Die rheumatoide Arthritis (RA) ist eine biologisch heterogene Erkrankung, bei der die Therapieauswahl nach Versagen eines ersten Tumornekrosefaktorinhibitors (TNFi) noch immer weitgehend auf empirischem Wechsel statt auf gewebebasierter Stratifizierung beruht. Die Synovialbiopsie hat sich schrittweise von einem Forschungsverfahren zu einem praktikablen und informativen Ansatz für den Zugang zum Zielorgan der Erkrankung entwickelt und ermöglicht die histologische, molekulare und funktionelle Charakterisierung der entzündeten Synovialis. Frühere biopsiebasierte Studien, darunter R4RA und STRAP, haben gezeigt, dass synoviale Signaturen Aufschluss über das Ansprechen auf die Therapie geben können, wobei sie gleichzeitig die Grenzen einfacher histologischer Klassifikatoren und die Notwendigkeit multidimensionaler gewebebasierter Modelle aufzeigen. In diesem Rahmen wurde die SQUEEZE-BioTest-Studie als beobachtende, prospektive, biopsiebasierte Plattform für Patientinnen mit RA und primärem Versagen einer Erstlinien-TNFi-Therapie konzipiert, die im Rahmen der Routineversorgung eine Behandlung mit einem TNFi, einem IL-6-Rezeptor-Inhibitor oder einem JAK-Inhibitor beginnen. Durch die Kombination einer ultraschallgeführten Synovialbiopsie zu Studienbeginn mit serieller klinischer Nachbeobachtung, Blutprofilierung, Einzelzell- und Multiomics-Analysen sowie Ex-vivo-Modellen zur Arzneimittelreaktion zielt BioTest darauf ab, synoviale Reaktionssignaturen zu validieren und die Entwicklung klinisch umsetzbarer Biomarkerplattformen zu unterstützen. Dieser Übersichtsartikel erörtert die Begründung, Struktur und potenziellen translationalen Auswirkungen von BioTest und positioniert die Studie als Modell für die nächste Phase der gewebegesteuerten Präzisionsmedizin bei RA.

OBJECTIVES:Methotrexate (MTX) is the first-line treatment for rheumatoid arthritis (RA), yet its immunological mechanisms remain incompletely understood. This study aimed to define the time-resolved cellular and molecular changes induced by MTX. METHODS:In a prospective longitudinal study, newly diagnosed patients with RA initiating MTX were systematically profiled over time. We applied an integrated single-cell multiomics approach combining high-parameter immunophenotyping and single-cell RNA sequencing of PBMCs (peripheral blood mononuclear cells) obtained from 6 patients at baseline and at sequential time points during therapy. Functional in vitro assays were conducted to evaluate direct effects of MTX. Key findings were validated in an independent cohort of 22 patients with RA, stratified by clinical response, and integrated with external transcriptomic datasets. RESULTS:MTX-induced rapid immune remodelling that was detectable as early as 3 weeks after treatment initiation and preceded clinical improvement. The earliest and most pronounced alterations occurred in T follicular helper (Tfh) cells and plasmablasts, which declined selectively in clinical responders but remained largely unchanged in nonresponders across independent cohorts and external datasets, underscoring their potential as early biomarkers of treatment response. Functional plasmablast differentiation assays further linked MTX treatment to impaired development. Transcriptomic network analyses identified novel regulatory hubs associated with MTX exposure, including LMNA, SCO2, GVQW3, and members of the GIMAP family. To promote data accessibility, we developed an interactive single-cell browser for community-driven exploration. CONCLUSIONS:Together, these findings reveal an unexpectedly rapid and coordinated immune reconfiguration after MTX initiation and position early alterations in Tfh cells and plasmablasts as biomarkers of treatment response.

