Background Clinical heterogeneity, a hallmark of systemic autoimmune diseases (SADs) impedes early diagnosis and effective treatment, issues that may be addressed if patients could be grouped into a molecular defined stratification. Methods With the aim of reclassifying SADs independently of the clinical diagnoses, unsupervised clustering of integrated whole blood transcriptome and methylome cross-sectional data of 918 patients with 7 SADs and 263 healthy controls was undertaken. An inception cohort prospectively followed for 6 and 14 months was studied to validate the results in early cases and analyze if cluster assignment was modified with time. Results Four clusters were identified Three aberrant clusters were ‘acute phase inflammatory’, ‘T cell immunity’, and ‘interferon’, each including all diagnoses, were defined by genetic, clinical, serological and cellular features. A fourth cluster showed no specific molecular pattern, to which 74% of healthy controls clustered with patients. The inception cohort showed that most patients were either assigned always to the same cluster or moved from the healthy-like cluster to a single aberrant cluster resembling the relapsing-remitting dynamic of these diseases, showing that single aberrant molecular signatures characterize each individual patient. Conclusions Patients with SADs share molecular signatures and can be therefore stratified into three disease clusters differentiating each patient into a specific molecular disease pathway. Such assignment is stable with time. These results have important implications for understanding disease progression and therapy design marking a paradigm shift in our view of SADs. Acknowledgment This work has been supported through a grant from the Innovative Medicines Initiative Joint Undertaking No. 115565 and in-kind and in-cash contributions from the EFPIA partners. G.B. is supported by the Instituto de Salud Carlos III (ISCIII, Spanish Health Ministry), through the Sara Borrell subprogram (CD18/00153). The authors would like to particularly express their gratitude to the patients, nurses and many others who helped directly or indirectly in the consecution of this study.

Objectives This phase 2 proof-of-concept study ( NCT02610543) assessed efficacy, safety and effects on salivary gland inflammation of seletalisib, a potent and selective PI3Kδ inhibitor, in patients with moderate-to-severe primary Sjögren’s syndrome (PSS) . Methods Adults with PSS were randomised 1:1 to seletalisib 45 mg/day or placebo, in addition to current PSS therapy. Primary endpoints were safety and tolerability and change from baseline in ESSDAI score at Week 12. Secondary endpoints included change from baseline at Week 12 in ESSPRI score and histological features in salivary gland biopsies.

OBJECTIVES This phase 2 proof-of-concept study (NCT02610543) assessed efficacy, safety and effects on salivary gland inflammation of seletalisib, a potent and selective PI3Kδ inhibitor, in patients with moderate-to-severe primary Sjögren's syndrome (PSS). METHODS Adults with PSS were randomized 1:1 to seletalisib 45 mg/day or placebo, in addition to current PSS therapy. Primary end points were safety and tolerability and change from baseline in EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) score at week 12. Secondary end points included change from baseline at week 12 in EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI) score and histological features in salivary gland biopsies. RESULTS Twenty-seven patients were randomized (seletalisib n = 13, placebo n = 14); 20 completed the study. Enrolment challenges led to early study termination with loss of statistical power (36% vs 80% planned). Nonetheless, a trend for improvement in ESSDAI and ESSPRI [difference vs placebo: -2.59 (95% CI: -7.30, 2.11; P=0.266) and -1.55 (95% CI: -3.39, 0.28), respectively] was observed at week 12. No significant changes were seen in saliva and tear flow. Serious adverse events (AEs) were reported in 3/13 of patients receiving seletalisib vs 1/14 for placebo and 5/13 vs 1/14 discontinued due to AEs, respectively. Serum IgM and IgG concentrations decreased in the seletalisib group vs placebo. Seletalisib demonstrated efficacy in reducing size and organisation of salivary gland inflammatory foci and in target engagement, thus reducing PI3K-mTOR signalling compared with placebo. CONCLUSION Despite enrolment challenges, seletalisib demonstrated a trend towards clinical improvement in patients with PSS. Histological analyses demonstrated encouraging effects of seletalisib on salivary gland inflammation and organisation. TRIAL REGISTRATION https://clinicaltrials.gov, NCT02610543.

