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To determine the tolerability, safety and yield of synovial tissue in an early arthritis cohort using a minimally invasive, ultrasound (US)-guided, synovial biopsy technique in small, medium and large joints. 93 sequential biopsy procedures were assessed from a total of 57 patients (baseline and 36 repeat biopsies at 6 months) recruited as part of the 'Pathobiology of Early Arthritis Cohort' study. Patients completed a tolerability questionnaire prior to and following the synovial biopsy procedure. The synovial biopsy was performed under US guidance with US images of the joint recorded prior to each procedure. Synovial tissue was harvested for immunohistochemistry and RNA extraction. Five different joint sites were biopsied (knee, elbow, wrist, metacarpal phalangeal and proximal interphalangeal). No significant complications were reported following the procedure. No difference in pain, swelling and stiffness of the biopsied joint from before and after the procedure was demonstrated. A median of 14 biopsy samples was retrieved from each procedure with 93% of biopsy procedures yielding good quality tissue. RNA yield was good in all joints and in repeat biopsies. Multivariant analysis demonstrated a significantly greater yield of RNA and graded tissue in relation to a high prebiopsy, grey-scale synovitis score (0-3, semiquantitative). A minimally invasive approach to synovial tissue harvesting, using US guidance, is both safe and well-tolerated by patients. Tissue quality/RNA yield is preserved in subsequent biopsies following therapeutic intervention. A high US grey-scale synovitis score is a predictor of good quality/quantity of tissue and RNA.

Background:Rheumatoid Arthritis (RA) is characterized by formation of synovial ectopic lymphoid structures, where we previously showed accumulation of Epstein Barr virus (EBV)-infected B-cells. RA synovial fibroblasts (RA-SFs) can sustain B-cells activation and maturation. In more recent work, we showed that RA-SFs activate Interferon type I signalling (IFN-I) upon B-cells co-culture and preferentially support EBV+B-cells clones expansion.Objectives:1) to characterize B-cells after RA-SFs co-culture 2) to dissect the molecular mechanisms behind B-cells-SFs crosstalk.Methods:RA B-cells were co-cultured with RA-SFs for 28 days and then characterized through flowcytometry for CD58 and CD23 expression. STING (stimulator of interferon genes) activation/expression in B cells were evaluated through RT-PCR, immunoblot and immunocytofluorescence. RA synovium was characterized by immunofluorescence. CRISPR-Cas9 was employed to generate stable STING-KO EBV+CD58/CD23+ B-cells.Results:After RA-SFs co-culture, B-cells were organized in two subpopulations: CD58+/CD23high and CD58+/CD23low cells, previously described in EBV-infected B-cells. EBV+CD58/CD23+ B-cells express STING, physiologically absent in B-cells. The analysis of STING alternative RNA splicing isoforms revealed the expression of IFN-I inactive variants in B-cells, while EBV+CD58/CD23+ B-cells express the fully active protein. STING activation in EBV+CD58/CD23+ B-cells promotes the phosphorylation/activation of TBK1 and IRF3, and subsequent INFα and IFNβ production. CD58/CD23+ B-cells are expanded in RA synovium, showing STING expression and different levels of CD23 among aggregates. STING activation promotes the accumulation of CD58+/CD23low cells. STING-KO EBV+CD58/CD23+ B-cells confirms reduced percentages of CD58+/CD23low at basal level and upon STING activation.Conclusion:We demonstrated that RA-SFs are able to preferentially induce the selection/proliferation of EBV+CD58/CD23+ B-cells, with dysregulation of alternative RNA splicing mechanisms, promoting the expression of STING. These cells can be identified in the synovium of RA patient. STING expression and activation in EBV+CD58/CD23+ B-cells not only regulate IFN-I cytokines production, but are involved in CD23 and CD58 expression and in the organization of two functional subpopulations.REFERENCES:[1] Croia C, et al. Epstein-Barr virus persistence and infection of autoreactive plasma cells in synovial lymphoid structures in rheumatoid arthritis. Ann Rheum Dis 2013.[2] Bombardieri M, et al. A BAFF/APRIL-dependent TLR3-stimulated pathway enhances the capacity of rheumatoid synovial fibroblasts to induce AID expression and Ig class-switching in B cells. Ann Rheum Dis 2011.[3] Megyola C et al. Identification of a sub-population of B cells that proliferates after infection with epstein-barr virus. Virol J 2011.Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:Rheumatoid Arthritis (RA) is a systemic autoimmune disease with a prevalence of 0.5-1% worldwide1. Anti-cytokine antibodies, especially anti-Tumor Necrosis Factor (TNF) antibodies are considered the gold standard for RA therapy. However, there are some concerns regarding their lack of therapeutic efficacy in a significant proportion of patients2 and their potential systemic implications such as the risk of serious infections3. Developing novel agents with synovial targeting specificity might help to increase the therapeutic index while reducing systemic side effects of RA therapeutics.Objectives:Our work aims to develop a novel bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein combining synovium specificity with anti-TNFα activity. The potential advantage of this construct is a reduced systemic TNF-binding activity and increase delivery and activation of the TNF-neutralising capacity at the inflamed joints.Methods:The therapeutic tandem scFv-Fc fusion protein comprises two external arms with synovium specific targeting ability linked through a metalloproteinase (MMP) cleavable linker to the anti-TNFα variable fragments of Adalimumab fused to the CH2 and CH3 domains of IgG1 (Figure 1). The external scFv regions with synovium specificity were previously identified by in vivo phage display using a SCID mouse model transplanted with human synovium4. The construct was tested for its ability to bind and neutralize in vitro and ex vivo targets.Results:The fusion protein was tested by immunohistochemistry staining on RA synovium biopsies and an array of non-inflamed human tissues showing specific targeting of synovial microvasculature without no reactivity to the non-inflamed tissues. The TNFα binding and blocking capacity of the fusion protein was measured respectively by ELISA and cell assays measuring NF-κB activation, and it showed a two-fold decreased activity compared to the control antibody Adalimumab prior to detachment of the cleavable targeting fragment shielding the active anti-TNF fragment. Human synovial fluid and recombinant human MMP-1 efficiently cleaved the external arms of the antibody, releasing the anti-TNF scFv-Fc. The cleaved construct, detached from the synovium targeting arms, showed the same binding and anti-TNF inhibitory capacity/potency as Adalimumab.Conclusion:The novel bispecific tandem scFv-Fc demonstrated specific synovium targeting ability and intended reduced anti-TNF activity in its intact form prior to reaching the joint. Following MMPs-induced cleavage present in RA synovial fluid the therapeutic activity was restored to the same level as Adalimumab. Overall, this construct has the potential of decreasing the anti-TNF off-site activity and consequently, reduce systemic toxicity while maintaining high on-site activity. Also, the presence of a synovium targeting domain has the advantage of increasing the delivery and retention within the inflamed synovium and possibly increase the therapeutic index of this anti-TNF therapeutics.Figure 1.Schematic diagram of the bispecific tandem scFv-Fc fusion protein.[Figure omitted. See PDF]References:[1]Silman, A. J. & Pearson, J. E. Epidemiology and genetics of rheumatoid arthritis. 265–272 (2002).[2]Balogh, E. et al. Comparison of remission criteria in a tumour necrosis factor inhibitor treated rheumatoid arthritis longitudinal cohort: patient global health is a confounder. Arthritis Res. Ther. 15, R221 (2013).[3]Galloway, J. B. et al. Anti-TNF therapy is associated with an increased risk of serious infections in patients with rheumatoid arthritis especially in the first 6 months of treatment: updated results from the British Society for Rheumatology Biologics Register with special emp. Rheumatology (Oxford). 50, 124–131 (2011).[4]Kamperidis, P. et al. Development of a novel recombinant biotherapeutic with applications in targeted therapy of human arthritis. Arthritis Rheum. 63, 3758–3767 (2011).Disclosure of Interests:None declared

Human/SCID mouse chimeras have become a powerful tool to investigate immunological and inflammatory mechanisms involved in the pathogenesis of RA synovitis in leaving human synovial tissues and cells. In this model, grafted human synovium or its cellular components have been shown to retain features of the diseased tissue with operational cellular and molecular systems, as well as vascularisation and lymphatic drainage that can be investigated by examining/targeting specific pathways within the grafts. In particular, it has been shown that: a) grafts remain viable and become vascularized by mouse subdermal vessels as early as 7 days post-implantation; b) the graft vasculature forms chimeric anastomoses (human/mouse) at the edge of the transplants, while the transplanted tissue itself maintains expression of human adhesion molecules; c) the anastomoses are patent and functional allowing for the delivery of human cells, antibodies and pharmacological moieties; d) human adhesion molecules can be modulated by intra-graft injection of TNFα correlating with increased migration of human cells into the transplants; e) Chemokines e.g.SDF-1α are functional in this model and human cell migration into the graft can be inhibited by blocking the cognate CXCR4 receptor; f) VEGF induces a dose dependent blood vessel proliferation within synovial grafts; g) by TaqMan QT-PCR grafts can be examined for modulation of human pro-inflammatory cytokines in response to therapies such as biologics or conventional synthetic drugs. Recently we have used this model to gain direct functional evidence that activation induced cytidine deaminase (AID) positive synovial germinal centre-like structures (GC-LS) support class switch recombination (CSR) and in situ manufacturing of class-switched IgG anti-CCP antibodies in the human grafts, which can be detected in mouse sera. Thus, the functional information that this model provides in vivo is of great value to increase our