OBJECTIVE:Idiopathic inflammatory myopathies (IIM) are autoimmune disorders characterised by muscle inflammation, high creatine kinase (CK) levels and disability. Despite immunomodulating therapies, patients often experience progressive disease, resulting in refractory conditions or dependence on glucocorticoids (GC). This study addresses the efficacy and safety of immunoadsorption (IAS) in refractory IIM. METHODS:A retrospective, monocentric study was conducted on patients with highly active IIM subjected to IAS between January 2000 and September 2021. Inclusion criteria were adult patients with confirmed IIM unresponsive to standard therapies, including GC and at least one disease-modifying antirheumatic drug. Primary end point was defined as minor improvement, requiring a ≥ 20% reduction in both daily GC dosage and CK levels at week 12, alongside patient comparative self-assessment (PCSA) rated as 'no change' or 'better'. Secondary endpoints included moderate (≥40%) and major (≥60%) improvements at weeks 4, 8 and 12. Relapse rates were assessed over a 3-month period following the last IAS procedure. RESULTS:The study included 23 refractory IIM patients treated with IAS. Primary end point was reached by 52.2% (n = 12) patients. Moderate improvement was observed in 8.7% (n = 2), 26.1% (n = 6) and 34.8% (n = 8) and major improvement in 8.7% (n = 2), 17.4% (n = 4) and 30.4% (n = 7) at weeks 4, 8 and 12, respectively. No unexpected adverse events were reported, with low relapse rates within 3 months post-IAS (18%). CONCLUSION:IAS may be an effective adjunctive therapy in patients refractory to GC and conventional treatments, leading to rapid CK level reductions, decreased GC usage and improvements in PCSA and muscle function.

Rheumatoid arthritis (RA) is characterized by immune dysregulation, including alterations in peripheral blood mononuclear cell (PBMC) populations and aberrant cytokine signaling. Methotrexate (MTX) is the preferred first-line treatment for RA, yet its precise mechanisms of action remain incompletely understood. This study employed a multi-omics strategy-combining single-cell RNA sequencing (scRNA-seq) and immunophenotyping-to identify key effector peripheral immune cells and their cellular responses in RA patients over 12 weeks of MTX treatment. In our study, MTX was associated with significant immune modulation, including the restoration of naive T and B cells and reductions in T cell memory subsets with these effects detectable as early as three weeks post-treatment. Plasmablast levels also emerged as a potential biomarker for early therapeutic response, reflecting MTX's impact on immune homeostasis. Transcriptional analysis revealed modulation of key pathways, including TNF-alpha; signaling, B cell receptor signaling, and T cell receptor-mediated apoptosis. Network analysis identified critical regulatory hubs, such as EGR1, JAK2, and SOCS1, in monocytes and CD4 memory T cells, highlighting these cell types as key mediators of MTX's effects. In conclusion, these findings advance our understanding of MTX's effects on immune cell dynamics at different stages of treatment, showing for the first time the early cellular changes leading to immune modulation in RA. Altogether, our results provide the foundation for further mechanistic investigations into MTX. ### Competing Interest Statement MaB and TP received funding from Lilly. MiB received grants from AlphaSigma. DA received grants and consulting fees from AbbVie, Amgen,Lilly, Merck, Novartis, Pfizer, Roche and Sandozand.

OBJECTIVES AND DESIGN:To further elucidate the effects of rare systemic autoimmune rheumatic diseases (SARD) and their treatment on antibody development after vaccination against SARS-CoV-2, we compared patients with and without immunosuppressive therapy to healthy controls in an observational cohort study. PARTICIPANTS AND SETTING:We enrolled 52 patients with SARD and 72 healthy subjects in a prospective, observational study at the Medical University of Vienna and measured the humoral response 6 months after two mRNA vaccinations and 2-6 weeks after a third dose. RESULTS:Patients with vasculitis showed significantly (p=0.02) lower antibody titres 6 months after vaccination (median 247 BAU/mL, IQR [185-437]), as compared with healthy controls (median 514 BAU/mL, [185-437], IQR 323; 928, vasculitis patients: 247, IQR [185; 437], p<0.05). Patients receiving 2-3 immunomodulatory medications showed significantly lower antibody levels. Of note, all patients with SARD, even those without immunomodulatory medication, developed lower antibody levels after the third dose compared with healthy controls (median 22 630, IQR [16 945; 43 200] in HC, 9510 IQR [3866; 14 215] in patients without immunosuppressive treatment (p<0.001), 7780 IQR [2203; 15 645] in patients receiving a single immunomodulatory drug (p<0.0001) and 14 320 IQR [2415; 35 400] in patients receiving combination therapy (p=0.081)). CONCLUSIONS:Patients with SARD displayed lower antibody development after booster vaccination, even if antibody levels after two immunisations were comparable to healthy controls. Our data may be limited due to sample size, but it provides pointers for a more individualised, antibody-titre-oriented approach and earlier booster vaccination in patients with SARD.