The delta isoform of phosphoinositide 3-kinase (PI3Kδ) regulates various lymphocyte functions. Considering the key pro-inflammatory role of IL-17A and IL-17F cytokines in psoriasis and spondyloarthritis (SpA), we investigated the potential of PI3Kδ blockade to suppress IL-17A, IL-17F and associated pro-inflammatory cytokines that could synergize with IL-17A and IL-17F. Using in vitro studies with primary human cells and ex vivo studies with inflamed target tissues, we assessed if seletalisib, a selective PI3Kδ inhibitor, suppresses cytokine production by T cells and innate-like lymphocytes, and if seletalisib modulates the inflammatory responses in stromal cell populations in psoriasis (human dermal fibroblasts (HDF)) and SpA (fibroblast-like synoviocytes (FLS)). In vitro, seletalisib inhibited the production of pro-inflammatory cytokines, including IL-17A and IL-17F, from peripheral blood mononuclear cells (PBMCs), T helper 17 (Th17) cells as well as γδ-T cells and mucosal-associated invariant T cells. This inhibition resulted in decreased inflammatory activation of HDF in co-culture systems. Seletalisib was also efficacious in inhibiting SpA PBMCs and synovial fluid mononuclear cells (SFMCs) from producing pro-inflammatory cytokines. Furthermore, supernatant derived from cultured seletalisib-treated Th17 cells showed reduced potency for activating inflammatory responses from cultured SpA FLS and decreased their osteogenic differentiation capacity. Finally, analysis of inflamed SpA synovial tissue biopsies revealed activation of the PI3K-Akt-mTOR pathway. We observed that ex vivo seletalisib treatment of inflamed synovial tissue reduced IL-17A and IL-17F expression. Collectively, inhibition of PI3Kδ reduces the production of pro-inflammatory cytokines from IL-17-producing adaptive and innate-like lymphocytes and thereby inhibits downstream inflammatory and tissue remodeling responses. PI3Kδ-targeting may therefore represent a novel therapeutic avenue for the treatment of IL-17-mediated chronic inflammatory diseases such as psoriasis and SpA.

Mutations in two genes can result in activated PI3Kδ syndrome (APDS), a rare immunodeficiency disease with limited therapeutic options. Seletalisib, a potent, selective PI3Kδ inhibitor, was evaluated in patients with APDS1 and APDS2. In the phase 1b study (European Clinical Trials Database 2015-002900-10) patients with genetic and clinical confirmation of APDS1 or APDS2 received 15-25 mg/d seletalisib for 12 wk. Patients could enter an extension study (European Clinical Trials Database 2015-005541). Primary endpoints were safety and tolerability, with exploratory efficacy and immunology endpoints. Seven patients (median age 15 years; APDS1 n = 3; APDS2 n = 4) received seletalisib; five completed the phase 1b study. For the extension study, four patients entered, one withdrew consent (week 24), three completed ≥84 wk of treatment. In the phase 1b study, patients had improved peripheral lymphadenopathy (n = 2), lung function (n = 1), thrombocyte counts (n = 1), and chronic enteropathy (n = 1). Overall, effects were maintained in the extension. In the phase 1b study, percentages of transitional B cells decreased, naive B cells increased, and senescent CD8 T cells decreased (human cells); effects were generally maintained in the extension. Seletalisib-related adverse events occurred in four of seven patients (phase 1b study: hepatic enzyme increased, dizziness, aphthous ulcer, arthralgia, arthritis, increased appetite, increased weight, restlessness, tendon disorder, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (aphthous ulcer). Serious adverse events occurred in three of seven patients (phase 1b study: hospitalization, colitis, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (stomatitis). Patients with APDS receiving seletalisib had improvements in variable clinical and immunological features, and a favorable risk-benefit profile was maintained for ≤96 wk.