understanding of disease pathogenesis and for drug development. This is of particular relevance for drugs in early phase of development when it might be important to obtain more supportive data before going into patients or target expression/modulation, tissue penetration as well as pharmacokinetics and pharmacodynamics. Currently, dose regimens are extrapolated to man from animal models and human cell in vitro systems, which may not be correct. Human-SCID chimeras permits a direct correlation between the dose regime and the degree of target tissue association, related to the effector response in the disease tissue, as well as validation of biomarkers/targets thus providing better understanding of pathobiology and some estimation of optimal dose regimen. Several groups worldwide have used this model to dissect various immunological/inflammatory mechanisms. Thus, from the literature and our own work it is clear that the human/SCID mouse transplant model represents a robust platform to investigate disease pathogenesis, autoimmunity and facilitate drug development. This presentation will review and discuss current available evidence. Disclosure of Interest None Declared

Background:Rheumatoid arthritis (RA) is a complex heterogenous autoimmune disease where 40% of patients are non-responsive to individual agents. Therefore, there is an urgent need to better strategise treatment through understanding the disease molecular mechanisms and its response to therapy. The biopsy driven clinical trial, R4RA, demonstrated that patients with low B cell signatures responded more to tocilizumab compared to rituximab[1]. Previous molecular analysis of the R4RA synovial biopsies using RNA-seq demonstrated immune response signatures which predicted future response to rituximab or tocilizumab as well as a stromal signature in refractory patients to both treatments[2]. However, how this translates to functional protein levels and active signalling pathways is not known and there is a paucity of phosphoproteome studies in RA.Objectives:We aimed to deepen our understanding on the potential pathways driving the different RA pathotypes and response to rituximab or tocilizumab in late-stage RA patients from the R4RA trial using phosphoproteome and total proteome analysis.Methods:33 baseline synovial biopsies before treatment from the trial were analysed using label-free mass spectrometry. All three pathotypes were represented: lymphoid (n=21), myeloid (n=7) and fibroid (n=5). 15 patients of the synovial biopsies went on to receive rituximab where 4 were responders and 11 were non-responders as determined by Disease Activity Score (DAS)-28-ESR < 3.2 and an improvement of > 1.2 after 16 weeks of treatment. The remaining 18 were treated with tocilizumab where 10 were responders and 8 were non-responders as determined by the same criteria as rituximab.Results:5,489 proteins and 12,989 phosphosites were quantified. The different pathotypes of the synovial biopsies were a key determinant of both phosphoproteome and proteome profiles. Immunological activity corresponded to the pathotypes with lymphoid being the most active, followed by myeloid and fibroid. For instance, the lymphoid pathotype had upregulated B cell related kinase activity, such as Mammalian Target of Rapamycin (MTOR, p=0.001), and enrichment of proteins regulated by interferon-regulated factor 8 (IRF8, q=0.02) and Signal Transducer and Activator Of Transcription 3 (STAT3, q=0.0003) (Figure 1). Characterisation of rituximab responders and non-responders using baseline synovial biopsies highlighted the enrichment of B cell related kinases in rituximab responders before treatment (BLK, LYN, SYK p=0.001 and MTOR p=0.007). For tocilizumab, there was a predicted enrichment of Mitogen Activated Protein Kinase (MAPK) activity in tocilizumab responders before treatment (e.g MAPK2K6 p=0.034 and MAP2K3 p=0.035), which may reflect the reciprocal interactions of MAPK and interleukin 6 (IL6) receptor signalling.Conclusion:This phosphoproteome study demonstrates the distinct baseline phosphoproteome profiles for each RA pathotype and the pre-treatment signalling pathways associated with drug response. Therefore, it highlights the importance of exploring the signalling network to improve personalised therapeutic strategies for managing RA.REFERENCES:[1] Humby et al Lancet 2021 397 (10271): 305-217.[2] Rivellese et al Nat Med 2022 28 (6): 1256-1268.Figure 1.Acknowledgements:The R4RA study was supported by the UK National Institute of Health Research (grant reference: 11/100/76) and core work associated with this project was supported by Versus Arthritis (Experimental Arthritis Treatment Centre, grant number 20022) and Barts and The London School of Medicine and Dentistry charity (grant number 523/819), MRC and Arthritis Research UK (ARUK) by their joint funding of Maximizing Therapeutic Utility in Rheumatoid Arthritis (MATURA) (grant numbers MR/K015346/1 and 20670 respectively), NIHR (grant 131575) and MRC TRACT-RA (MR/ V012509/1). This work acknowledges the support of the National Institute for Health Research Barts Biomedical Research Centre (NIHR 203330). The work of PC and VR is funded by Cancer Research UK (C15966/A24375 and C16420/A18066).Disclosure of Interests:None declared.