Background In systemic sclerosis (SSc), an autoimmune response leads to progressive fibrosis of the skin and internal organs, driven by aberrant activation of fibroblasts. The mechanisms dictating persistent dermal fibroblast (DF) activation and production of extracellular matrix (ECM) remain poorly understood. Nicotinamide N-methyltransferase (NNMT), a SAM-consuming enzyme that modulates cellular methylation potential, has been implicated in fibrotic tissue remodelling in metabolic and malignant diseases. Here, we identify NNMT as a key determinant in DF activation and fibrosis in SSc. Methods We analyzed bulk, single-cell RNA-Seq and spatial transcriptomics datasets from SSc skin. Functional studies were performed in TGFβ-activated primary human DFs using siRNA-mediated NNMT knockdown (KD) combined with RNA-Seq, metabolite profiling, ELISA, and western blotting. The role of NNMT-regulated transcription factors was assessed by QuantSeq 3′ RNA-Seq following ATF4, SOX9, or SRF KD. Findings NNMT was markedly upregulated in SSc skin and enriched in disease-expanded SFRP2⁺/COL8A1⁺ myofibroblast states. NNMT KD restored methylation balance by increasing the SAM/SAH ratio and H3K27me3 levels, and abrogated TGFβ-induced profibrotic programs regulating ECM production and collagen synthesis. Mechanistically, NNMT was required for TGFβ-induced upregulation of the transcription factors ATF4, SOX9, and SRF, which together orchestrate ECM gene expression and COL1A1 secretion. Interpretation These findings define a previously unrecognized TGFβ–NNMT-ATF4/SOX9/SRF axis that coordinates profibrotic transcriptional programs in DFs. Accordingly, NNMT functions as a central effector linking TGFβ signaling to DF activation and ECM remodelling. Targeting NNMT may thus represent a promising therapeutic strategy to attenuate skin fibrosis in SSc. ### Competing Interest Statement D.A. reports no conflicts of interest related to this work. For transparency, D.A. discloses receiving grants from Eli Lilly and speaker or consultancy fees from AbbVie, Gilead, Johnson & Johnson, Eli Lilly, MSD, Novartis, and Sandoz. The other authors declare no conflicts of interest.

Background: The pathogenesis of synovitis in systemic sclerosis (SSc) is unknown. Due to the lack of evidence-based therapies, SSc patients with synovitis are treated as rheumatoid arthritis (RA) patients, however with unknown effect. Previously, we showed that SSc synovitis differs from RA synovitis with a prevalent pauci-immune pathotype. Objectives: To decipher the histological, cellular, and transcriptional differences between SSc and RA synovitis, specifically in synovial fibroblasts (SF). Methods: We collected ultrasound-guided synovial biopsies from 11 SSc and 7 RA patients. All samples were analysed histologically, including Krenn synovitis score (0-9) and pathotype characterisation. Additionally, 7 SSc and 6 RA samples were dissociated and processed for single-cell RNA-sequencing (20,705 cells in SSc and 31,681 in RA). Due to the pauci-immune nature of the synovitis, we focused our analysis on the SF population (4,638 SF in SSc, 6,097 in RA). We performed differential gene expression analysis, over-representation pathway analyses and calculated signature expression scores between SSc and RA SF. Pathway analyses included publicly available datasets and bulk RNA sequencing data from SF stimulated in vitro with TNF-α, IFN-α/b, IFN-γ, and IL-6 from internal experiments and published data1 (Figure 1A). Results: The Krenn synovitis score was lower in SSc than in RA (2.1 ± 1.4 vs. 4.2 ± 1.0, p = 0.01). There was no difference in fibrosis (Elastica van Gieson staining) and vascularisation (CD31 staining) between SSc and RA synovitis. We confirmed a pauci-immune pathotype characteristic of SSc synovitis (91% vs. 43% of RA biopsies, p = 0.04). Consistently, neutrophils were more prevalent in RA as compared to SSc synovitis (p < 0.01). On the single-cell level, we identified seven SF cell states defined by the expression of the marker genes PRG4, CHI3L2, COMP, CXCL12, CXCL14, MFAP5, and POSTN consistent with the current literature in RA2. The proportion of SF clusters differed between both diseases with a higher enrichment of MFAP5+ (Fold-change (FC) = 3.81, false discovery rate (FDR) < 0.001) and COMP+ SF in SSc (FC = 2.28, FDR < 0.001), and POSTN+ (FC = 3.12, FDR < 0.001) and CXCL12+ SF in RA (FC = 1.70, FDR < 0.001). We identified 675 significant differentially expressed genes (FDR < 0.01, |log2FC| > 0.25) and several differentially activated signalling pathways between SSc and RA SF. In particular, SSc SF were enriched in IFN-α and IFN-γ (both FDR < 0.018) and complement and coagulation pathways (FDR < 0.008), especially in genes of the alternative complement pathway (Figure 1B). RA SF showed a strong enrichment in TNF-α signaling (FDR < 6.3e-12) and inflammatory response genes (FDR < 