Mutations in two genes can result in activated PI3K delta syndrome (APDS), a rare immunodeficiency disease with limited therapeutic options. Seletalisib, a potent, selective PI3K delta inhibitor, was evaluated in patients with APDS1 and APDS2. In the phase lb study (European Clinical Trials Database 2015-002900-10) patients with genetic and clinical confirmation of APDS1 or APDS2 received 15-25 mg/d seletalisib for 12 wk. Patients could enter an extension study (European Clinical Trials Database 2015-005541). Primary endpoints were safety and tolerability, with exploratory efficacy and immunology endpoints. Seven patients (median age 15 years; APDS1 n = 3; APDS2 n = 4) received seletalisib; five completed the phase lb study. For the extension study, four patients entered, one withdrew consent (week 24), three completed >= 84 wk of treatment. In the phase lb study, patients had improved peripheral lymphadenopathy (n = 2), lung function (n = 1), thrombocyte counts (n = 1), and chronic enteropathy (n = 1). Overall, effects were maintained in the extension. In the phase lb study, percentages of transitional B cells decreased, naive B cells increased, and senescent CD8 T cells decreased (human cells); effects were generally maintained in the extension. Seletalisib-related adverse events occurred in four of seven patients (phase lb study: hepatic enzyme increased, dizziness, aphthous ulcer, arthralgia, arthritis, increased appetite, increased weight, restlessness, tendon disorder, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (aphthous ulcer). Serious adverse events occurred in three of seven patients (phase lb study: hospitalization, colitis, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (stomatitis). Patients with APDS receiving seletalisib had improvements in variable clinical and immunological features, and a favorable risk-benefit profile was maintained for <= 96 wk.

Background: Seletalisib is a potent, selective oral inhibitor of phosphoinositide-3 kinase delta (PI3Kδ). Preclinical data have shown that the PI3Kδ pathway is upregulated within salivary glands of patients with PSS and contributes to disease pathogenesis.1 Objectives: To assess the efficacy and safety of seletalisib in patients with PSS. Methods: In this Phase II, double-blind, proof of concept study (NCT02610543), patients with PSS having an EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) score ≥5 were randomised 1:1 to seletalisib once daily or placebo (PBO) in addition to current PSS therapy for 12 weeks. The primary endpoint was change from baseline in ESSDAI at Week 12. The study was designed to have 80% power to detect a difference of 3.8 points in change from baseline in ESSDAI between seletalisib and PBO at Week 12 and required 58 patients to complete treatment. Other endpoints included EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI), salivary gland biopsy changes, Schirmer's I test, immunoglobulin concentrations and incidence of treatment-emergent adverse events (TEAEs). Results: The study was terminated early due to slow recruitment, which led to study power decreasing to 36%. Twenty of 27 patients randomised (seletalisib n=13, PBO n=14) completed treatment. Demographic characteristics were generally similar between groups. Mean (SE) change from baseline in ESSDAI at Week 12 was seletalisib –5.4 (1.7) vs PBO –2.8 (1.5); treatment difference vs PBO (95% CI) was –2.59 (–7.30, 2.11; p=0.266). The percentages of patients achieving a ≥3 point reduction in ESSDAI were seletalisib 66.7% vs PBO 54.5%. Post-hoc Bayesian analyses of treatment difference showed an 86.5% probability of being superior to PBO and a 48.8% probability of a >3 point difference from PBO. Clinically notable improvements in some secondary endpoints were also observed in the seletalisib group (Table 1). Minor salivary gland biopsies had broadly similar histological features across groups at baseline. At Week 12, seletalisib treatment led to a reduction in the size and cellular organisation of mononuclear inflammatory cell foci vs PBO (Table 2). TEAEs were reported by 13/13 (100.0%) seletalisib and 13/14 (92.9%) PBO patients; most frequently reported: diarrhoea (5/13 [38.5%] vs 0/14 [0%]) and headache (3/13 [23.1%] vs 2/14 [14.3%]). Serious TEAEs were reported by 3/13 (23.1%) vs 1/14 (7.1%), and discontinuations due to TEAEs by 5/13 (38.5%) vs 