline, Pfizer, Boehringer Ingelheim, Roche, Consultant of: MSD, Pfizer, Paul P. Tak Shareholder of: GlaxoSmithKline, Employee of: GlaxoSmithKline

Rheumatoid arthritis (RA) is a common immune-mediated inflammatory disease. Research on RA is increasingly focused on the earliest stages of the disease, and has provided strong evidence that clinical signs and symptoms may be preceded by a preclinical phase during which evidence of systemic autoimmunity may be present. To facilitate research in this area, a number of international initiatives have proposed definitions of the phases of disease leading up to RA. The first of these initiatives was the European League Against Rheumatism's (EULAR) set of recommendations on terminology in persons at risk for RA, which suggested that the "at-risk phases" be described in terms of patients variably having: (A) genetic risk factors for RA; (B) environmental risk factors for RA; (C) systemic autoimmunity associated with RA; (D) symptoms without clinical arthritis; and (E) unclassified arthritis. The phrase clinically suspect arthralgia (CSA) is now widely used and can be regarded as describing a subgroup of patients in phase D. A definition of CSA was recently proposed by a EULAR taskforce, and primary research has begun to explore the full range of symptoms, as well as their sensitivity and specificity alone and in combination with other factors, that characterize this phase. Similarly, immune abnormalities at mucosal and others sites that precede and/or are associated with the onset of musculoskeletal symptoms are being increasingly studied and understood. Whether some of these at-risk phases, in particular CSA, represent entities meriting their own classification criteria is an essential area for consensus and will be discussed.

Rheumatoid arthritis (RA) is a progressive, destructive autoimmune arthritis. Break of tolerance and formation of autoantibodies occur years before arthritis. Adaptive immunity is initiated in lymphoid tissue where lymph node stromal cells (LNSCs) play a crucial role in shaping the immune response and maintaining peripheral tolerance. Here we performed the first epigenomic characterization of LNSCs during health and early RA, by analyzing their transcriptome and DNA methylome in LNSCs isolated from lymph node needle biopsies obtained from healthy controls (HC), autoantibody positive RA-risk individuals and patients with established RA. Of interest, LNSCs from RA-risk individuals and RA patients revealed a common significantly differential expressed gene signature compared with HC LNSCs. Pathway analysis of this common signature showed, among others, significant enrichment of pathways affecting the extracellular matrix (ECM), cholesterol biosynthesis and immune system. In a gel contraction assay LNSCs from RA-risk individuals and RA patients showed impaired collagen contraction compared to healthy LNSCs. In RA LNSCs a significant enrichment was observed for genes involved in cytokine signaling, hemostasis and packaging of telomere ends. In contrast, in RA-risk LNSCs pathways in cancer (cell cycle related genes) were differentially expressed compared with HC, which could be validated in vitro using a proliferation assay, which indicated a slower proliferation rate. DNA methylation analyses revealed common and specific differentially methylated CpG sites (DMS) in LNSC from RA patients and RA-risk individuals compared with HC. Intriguingly, shared DMS were all associated with antigen processing and presentation. This data point toward alterations in cytoskeleton and antigen-processing and presentation in LNSC from RA-risk individuals and RA patients. Further studies are required to investigate the consequence of this LNSC abnormality on LNSC-mediated immunomodulation.

Lymph nodes (LNs) are highly organized structures where specific immune responses are initiated by dendritic cells (DCs). We investigated the frequency and distribution of human myeloid (mDCs) and plasmacytoid (pDCs) in LNs and blood during the earliest phases of rheumatoid arthritis (RA). We included 22 RA-risk individuals positive for IgM rheumatoid factor and/or anti-citrullinated protein antibodies, 16 biological-naïve RA patients and 8 healthy controls (HCs). DC subsets (CD1c+ mDCs and CD304+ pDCs) in LN tissue and paired peripheral blood were analyzed using flow cytometry and confocal microscopy. In blood of RA patients a significant decreased frequency of pDCs was found, with a similar trend for mDCs. In contrast, mDC frequencies were higher in RA compared with HCs and RA-risk individuals, especially in LN. Frequency of mDCs seemed higher in LNs compared to paired blood samples in all donors, while pDCs were higher in LNs only in RA patients. As expected, both mDCs and pDCs localized mainly in T-cell areas of LN tissue. In conclusion, compared with RA-risk individuals, mDCs and pDCs were enriched in the LN tissue of early-RA patients, while their frequency in RA-risk individuals was comparable to HCs. This may suggest that other antigen-presenting cells are responsible for initial breaks of tolerance, while mDCs and pDCs are involved in sustaining inflammation.