0.018). Using the transcriptional signature of in vitro stimulated SF as gene sets, we confirmed the IFN response in SSc SF, whereas TNF-α and IL-6 response signatures were more dominant in RA SF (Figure 1C). Interestingly, IFN-α/β and IFN-γ stimulation of SF in vitro led to an activation pattern of alternative complement pathway genes similar to that observed in SSc SF (Figure 1D). IFN and complement signature scores were significantly higher in sublining CXCL12+ SF in SSc compared to RA (both p < 2.22e-16). To identify the drivers of the IFN signature in SSc SF, we assessed the expression of the different IFN genes in synovial cell populations. Only IFNG was expressed, mainly by T cells, which is consistent with the preferential expression of IFN response genes in the sublining synovium. Conclusion: This first in-depth characterization of the SSc synovium revealed hallmark tissue, cellular and molecular changes between SSc and RA synovitis. We found a strong enrichment of the IFN signalling pathway in SSc SF, possibly leading to a dysregulation of the alternative complement pathway. Our findings provide the basis for the development of specific targeted therapies for synovitis in SSc. REFERENCES: [1] Tsuchiya, Haruka, et al. Annals of the Rheumatic Diseases 80.4 (2021): 440-450.[2] Zhang, Fan, et al. Nature (2023): 1-9. Acknowledgements: This project is funded by FOREUM – Foundation for Research in Rheumatology. We thank our patient research partners Joëlle Messmer & Andreas Eisenring. Disclosure of Interests: Celina Geiss: None declared, Miranda Houtman: None declared, Raphael Micheroli: None declared, Yannis Djeffal: None declared, Mojca Frank Bertoncelj: None declared, Sam G. Edalat: None declared, Kristina Buerki: None declared, Chantal Pauli: None declared, Michael Bonelli GSK, Galapagos, Thomas Karonitsch: None declared, Oliver Distler 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant, Amgen, AnaMar, Argenx, Arxx, AstraZeneca, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galderma, Galapagos, Glenmark, Gossamer, Horizon, Janssen, Kymera, Lupin, Medscape, Merck, Miltenyi Biotec, Mitsubishi Tanabe, Novartis, Orion, Prometheus, Redxpharma, Roivant, Topadur and UCB, Co-founder of CITUS AG, 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant, Amgen, AnaMar, Argenx, Arxx, AstraZeneca, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galderma, Galapagos, Glenmark, Gossamer, Horizon, Janssen, Kymera, Lupin, Medscape, Merck, Miltenyi Biotec, Mitsubishi Tanabe, Novartis, Orion, Prometheus, Redxpharma, Roivant, Topadur and UCB, 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant, Amgen, AnaMar, Argenx, Arxx, AstraZeneca, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galderma, Galapagos, Glenmark, Gossamer, Horizon, Janssen, Kymera, Lupin, Medscape, Merck, Miltenyi Biotec, Mitsubishi Tanabe, Novartis, Orion, Prometheus, Redxpharma, Roivant, Topadur and UCB, Caroline Ospelt: None declared, Muriel Elhai: None declared.Figure 1Abbreviations: log2FC = log2 fold-change, RA = rheumatoid arthritis, SF = synovial fibroblasts, SSc = systemic sclerosis, stim = stimulation. Panel A created with BioRender.

Objective: We analyzed the impact of amino acid (AA) availability on the inflammatory response in arthritis.Methods: We stimulated rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) with tumor necrosis factor (TNF) in the presence or absence of proteinogenic AAs and measured their response by QuantSeq 3' messenger RNA sequencing, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. Signal transduction events were determined by Western blot. We performed K/BxN serum transfer arthritis in mice receiving a normal and a low-protein diet and analyzed arthritis clinically and histologically.Results: Deprivation of AAs decreased the expression of a specific subset of genes, including the chemokines CXCL10, CCL2, and CCL5 in TNF-stimulated FLSs. Mechanistically, the presence of AAs was required for the TNF-induced activation of an interferon regulatory factor 1 (IRF1)-STAT1 signaling circuit that drives the expression of chemotactic factors. The expression of IRF1 and the IRF1-dependent gene set in FLSs was highly correlated with the presence of inflammatory cells in human RA, emphasizing the important role of this AA-dependent pathway in inflammatory cell recruitment to the synovial tissue. Finally, we show that mice receiving a low-protein diet expressed less IRF1 in the inflamed synovium and consequently developed reduced clinical and histologic signs of arthritis.Conclusion: AA deprivation reduces the severity of arthritis by suppressing the expression of IRF1-STAT1-driven chemokines, which are crucial for leukocyte recruitment to the arthritic joint. Overall, our study provides novel insights into critical determinants of inflammatory arthritis and may pave the way for dietary intervention trials in RA.