Background:Despite the availability of effective treatments, up to 10-20% of patients with Rheumatoid Arthritis (RA) do not respond to multiple targeted medications and can be classified as multi-drug resistant (MDR). MDR-RA has been linked to both extrinsic factors, such as comorbidities and non-compliance and, intrinsic factors related to the molecular pathology of the diseased tissue. For example, recently, a stromal synovial signature has been associated to multi-drug resistance. However, the role of immune cells and pathways in mediating multi-drug resistance is unknown.Objectives:To investigate the contribution of specific synovial immune cells in the pathogenesis of MDR-RA.Methods:In The R4RA trial, TNF-inhibitors inadequate responders RA patients were randomised to Tocilizumab or Rituximab following a synovial biopsy. At week 16, CDAI50 non-responders were switched to the alternative medication. At the end of the trial, we identified patients who did not respond to at least 3x bDMARDs, defined as MDR-RA (n=39), and compared with first-line (16 weeks) responders to either Rituximab or Tocilizumab (n=65), for baseline demographics, clinical characteristics and synovitis assessed by immunohistochemistry. In silico deconvolution (xCell) and differentially expressed gene (DEG) analyses were used to identify cell types and molecular signatures associated with treatment response/refractoriness specific to the lympho-myeloid population, i.e. with synovial infiltration of B and T cells.Results:The synovial pathotype distribution was similar in MDR-RA patients and first-line responders, with a comparable prevalence of the lympho-myeloid pathotype, characterised by an abundance of B and T cells—specifically 16 (41%) in MDR-RA and 34 (52.3%) in first-line responders. However, within this highly-inflamed lympho-myeloid population, MDR-RA patients showed significantly higher disease activity (CDAI 37.0 [33.9-41] vs 25.5 [17.6-35.3]; p=0.02), tender joint count (16.0 [12.3-22.3] vs 9.5 [4.0-15.5]; p=0.02), circulating white blood cells (10.3 [7.8-12.3] vs 8.2 [6.7-9.1]; p=0.02) and neutrophils (7.2 [5.6-9.3] vs 5.2 [4.4-6.5]; p=0.004) (Table 1). Molecular analysis showed 39 Differentially Expressed Genes in the MDR-RA lympho-myeloid population, particularly genes related to the innate immunity, such as CXCR1, CXCR2, S100A12, MS4A3, and TNFRSF10C. Accordingly, pathway analysis highlighted pathways related to granulocyte chemotaxis and migration upregulated in MDRA-RA lympho-myeloid patients. In silico deconvolution using xCell showed significantly higher levels of neutrophils, eosinophils, and mast cells in MDR-RA lympho-myeloid patients (Figure 1).Conclusion:Our study highlights a considerable heterogeneity in synovial inflammation among MDR-RA patients, pointing to diverse mechanisms underlying treatment resistance. Particularly within the lympho-myeloid population, our findings unveil an innate immune signature linked to MDR-RA, suggesting a potential role for innate immune cells in driving treatment resistance through potential crosstalk with adaptive immunity.REFERENCES:[1] Rivellese et al, Nat Med 28, 1256–1268 (2022).Table 1.MDR-RA vs First line responders (lympho-myeloid patients)MDR-RA(n=16)1st line Responders(n=34)p-valueAge56.2 (50.7-68.1)58.1 (52.3-65.4)0.99Gender (F)13 (81.3)24 (70.6)0.64BMI26.1 (20.4-30.0)26.4 (22.6-32.1)0.43WBC10.3 (7.8-12.3)8.2 (6.7-9.1)0.02Platelets335 (254-405)290 (241-263)0.08Neutrophils7.2 (5.6-9.3)5.2 (4.4-6.5)0.004Lymphocytes1.6 (1.4-2.3)1.7 (1.4-2.4)0.97RA duration9.0 (3.8-25.3)10.0 (4.5-16.8)0.93TJC 2816.0 (12.3-22.3)9.5 (4.0-15.5)0.02SJC 287.5 (6.0-9.5)4.5 (2.0-9.0)0.08ESR42.0 (29.0-59.6)28.5 (18.0-43.5)0.15CRP19.5 (6.7-47.0)10.5 (5.6-28.8)0.32VAS Pain66.0 (54.3-81.8)73.5 (54.0-88.8)0.45Patient Global Assessment75.5 (55.5-81.3)71 (54.3-85.5)0.84CDAI37.0 (33.9-41)25.5 (17.6-35.3)0.02DAS28 CRP5.91 (5.26-6.45)5.25 (4.16-6.02)0.04DAS28 ESR6.6 (5.9-6.8)5.4 (4.4-6.5)0.04Data shown as median (IQR) or n (%)Figure 1.Acknowledgements:We thank all patients participating in the trial and the Precision Medicine Patient Advisory Group (PM-PAG) for their continuous support. The R4RA trial was funded by the Efficacy and Mechanism Evaluation (EME) Programme, a partnership between the Medical Research Council (MRC) and the National Institute for Health and Care Research (NIHR) (grant no. 11/100/76). This study was further supported by NIHR (grant 131575) and MRC (MR/V012509/1) and acknowledges the support of the National Institute for Health Research Barts Biomedical Research Centre (NIHR203330).Disclosure of Interests:None declared.

Rheumatoid arthritis (RA) is a chronic systemic inflammatory autoimmune disease which primarily affects the joints [1]. Numerous agents are currently used to modify the natural course of the disease, however, several RA patients still do not reach clinical remission[2]. A recently developed quinoxaline analogue named Rabeximod (RBM) showed clinical benefits comparable to TNF-α blockers in a mouse model of collagen antibody-induced arthritis[3]. Moreover, in a phase IIa clinical trial a significantly higher clinical efficacy of RBM + Methotrexate versus Methotrexate alone has also been confirmed. Since RBM's exact biologic target has not been entirely established yet, our objective is to investigate its mode of action at the cellular and molecular level. Two in vitro models were developed: i) Toll-like receptor (TLR)-4 and TLR-2–stimulated peripheral blood mononuclear cells (PBMCs) cultured for 24-hours and ii) Monocytes derived-macrophages (MDMs), cultured for 96-hours in persisting inflammation conditions mimicking the different phases of ACPA-positive rheumatoid synovial inflammation: recruitment, initiation, development, and persistence of the inflammatory process. PBMCs-transcriptomic analysis was performed using a NanoString nCounter SPRING Profiler (customised panel). Cytokine production was measured by ELISA. RBM induced dose-dependent cell mortality. Among PBMCs, monocyte viability was preferentially affected compared to lymphocytes. RBM treatment reduced IL-6 and TNF-α production both in TLR4/TLR2-stimulated PBMCs and MDMs supernatants at 24h (PBMCs) and 10-, 24-, 48- and 72-hour time-points (MDMs). RBM-induced down-regulation of IL-6 and TNF-α in PBMCs was also obtained when PBMCs were pre-incubated for 7 hours prior to TLR activation (without additional RBM), suggesting an internalisation process. After 24h, RBM induced the downregulation of several pro-inflammatory genes in TLR-4 and-2 stimulated PBMCs, including IL-6, TNF-α, CSF1, FCN1, and SPP1. Finally, after 24 hours, MDMs treated with RBM developed an altered morphology independently of the frequency of treatment exposure and the stage of inflammation. Overall, our data suggest that RBM acts predominantly on monocytes/macrophages and can inhibit the production of pro-inflammatory molecules playing a crucial role in RA. [1]Scherer HU, Häupl T, Burmester GR. The etiology of rheumatoid arthritis. J Autoimmun. Published online 2020. doi:10.1016/j.jaut.2019.102400 [2]Guo Q, Wang Y, Xu D, Nossent J, Pavlos NJ, Xu J. Rheumatoid arthritis: Pathological mechanisms and modern pharmacologic therapies. Bone Res. 2018;6(1). doi:10.1038/s41413-018-0016-9 [3]Hultqvist M, Nandakumar KS, Björklund U, Holmdahl R. Rabeximod reduces arthritis severity in mice by decreasing activation of inflammatory cells. Ann. Rheum Dis. 452010;69(8):1527-1532. doi:10.1136/ard.2009.121178 NIL. Giulia Maria Ghirardi: None declared, Alessandra Nerviani: None declared, Liliane Fossati-Jimack: None declared, Elena Pontarini: None declared, Paola Italiani: None declared, Federico Pratesi: None declared, Sally Abdelmoaty Employee of: Cyxone AB, Carl Högerkorp Employee of: Cyxone AB, Costantino Pitzalis: None declared.