Abstract Purpose While the aetiology of rheumatoid arthritis (RA) remains unclear, many of the inflammatory components are well characterised. For diagnosis and therapy evaluation, in vivo insight into these processes would be valuable. Various imaging probes have shown value including dynamic contrast-enhanced (DCE) MRI and PET/CT using 18F-fluorodeoxyglucose (18F-FDG) or tracers targeting the translocator protein (TSPO). To evaluate 18F-GE-180, a novel TSPO PET tracer, for detecting and quantifying disease activity in RA, we compared 18F-GE-180 uptake with that of 18F-FDG and DCE-MRI measures of inflammation. Methods Eight RA patients with moderate-to-high, stable disease activity and active disease in at least one wrist were included in this study (NCT02350426). Participants underwent PET/CT examinations with 18F-GE-180 and 18F-FDG on separate visits, covering the shoulders and from the pelvis to the feet, including hands and wrists. DCE-MRI was performed on one affected hand. Uptake was compared visually between tracers as judged by an experienced radiologist and quantitatively using the maximum standardised uptake value (SUVmax). Uptake for both tracers was correlated with DCE-MRI parameters of inflammation, including the volume transfer coefficient K trans using Pearson correlation (r). Results PET/CT imaging with 18F-GE-180 in RA patients showed marked extra-synovial uptake around the affected joints. Overall sensitivity for detecting clinically affected joints was low (14%). 18F-GE-180 uptake did not or only weakly correlate with DCE-MRI parameters in the wrist (r = 0.09–0.31). 18F-FDG showed higher sensitivity for detecting symptomatic joints (34%), as well as strong positive correlation with DCE-MRI parameters (SUVmax vs. K trans: r = 0.92 for wrist; r = 0.68 for metacarpophalangeal joints). Conclusions The correlations between DCE-MRI parameters and 18F-FDG uptake support use of this PET tracer for quantification of inflammatory burden in RA. The TSPO tracer 18F-GE-180, however, has shown limited use for the investigation of RA due to its poor sensitivity and ability to quantify disease activity in RA.

Objectives. The exact underlying mechanism of rituximab treatment in patients with RA is poorly defined and knowledge about the effect of B cell depletion on immune cells in secondary lymphoid organs is lacking. We analysed lymphoid tissue responses to rituximab in RA patients. Methods. Fourteen RA patients received 2 x 1000 mg rituximab intravenously, and lymph node (LN) biopsies were obtained before and 4 weeks after the first infusion. Tissues were examined by flow cytometry, immunohistochemistry and quantitative PCR. LN biopsies from five healthy individuals (HC) served as controls. Results. LN biopsies of RA patients showed increased frequencies of CD21(+)CD23(+)IgD(high)IgM(variable) follicular B cells and CD3(+)CD25(+)CD69(+) early activated, tissue resident T cells when compared with HCs. After treatment, there was incomplete depletion of LN B cells. There was a significant decrease in CD27(-)IgD(+) naive B cells, and CD27(+)IgD(+)unswitched memory B cells including the CD27(+)IgD(+)IgM(+) subset and follicular B cells. Strikingly, CD27(+)IgD(-) switched memory B cells persisted in LN biopsies after rituximab treatment. In the T cell compartment, a significant decrease was observed in the frequency of early activated, tissue resident T cells after rituximab treatment, but late activated T cells persisted. B cell proliferation inducing cytokine IL-21 was higher expressed in LN biopsies of RA patients compared with HC and expression was not affected by rituximab treatment. Conclusion. Rituximab does not cure RA, possibly due to persistence of switched memory B cells in lymphoid tissues suggesting that factors promoting B cell survival and differentiation need to be additionally targeted.

Career situation of first and presenting author Student for a master or a PhD. Introduction In rheumatoid arthritis (RA) the cause for loss of tolerance and anti–citrullinated protein antibody (ACPA) production remains unidentified. Mouse studies showed that peripheral tolerance can be maintained through presentation of peripheral tissue antigens (PTAs) by lymph node stromal cells (LNSCs). We hypothesize that deregulation of peripheral tolerance mechanisms mediated by LNSCs might underlie pathogenesis of RA. Here we investigated the expression of PTAs, citrullinated proteins and immunomodulatory molecules by human LNSCs during health and autoimmunity. Methods LN tissue sections and LNSCs were prepared from freshly collected lymph node needle biopsies obtained from 24 patients with RA, 23 individuals positive for autoantibodies but without clinical apparent disease (RA-risk group) and 14 seronegative healthy controls. Expression of PADI enzymes, citrullinated proteins, DEAF1, AIRE and PTAs was investigated at mRNA and protein level. Expression of immunomodulatory molecules in LNSCs was assessed after stimulation with IFNγ (n=15). Results Citrinullated proteins, targeted by ACPA, were found in human LN tissue as well as in cultured LNSCs. In addition, we observed the expression of transcription factors AIRE and DEAF1 as well as disease-related PTAs in LNSCs with some PTAs showing a distinct expression pattern in autoimmune LNSCs compared to healthy controls. TGF-β was constitutively expressed by LNSCs while CD86 or IL-10 were not detected. Upon IFNγ stimulation LNSCs upregulated MHC class II, co-stimulatory molecules CD40 and CD80 as well as T cell negative regulators CD274, NOS2 and IDO. Overall, no clear differences between donor groups were observed for these markers with exception of a slightly lower induction of CD40 and NOS2 in RA LNSCs. Conclusions We present for the first time that human LNSCs express several PTAs and the transcription factors AIRE and DEAF1, driving PTA expression. Additionally, human LNSCs express molecules involved in citrullination, antigen presentation and immunomodulation. Moreover, antigens targeted by ACPAs are present in LN tissue and LNSCs. These data suggest that LNSCs have the machinery to regulate peripheral tolerance. Disclosure of Interest None declared.