Background: Idiopathic inflammatory myopathies (IIM) comprise a heterogenous group of autoimmune diseases characterised by inflammation of muscle and affection of other organs, such as lung or skin. A significant proportion of IIM patients have progressive weakness of the skeletal muscle as the predominant or sole clinical manifestation despite therapy. Response to treatment with glucocorticoids (GC) and immunosuppressive drugs varies substantially among patients and IIM-subtypes. However, some patients do not respond sufficiently to these therapies and, therefore, other treatment modalities have to be considered. There are only limited data on efficacy and safety of immunoglobulin removal by immunoadsorption (IAS) in patients with refractory IIM. Objectives: We aimed to assess IAS efficacy and safety in management of therapy-refractory IIM-patients. Methods: In this monocentric retrospective study, we evaluated the effects of IAS in IIM patients with predominantly muscular involvement, and previous non-response to GC or/and ≥1 immunomodulating drug. IAS was performed via columns using Protein A, Globaffin or Ig Therasorb columns with polyclonal sheep antibodies to eliminate immunoglobulin G from circulation. In a single treatment session, a total plasma volume of 6000-8000 ml was processed. The frequency of IAS sessions was on average 2.3 sessions per week. The primary endpoint was a minor improvement, defined as ≥20% reduction in daily GC dosage and ≥20% decline in creatine kinase (CK) after 4 weeks. Secondary endpoints included moderate and major improvement (reduction in daily GC dosage and CK-values of ≥40% and ≥60%, respectively) at weeks 4, 8, and 12. We further assessed relapse rates during the 3-month period following the last IAS procedure. A sensitivity analysis was performed using a last observation carried forward method addressing missing values. Results: Between 2000 and 2021, 25 IIM patients treated with IAS were identified; 23 of them fulfilled the inclusion criteria and were evaluated. Patient characteristics at start of IAS are shown in Table 1. At week 4, 43.5% (n=10) of IIM patients achieved the primary endpoint of minimal improvement. Secondary endpoints, such as moderate improvement was observed in 10 patients (43.5%) at week 4, and a major improvement was noted in 12 patients (52.2%) at week 4. Further rates of clinical response are shown in Figure 1. The daily dosage of oral glucocorticoids decreased by 14.3% at week 4 as well as week 8, and by 20% at week 12 after initiation of IAS. Reduction from baseline in creatine kinase levels was maximal at week 8 (84.3%). Among the 6 seronegative patients, 5 (83%) were non-responders, while 53% of the 17 patients presenting with any autoantibody were responders. No unexpected adverse safety or tolerability issues were observed. Relapses occurred only in about 20% within three months after cessation, however the rate of relapses was higher in those without initial response at week 4. Conclusion: IAS may serve as efficacious adjunctive therapy in seropositive IIM-patients who are refractory to GC and conventional treatments, leading to rapid reduction CK levels and allowing a decrease of GC dosage despite their previously non-responsive state.Table 1. Cohort descriptives REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: None declared.Figure 1Clinical response categories. Red (minor improvement), yellow (moderate improvement), blue (major improvement)

Background: Rheumatoid arthritis (RA) is a chronic inflammatory disease which is characterized by synovitis. During inflammation, the synovium transforms into the RA-typical hyperplastic membrane which is composed of fibroblast-like synoviocytes (FLS) and activated resident as well as infiltrating immune cells. By attacking adjacent structures, the hyperplastic membrane drives the destruction of bone and cartilage. Interactions and reciprocal cross-talks between FLS and immune within the synovium promote the pathogenic course of RA. Objectives: Understanding of molecular and cellular mechanisms in disease pathogenesis has led to great advances in the treatment of RA. The role of cell-to-cell interaction in the chronic course of RA however remains largely unknown. In this study, we investigated FLS – immune cell interaction patterns in an inflammatory setting and its consequences on synovial inflammation which will advance clinical biomarker development. Methods: Human FLS were isolated and treated with different inflammatory cytokines to mimic the inflamed joint. After 