BackgroundA consistent connection between rheumatoid arthritis (RA) and type 2 diabetes (T2D) has been recently reported highlighting interleukin-1β (IL-1β) as shared pathogenic pathway and therapeutic target [1]. In a clinical trial enrolling patient with RA and T2D, we demonstrated that IL-1 inhibition led to a marked reduction of glycated haemoglobin together with a decrease of RA disease activity [2].ObjectivesTo evaluate the synovial expression of IL-1 related genes and the relationship to the ubiquitin-proteasome system in the synovial tissues of RA patients with and without T2D. To assess the effects of high concentration of insulin in vitro, mimicking the hyperinsulinism of the early phases of T2D, on ubiquitinated proteins in fibroblast-like synoviocytes (FLSs).MethodsEarly (<1 year) treatment-naïve RA patients with T2D (RA/T2D n=16) were compared with age- and gender-matched RA patients without T2D (n=16) enrolled in the Pathobiology of Early Arthritis Cohort (PEAC) [3]. Synovial tissue biopsies obtained under ultrasound guidance underwent RNA-sequencing as previously described [3], and we compared IL-1 pathway genes in patients without and with T2D. The synovial expression of ubiquitin in macrophages and synovial lining fibroblasts was assessed by immunohistochemistry/immunofluorescence and correlated with synovial pathotypes [4]. Finally, FLSs from RA patients (n=5) were isolated and treated with human insulin (200 and 500 nM) and ubiquitinated proteins were assessed by western blot.ResultsSynovial RNA-sequencing showed that one third of IL-1 pathway genes (41/138) were significantly different in RA/T2D patients compared to RA patients without T2D. In parallel, synovial tissues of RA/T2D patients were characterised by a consistent reduced expression of ubiquitin-proteasome genes. More specifically, ubiquitin genes (UBB, UBC, and UBA52) were significantly lower in T2D/RA patients. Furthermore, 22 genes codifying proteasome subunits were significantly lower in RA/T2D patients (PSMA2, PSMA6, PSMA7, PSMB1, PSMB3, PSMB4, PSMB6, PSMB7, PSMB8, PSMB9, PSMB10, PSMC1, PSMC3, PSMC5, PSMC13, PSMD4, PSMD7, PSMD8, PSMD9, PSME1, PSME2, and PSMF1). Additionally, several genes regulating ubiquitin and proteasome system were significantly different in the synovial tissue of RA/T2D patients. Specifically, APP, BAG4, and BTRC were upregulated in RA/T2D patients. Conversely, RACK1, RBX1, RPS27A, SEM1, SHARPIN, and SIGIRR were significantly downregulated in RA/T2D patients.Immunohistochemistry showed a significant reduction of the percentage of ubiquitin-positive cells in synovial tissues of RA/T2D patients. The percentage of ubiquitin-positive cells was also increased in patients with a lympho-myeloid pathotype compared to diffuse myeloid or pauci-immune-fibroid. Despite its widespread expression in synovia, immunofluorescence showed that ubiquitin mainly colocalized with synovial macrophages and lining fibroblasts.Finally, in vitro experiments showed a reduction of ubiquitinated proteins in RA-FLSs treated with a high concentration of insulin (500 nM).ConclusionIncreased IL-1 gene expression was observed in the synovial tissues of RA/T2D patients linked with decreased ubiquitin-proteasome system. These findings may provide a mechanistic explanation of the observed clinical benefits of IL-1 inhibition in patients with RA and concomitant comorbid T2D, as the reduction of the ubiquitin-proteasome system may enhance the levels of precursor IL-1β and the production of bioactive IL-1β.References[1] Giacomelli R, et al. Expert Rev Clin Immunol. 2016;12:849-55[2] Ruscitti P, et al. PLoS Med. 2019;16:e1002901[3] Lewis MJ, et al. Cell Rep. 2019;28:2455-70[4] Humby F, et al. Ann Rheum Dis. 2019;78:761-72Acknowledgements:NIL.Disclosure of InterestsNone Declared.