1/14 (7.1%) seletalisib and PBO patients, respectively. Conclusion: Although this Phase II PSS study was terminated early due to slow recruitment, seletalisib demonstrated a trend to clinical improvement in patients with PSS and acceptable safety and tolerability. Histological analyses demonstrated encouraging effects of seletalisib on the organisation and extent of salivary gland lymphocytic infiltration in patients with PSS. References [1] Nayar S, et al. Ann Rheum Dis. 2019;78:249–260 Acknowledgement: Funded by UCB Pharma Disclosure of Interests: Maria Juarez Shareholder of: UCB Pharma, Employee of: UCB Pharma, Nieves Diaz Shareholder of: UCB Pharma, Employee of: UCB Pharma, Geoffrey I. Johnston Shareholder of: UCB Pharma, Pfizer, AstraZeneca, Employee of: UCB Pharma (and AstraZeneca + Pfizer previously), Saba Nayar: None declared, Andrew Payne Employee of: UCB Pharma, Eric Helmer Employee of: Galapagos, UCB Pharma, Takeda, Johnson & Johnson, Dionne Cain Employee of: UCB Pharma, Paulette Williams Shareholder of: UCB Pharma, Employee of: UCB Pharma, Wan Fai Ng: None declared, Benjamin Fisher Consultant for: Novartis, Roche, MedImmune, Bristol-Myers Squibb, Jacques-Eric Gottenberg Grant/research support from: Bristol-Myers Squibb, Grant/research support from: Bristol-Myers Squibb, Consultant for: Bristol-Myers Squibb, Lilly, Pfizer, Sanofi-Genzyme, UCB Pharma, Consultant for: Bristol-Myers Squibb, Eli Lilly, UCB, Sanofi-Genzyme, Pfizer, Giuliana Guggino Grant/research support from: Laborest, Pfizer, Consultant for: Novartis, Abbvie, Speakers bureau: Sandoz, Xavier Mariette Grant/research support from: Servier, Consultant for: AstraZeneca, Bristol-Myers Squibb, GlaxoSmithKline, Janssen, Pfizer, UCB Pharma, Marika Kvarnström: None declared, Valerie Devauchelle-Pensec Grant/research support from: Roche-Chugai, Speakers bureau: MSD, BMS, UCB, Roche, Jose Rosas Consultant for: Abbvie, Amgen, Bristol, Janssen, Lilly, Merck Sharp & Dohme, Pfizer, UCB Pharma, Speakers bureau: Abbvie, Amgen, Bristol, Janssen, Lilly, Merck Sharp & Dohme, Pfizer, UCB Pharma, Juan Sanchez-Burson Speakers bureau: Lilly, Janssen, Pfizer, Roberto Giacomelli Grant/research support from: Pfizer, Actelion, Speakers bureau: Actelion, Bristol-Myers Squibb, Merck Sharp & Dohme, Abbvie, Pfizer, Sobi, Roche, Francesca Barone Grant/research support from: GlaxoSmithKline, Roche, UCB Pharma, Actelion, ONO Pharmaceutical, Consultant for: GlaxoSmithKline, Roche, Actelion, ONO Pharmaceutical, Simon J. Bowman Grant/research support from: Previously UCB Pharma (to University of Birmingham) and Roche, Consultant for: 2016-7: Novartis, Mitsubishi Tanabe Pharma 2017-8: AstraZeneca, MedImmune, GFK, Xtlbio, ONO Pharmaceutical 2018-9: Novartis, AstraZeneca, UCB Pharma

Background Autoantibodies directed against antigens with post-translational modifications (PTMs), such as citrullination (ACPA), are a hallmark of rheumatoid arthritis (RA)1. ACPA titres increase prior to disease onset, but are thought to be relatively stable after symptomatic inflammation is established2. The temporal profile of antibodies against acetylated (AAPA) and carbamylated (ACarPA) peptides has not been so comprehensively characterised following the onset of joint swelling3. Objectives We aimed to track serum levels of anti-PTM antibodies over 18 months in patients with newly-presenting RA in our prospective observational cohort. Methods Patients with treatment-naïve inflammatory arthritis donated serum at baseline, 6 and 18 months. 103 patients satisfying ACR/EULAR 2010 criteria for RA underwent testing for IgG and IgA antibodies against peptides with citrulline (ACPA), carbamylated lysine (ACarPA), and acetylated lysine (AAPA) PTMs using ELISA as previously described4. Results 57% of participants were female, and 48% and 50% patients were anti-CCP2 or rheumatoid factor positive respectively. Mean age was 56 years (s.d. 15.2), symptom duration 55 days (s.d 22.4), and DAS28CRP 4.4 (s.d. 1.3) at enrolment. Comparing baseline and 18 month median antibody levels measured by optical density, a decrease was observed over time for IgG (0.26 vs 0.17, p<0.0001) and IgA AAPA (0.23 vs 0.09, p<0.0001), as well as both IgG and IgA antibodies against citrullinated peptides (0.47 vs 0.40, p<0.0001 and 0.16 vs 0.12, p<0.0001 respectively). Significant reductions occurred between baseline and 6 months for both IgG (p<0.0001) and IgA (p<0.0001) AAPA antibodies, but not between 6 and 18 month timepoints after correction for multiple testing. A similar pattern was observed for IgA ACPA antibody levels, although IgG ACPA levels did not vary significantly comparing baseline with 6, and 6 with 18 month timepoints. Antibody levels were not influenced by exposure to therapy. Patients seropositive for anti-CCP2 at baseline demonstrated less labile antibody levels, with the exception of sustained increases in IgG ACarPA reactivity compared with seronegative individuals. Abstract THU0104 – figure 1 demonstrates bidirectional changes in anti-PTM antibody reactivity at the patient level. Conclusions Median AAPA and, to a lesser extent, ACPA levels fell over time, regardless of therapy. This was most marked for the IgA isotype. Differential isotype effects may represent maturation of the autoantibody repertoire from mucosal IgA antibodies involved in the breach of tolerance. Lability of AAPA levels may reflect the relative reversibility of acetylation of a lysine amino acid residue by comparison with citrullination and carbamylation modifications. By contrast, and as observed previously3, IgG ACarPA levels increased over time, perhaps reflecting the tendency of the human proteome to accrue carbamylation modifications due to ageing, and metabolic or inflammatory stress. References [1] Trouw LA, et al. Nat Rev Rheumatol2017;13: 331–9. [2] Modi S, et al. Clin Exp Immunol2013;173(1):8–17. [3] Spinelli FR, et al. J Rheum Dis Treat2015;1:13. [4] Juarez M, et al. Ann Rheum Dis2016;75:1099–1107. Acknowledgements This work was funded via an EU FP7 grant (EuroTeam). KR is funded by the Birmingham NIHR Biomedical Research Centre. Disclosure of Interest None declared

Background PI3Kδ is predominantly expressed in lymphocytes; the role it plays in immune disease has encouraged the development of inhibitors targeting its kinase activity. Inhibitors of PI3Kδ have been approved or are in clinical development for the treatment of B cell malignancies; their therapeutic potential in inflammatory and autoimmune disease is being explored. Seletalisib is a selective and potent PI3Kδ inhibitor that has been profiled in preclinical and early clinical studies. Objectives To assess the therapeutic potential of seletalisib in inflammatory and autoimmune disease. Methods In vitro cell-based studies were performed on blood samples taken from healthy volunteers (HV) or from patients with primary Sjogren9s Syndrome (pSS) and/or psoriasis. In addition, samples were analysed from a single ascending dose study in HV (NCT02207595) and a multiple ascending dose study in HV and psoriasis patients (NCT02303509). T and B cell responses in the presence and absence of seletalisib were assessed by flow cytometry or a Meso-Scale discovery assay (Meso-Scale Diagnostics, MD, USA), following activation of the T cell- or B cell-receptor by receptor cross-linking or via stimulation with specific antigens. The functional selectivity of seletalisib was assessed in the BioMap system (DiscoverX, CA, USA). Translational studies used flow cytometry and/or immunofluorescence to demonstrate the presence of active PI3K signalling in diseased tissue, through expression of phosphorylated-AKT (pAKT) or -ribosomal protein S6 (pS6). The effect of seletalisib on PI3K signalling was determined by phospho-flow cytometry. Target engagement was determined through measurement of basophil degranulation in healthy subjects, and assessment of lesion severity score in skin biopsies from patients with psoriasis that were treated with seletalisib. Results Preclinical studies showed seletalisib potently inhibited T cell differentiation and function (IC50 range: 2–31 nM). Further, it blocked activation and proliferation of B cells (IC50 range: 16–49 nM). When profiled in a wide range of primary cell assay systems, including fibroblasts, epithelial, endothelial and vascular smooth muscle cells, seletalisib showed significant activity only in those systems containing lymphocytes, demonstrating its functional selectivity towards PI3Kδ-expressing cells. Expression of the PI3K pathway in lymphocytes was shown at the site of disease in clinical samples both from patients with psoriasis and pSS. Seletalisib inhibited PI3K signalling, measured by a reduction in pAKT and pS6 expression, in T cells derived from patients with psoriasis. In first-in-man studies, mean seletalisib plasma concentration-time profiles increased with increasing dose after single and multiple dosing, with no major deviations from dose proportionality. There was no unexpected accumulation or loss of exposure after multiple dosing (time-independent pharmacokinetic (PK) profile) and apparent t1/2 values (approx. 