The presence of circulating rheumatoid factor (RF) and the formation of ectopic lymphoid structures (ELS) in labial salivary glands (SG) of patients with Sjögren's Syndrome (SS) have been reported as independent risk factors associated with the development of SG B-cell MALT lymphoma (MALT-L). Neoplastic MALT-L B-cells express highly hypermutated B-cell receptors bearing RF immunoreactivity in up to 50% of the cases, but whether their maturation and proliferation is dependent on ELS or is also induced by extrafollicular responses in the SG is currently unclear. The definition of ELS and their association with circulating autoantibodies has so far relied on SG histopathology which bears significant limitations. Conversely, molecular pathology analysis through whole-tissue RNA Sequencing (RNASeq) has allowed a better definition of disease heterogeneity and disease taxonomy. To perform transcriptomic profiling of SS minor SG tissue characterised by different degrees of inflammatory aggregate organization and to identify transcriptomic clusters and gene signatures associated with peripheral and histological biomarkers of disease. Labial SG were obtained from 99 patients including SS with and without ELS (respectively ELS+ and ELS-, as assessed by immunohistochemistry) and non-specific chronic sialadenitis (Sicca). Total RNA was extracted, complementary DNA libraries were prepared and sequenced. Differentially expressed genes (DEG), deconvolution and pathway analysis were performed. Unsupervised gene clustering by differential expression between sicca and SS confirmed a clear transcriptome segregation between the two diagnoses. As expected, in SS SG expression of genes associated with inflammation and adaptive immune responses was upregulated (e.g. CCR7, CD19, CR2, CXCL13, CXCL9, CXCR5, FCRL3, FCRL4, IL21R, MS4A1, PAX5, SLAMF6, TLR10) (Figure A). Bulk RNAseq cell deconvolution confirmed immune cell enrichment (Th, B and plasma cells) in SS SG, especially those ELS+. A three-way comparison among sicca, ELS+ and ELS- SG, showed only a few genes specifically associated to ELS- and sicca, whereas most of the DEGs were either ELS+ specific or common to all SS (Figure B). Results of pathway analysis on ELS+ and SS-associated DEGs showed very similar profiles characterised by adaptive and interferon-associated pathway activation. Weighted Gene Co-Expression Network analysis showed higher activation of anti-viral pathways in ELS- SG, where ELS+ SG were enriched in adaptive immune pathways. Principal component analysis on SS SG mRNA showed that a significant proportion of variability was associated with the presence of RF in the serum of patients independently of the presence of ELS. Surprisingly, transcriptome clustering was closer between ELS+/RF+ and ELS-/RF+ SG than the clustering of ELS+/RF- with ELS-/RF- SG. Accordingly, SG of RF+ patients showed significant upregulation of B cell-associated genes (e.g. CXCL13, MS4A1). However, while SG transcriptomes of ELS+/RF+ were characterised by genes associated with germinal centre formation (e.g IL21, AICDA, LTB), transcriptomes from ELS-/RF+ SG displayed a unique set of DEGs such as BCL2, TRIM22, XAF1, DDX58, DDX60 and IFIT1. Conversely, a similar analysis performed based on anti-Ro/SSA and/or anti-La/SSB did not yield any significant differences in gene expression. A comprehensive bulk RNA sequencing analysis of SS and sicca patients SG showed that higher tissue inflammation with features of functional germinal centres is associated to the presence of both RF and ELS, rather than ELS alone. Furthermore, the existence of a RF-driven SG transcriptome independent of the presence of ELS suggest that both follicular and extra-follicular responses support the selection of B-cells with RF immunoreactivity within the SG of SS patients and could be involved in B-cell lymphomagenesis. [Display omitted] NIL. NIL. Elena Pontarini: None declared, Davide Lucchesi: None declared, Katriona Goldmann: None declared, Myles Lewis: None declared, Qingxuan Song Employee of: Janssen Pharmaceuticals, Inc., Spring House, PA, USA, Elizabeth Thomas Employee of: Janssen Pharmaceuticals, Inc., Spring House, PA, USA, Ling-Yang Hao Employee of: Janssen Pharmaceuticals, Inc., Spring House, PA, USA, Kathy Sivils Employee of: Janssen Pharmaceuticals, Inc., Spring House, PA, USA, Costantino Pitzalis: None declared, Michele Bombardieri Speakers bureau: UCB, Janssen, Consultant of: GSK, Janssen, MedImmune, Grant/research support from: Janssen, MedImmune.