20h) were supportive of once-daily dosing. Inhibition of basophil degranulation in healthy subjects and effects on the cellular composition in lesional skin biopsies from patients with psoriasis, provided indications of target engagement, following treatment with seletalisib. Conclusions Seletalisib is a potent, selective PI3Kδ inhibitor with an attractive preclinical and human PK profile; clinical studies are ongoing. Disclosure of Interest A. Payne Shareholder of: UCB Pharma, Employee of: UCB Pharma, M. Juarez Shareholder of: UCB Pharma, Employee of: UCB Pharma, G. Johnston Shareholder of: UCB Pharma, Employee of: UCB Pharma, E. Helmer Shareholder of: UCB Pharma, Employee of: UCB Pharma, M. Thomson Shareholder of: UCB Pharma, Employee of: UCB Pharma

Early treatment of rheumatoid arthritis (RA) results in more effective disease suppression and can be key to a successful patient response. However, not all people who exhibit early synovitis develop RA; for example, in some, synovial inflammation resolves spontaneously.1 The factors that drive RA development remain unclear and clinical tools to predict RA development are imperfect. Tenascin-C is a proinflammatory matrix molecule that is absent from healthy joints but highly expressed in the joints of patients with RA.2 ,3 We identified an immunodominant peptide in citrullinated tenascin-C, cTNC5, antibodies against which are detected in around half of the patients with RA, and can be found years before disease onset in some individuals.4 Here, we sought to determine if anti-cTNC5 antibodies can discriminate among people with early synovial inflammation those who develop RA and those with other outcomes. Sera from 263 patients in the Birmingham early arthritis cohort were analysed. Patients were disease-modifying antirheumatic drug (DMARD) naive with clinically apparent synovitis of ≥1 joint and with inflammatory joint symptoms of ≤3 months’ duration. Patients were followed for 18 months to ensure development of full disease phenotype and to allow any resolving arthritis time to resolve. At 18 months, patients were assigned to the following outcome categories: persistent RA according to the American College of Rheumatology (ACR) 2010 criteria5 (RA, n=101), persistent non-RA arthritis (PNRA, n=66) and resolving arthritis (no clinically apparent joint swelling, no DMARD/steroid use in the previous 3 months, n=96). …

Background: Synovial fibroblasts play a key role in joint destruction and regulation of the inflammatory infiltrate in established rheumatoid arthritis (RA).The mechanisms by which this occurs in the earliest stages of RA are largely unknown.We investigated the role of Dickkopf-related protein 1 (DKK1) produced by synovial fibroblasts of patients with very early rheumatoid arthritis (VeRA).Methods: Fibroblasts were isolated from the disease-modifying anti-rheumatic drug-naive Birmingham early arthritis cohort of patients with new onset of clinically apparent arthritis and inflammatory symptoms of ≤12 weeks' duration, who at follow-up had either resolving arthritis or RA.Endothelial fibroblast co-cultures were formed using porous filters, and lymphocyte adhesion to co-cultures was assessed using phase-contrast microscopy.DKK1 gene expression and secretion were quantified by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively.Results: Synovial fibroblasts from patients with VeRA expressed significantly higher levels of DKK1 messenger RNA than those from patients with resolving arthritis.A similar trend was observed for DKK1 protein secretion.In co-culture constructs, more DKK1 tended to be secreted in co-cultures incorporating fibroblasts from VeRA than in co-cultures from non-inflamed joints and resolving arthritis.DKK1 secretion during co-culture positively correlated with lymphocyte adhesion.Conclusions: Alterations in DKK1 could be involved in the pathogenesis and perpetuation of the inflammatory response in the earliest clinically apparent stages of RA.

Background and objectives For our understanding of the pathogenesis of rheumatoid arthritis (RA), it is important to elucidate the mechanisms underlying early stages of synovitis. Here, synovial cytokine production was investigated in patients with very early arthritis.Methods Synovial biopsies were obtained from patients with at least one clinically swollen joint within 12 weeks of symptom onset. At an 18-month follow-up visit, patients who went on to develop RA, or whose arthritis spontaneously resolved, were identified. Biopsies were also obtained from patients with RA with longer symptom duration (>12 weeks) and individuals with no clinically apparent inflammation. Synovial mRNA expression of 117 cytokines was quantified using PCR techniques and analysed using standard and novel methods of data analysis. Synovial tissue sections were stained for CXCL4, CXCL7, CD41, CD68 and von Willebrand factor.Results A machine learning approach identified expression of mRNA for CXCL4 and CXCL7 as potentially important in the classification of early RA versus resolving arthritis. mRNA levels for these chemokines were significantly elevated in patients with early RA compared with uninflamed controls. Significantly increased CXCL4 and CXCL7 protein expression was observed in patients with early RA compared with those with resolving arthritis or longer established disease. CXCL4 and CXCL7 co-localised with blood vessels, platelets and CD68(+) macrophages. Extravascular CXCL7 expression was significantly higher in patients with very early RA compared with longer duration RA or resolving arthritisConclusions Taken together, these observations suggest a transient increase in synovial CXCL4 and CXCL7 levels in early RA.

Objectives The success of B cell targeting therapies has highlighted the importance of B cells in rheumatoid arthritis pathogenesis. We have previously shown that B cells in the RA synovium are capable of producing pro-inflammatory and bone-destructive cytokines including RANKL. Here we sought to characterise the nature and functional relevance of the RANKL-producing B cell subset in the RA synovium. Methods Synovial fluid and peripheral blood B cells from patients with RA were analysed by flow cytometry for markers of B cell differentiation and activation and for chemokine receptors. FcRL4+ and FcRL4− B cells sorted from synovial fluid were analysed for cytokine expression using Taqman low-density arrays. Synovial tissue biopsies obtained from patients with RA were analysed by immunofluorescence for CD20, RANKL and FcRL4. FCRL4 mRNA expression was determined in synovial tissue of RA patients and non-inflammatory control subjects by real-time PCR. Results RANKL-producing B cells in RA synovial tissue and fluid were identified as belonging to a distinct subset of B cells defined by expression of the transmembrane protein FcRL4. FcRL4+ B cells express a distinct combination of cytokines and surface proteins indicating a function distinct from that of FcRL4− B cells. Notably, FcRL4+ B cells expressed high levels of TNF-α and RANKL mRNA. Conclusions We have identified a novel pro-inflammatory B cell population in the RA synovium which is defined by expression of FcRL4 and responsible for RANKL production. This B cell population expresses high levels of CD20, and its removal by rituximab may contribute to the anti-inflammatory effect of this drug.