Background:Tumor Necrosis Factor inhibitors (TNFi) are the first line biologic treatment for RA. However, about 40% of RA patients do not respond to TNFi, highlighting the need for predictive biomarkers.Objectives:We integrated synovial and blood DNA methylation data with RNA-sequencing and genotype data to identify drivers of synovial gene expression, markers of disease activity and optimal pre-treatment predictors of TNFi response in RA.Methods:Differential DNA methylation and gene expression analysis was performed on the pre-treatment baseline synovial biopsy samples (n=54) looking at differences between EULAR DAS28-ESR good responders/non-responder to etanercept and lymphoid/fibroid pathotypes classified based on synovial histology. Hierarchical clustering of gene expression and DNA methylation profiles covering key inflammatory pathways related to TNF, IL-6, B-cells (CD20), and Fibroblast Growth Factor (FGF) was performed. A machine learning model with 10x10-fold nested cross-validation (CV) with 25 repeats utilizing DNA methylation probe data as input was constructed to predict TNFi response.Results:We identified 44 differential DNA methylation regions (DMRs) across 48 genes that differentiate good responders from non-responders. Notable genes included IL32, linked to RA disease activity, and RA susceptibility genes ARID5B and RUNX3, involved in immune regulation and T cell development. Response analysis revealed 45 significantly differentially expressed genes (DEGs), including TNFRSF13B and B cell signalling genes (BLK, CD19). Lymphoid and fibroid pathotypes are linked to differential treatment response to TNF-inhibitors. Analysis showed distinct methylation and gene expression patterns between pathotypes, with 6389 differentially methylated probes (DMPs) and 3681 DEGs identified, of which 688 genes had 2680 DMPs located in their promoter regions, demonstrating that differential methylation influences differential gene expression. Network analysis highlighted 378 DEGs related to the immune system, 70 in lymphoid cell interactions, and 11 in the TNF-α signalling pathway (TNFSF11, BIRC3, LTB). Hierarchical clustering of pre-treatment DNA methylation revealed a subgroup (n=12, 83% good responders) with hypomethylation of TNF and IL6 module genes and FGF module hypermethylation. RNA-seq showed a similar subgroup (n=10, 90% good responders) with increased TNF/IL6/CD20 gene module expression and reduced FGF expression. These findings indicate that molecular patterns can distinguish responders at baseline, with specific subgroups showing varying response likelihoods to etanercept. Machine learning fitted using nested CV with a glmnet penalised regression model predicted a target DAS28<3.2 after 16 weeks of treatment with AUC of 0.81, suggesting that baseline DNA methylation data can accurately predict treatment response. Expression quantitative trait loci (eQTL) analysis highlighted 3954 genes, of which 8 eQTL driven genes, including B-cell specific genes BLK and FCLR1, showed a significant association with response to treatment, underscoring the role of genotypic control over expression in affecting treatment responsiveness.Conclusion:This study substantiates the predictive capacity of DNA methylation and multi-omic profiling in forecasting TNFi response in RA. The machine learning model demonstrates the robustness of epigenetic markers as predictors of treatment response, enhancing the prospects for personalized treatment strategies. Clinically, these findings have the potential to refine patient stratification and guide precision therapy, potentially transforming RA management by pre-emptively identifying individuals likely to benefit from anti-TNF therapy, thereby improving patient prognosis and optimizing healthcare resource allocation.REFERENCES:NIL.Acknowledgements:We thank all patients participating in the trial and the Patient Advisory Group. The STRAP trial was part of the Maximising Therapeutic Utility in RA (MATURA) programme, jointly funded by the UK Medical Research Council (MRC) and Versus Arthritis (grant reference MR/K015346/1). Infrastructure support was also provided by Versus Arthritis (Experimental Arthritis Treatment Centre grant, number 20022). We also acknowledge support from Barts Charity (grant number 523/819), NIHR (grant 131575) and MRC TRACT-RA (MR/V012509/1). This work acknowledges the support of the National Institute for Health Research Barts Biomedical Research Centre (NIHR 203330).Disclosure of Interests:None declared.