Background: Serum autoantibodies are measured in RA patients for diagnostic and prognostic purposes. Antibodies against citrullinated peptides are directed against citrulline that results from post-transla- tional modification (PTM) of arginine residues. Antibodies against another PTM residue, homocitrulline, have recently been described in RA patients’ sera. Lysine acetylation is another important PTM. Proteins carrying this modification regulate metabolic pathways and host protein acetylation is a mechanism through which the microbiome affects its host. Recent sequencing of the intestinal microbiome revealed a high prevalence of a specific bacterium, Prevotellacopri , inpatients with early RA suggesting a pathogenic function. This function may be exerted through protein acetylation. To date there have been no studies of immunity against acetylated proteins in RA. Objective: To investigate antibody reactivity against acetylated, citrullinated and homocitrullinated vimentin peptides in patients with early inflammatory arthritis. Methods: Microtitre plates were coated with vimentin derived peptides identical in length and composition except at one amino acid that was changed to introduce a citrullinated, carbamylated or acetylated residue. By using a peptide of known sequence and introducing one modification at a time, we were able to determine specific antibody reactivities. Sera of 268 treatment naive patients with early inflammatory arthritis and symptoms (cid:3) 3 month’s duration were tested. Patients were assigned to outcome categories at 18 month follow up. Anti-CCP antibody levels were determined using a commercial anti-CCP2 antibody assay (ELiA Background: Early diagnosis of RA improves outcomes but is challenging, particularly among ACPA-negative individuals. Previously we identified an IL-6-mediated CD4 þ T cell transcriptional signature, enriched for signal transduction and activation of transcription-3 (STAT3) target genes, which had discriminatory value for this purpose. In the present work we sought independent replication of those findings, the development of a more readily applicable diagnostic assay and insight into mechanisms of disease induction. Methods: Among 210 early arthritis patients attending the Newcastle Early Arthritis Clinic (NEAC) who were naive to immunomodulatory treatment, serum cytokine levels were ascertained by immuno-assay. Constitutive and IL-6-induced expression of phosphorylated STAT1 and 3 (pSTAT1/3) were determined in paired fresh circulating lymphocytes using flow cytometry. Finally, contemporaneous CD4 þ T cell gene expression was measured in highly purified, fresh CD4 þ T cells by hybridizing high-integrity RNA isolates to Illumina Human HT12 BeadChips, and employing appropriate microarray normalization algorithms. Patients were followed up for > 12 months and diagnostic outcomes confirmed. Analyses included non-parametric ANOVA (Dunn’s post-hoc group comparisons), Spearman’s rank correlation, multiple regression and hierarchical clustering. Results: Consistent with previous observations, serum IL-6 levels were significantly higher in early ACPA-negative RA patients ( n ¼ 29) than in Background: RA occurs when genetically predisposed individuals are exposed to environmental factors. The dominant RA environmental risk factor in European populations is smoking. Drinking alcohol appears to protect against RA in Europeans. The relevance of these risk factors to non-European populations is uncertain. We undertook a case–control study to evaluate the associations between smoking, drinking and RA in Black African and Black Caribbean ethnicity individuals. Methods: We evaluated 180 RA cases enrolled to the GENetics of RA in individuals of African ancestry (GENRA) study. All met the 1987/2010 ACR RA classification criteria and were of self-reported black or black Caribbean Patients were recruited from South London Rheumatology Smoking and alcohol intake data were captured by The study was ethically approved; all participants provided consent. Controls were evaluated from the 2004 Health England (focussing on the health of Ethnic minorities). 1812 controls of black African or black Caribbean ethnicity a history of arthritis, rheumatism fibrositis were Smoking and alcohol intake data were cases. Associations (ever vs . never), . case-control logistic for age and case-control (cid:4) pack-years) RA risk never-smokers/ pack-years). Results: In black individuals, smoking risk Ever-smoking rates lower in cases than controls adjusted odds ratio (OR) . black African ethnicity individuals. Our findings suggest that environ- mental risk factors for RA differ between European and African populations. They also indicate that research into RA risk factors needs undertaking separately in African and Caribbean ethnicity individuals. Disclosure statement: is an Research UK Clinical Research Fellowship NIHR Lectureship. All authors

balance between leukocyte recruitment, proliferation, emigration and death. Activated RASFs produce inflammatory chemokines (IL-8, CXCL5, CXCL1) implicated in leukocyte recruitment to diseased synovium, survival factors (type I interferon, IL-15, BAFF) and constitutive chemokines (e.g. CXCL12, CCL21). The net result is the chronic accumulation, survival and retention of leukocytes at sites of disease.