24h, cytokines were washed off and autologous peripheral blood mononuclear cells (PBMCs) were added to the pre-treated FLS. Automated high-content microscopy of co-cultures after 24h followed by downstream bioinformatic analysis of image data enabled visualisation and quantification of FLS – immune cell interactions. Spectral cytometry analysis of PBMCs re-isolated from co-culture allowed to determine the activation state of PBMC subsets. Sort-purification of PBMC subsets followed by RNA-seq enabled deep phenotyping and profiling of transcriptional changes in monocytes, B-cells, memory CD4 T-cells, and memory CD8 T-cells after co-culture with FLS. Results: Bioinformatic analysis of high-content fluorescence microscopy data showed cell type-specific interaction patterns between FLS and PBMC subsets. Cytokine pre-treatment of FLS had a profound cytokine-specific impact on FLS-immune cell interaction patterns. We also observed enhanced activation of immune cell subsets after co-cultures of FLS and PBMCs. In line with microscopy data, cytokine pre-treatment of FLS modulated their capacity to activate immune cells. IFNγ pre-treatment of FLS increased whereas TGFβ repressed the immune cell activation state. RNA-seq of sort-purified PBMCs after co-culture with FLS revealed a pro-inflammatory phenotype induced in monocytes by IFNγ pre-treatment of FLS. We integrated in vitro generated monocyte signatures of the IFNγ condition into synovial biopsy-derived inflammatory monocyte transcriptomic data which revealed a large overlap of activated genes. Conclusion: This research highlights the important role of FLS in orchestrating synovial inflammation via their interaction with immune cells and further underlines the importance of the synovial cytokine milieu in shaping immune cell development in RA. It further shows the potential of our 2D co-culture model system as a clinical biomarker. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: Mirjam Dellinger: None declared, Anela Tosevska: None declared, Felix Kartnig: None declared, Teresa Preglej: None declared, Leonhard Heinz: None declared, Marie Brinkmann: None declared, Bianca Luckerbauer: None declared, Günter Steiner: None declared, Daniel Aletaha AbbVie, Amgen, Lilly, Merck, Novartis, Pfizer, Roche and Sandoz, AbbVie, Amgen, Lilly, Merck, Novartis, Pfizer, Roche and Sandoz, Myles Lewis: None declared, Costantino Pitzalis: None declared, Thomas Karonitsch: None declared, Michael Bonelli Eli-Lilly, Galapagos

Background Patients suffering from systemic autoimmune rheumatic disease (SARD) display poor antibody development after two doses of mRNA vaccinations leaving these patients with only limited humoral protection against severe SARS-CoV-2 disease courses. Of key interest is the effect of conventional synthetic (csDMARD) and biological/ targeted drugs (b/tsDMARDs) disease modifying antirheumatic drugs on the time of protection. Objectives To compare antibody titer development in patients with vasculitis and connective tissue disease (CTD) with healthy controls 6 months after two mRNA vaccinations and after third immunization. To analyze factors, that affect the velocity of titer decline, well as qualitative humoral response. Methods Patients with SARD were enrolled and matched for gender and age with healthy control subjects (HC) and the humoral response after 6 months to two doses of mRNA vaccine BNT162b2 in terms of SARS-COV-2 antibody titer was assessed. In addition to binding antibody units (BAU) we also analyzed neutralizing antibodies. Patients receiving B-cell depleting therapy and those with prior SARS-CoV-2 infection (via detection of nucleocapsid antibodies) were excluded. Differences between two groups were calculated with Wilcoxon signed-rank test. Results A total of 53 patients with SARD (42 patients suffering from connective tissue disease and 11 with vasculitis respectively) and 73 HC were analysed. Interestingly only patients receiving a combination therapy of different csDMARDs/ b/tsDMARDs demonstrated diminished antibody titers 6 months after two doses of mRNA vaccine (p-value p-value<0,001), whereas patients receiving only csDMARD as monotherapy displayed comparable antibody levels to healthy controls. This effect was equalized after a third booster vaccination (p-value=0,13). Concerning disease entities, patients with vasculitis seemed to have lower BAU than HC (p-value<0,05) and patients suffering from CTD. After third vaccination both patient groups had lower antibody levels than HC (vasculitis: p-value <0,0001; CTD: p-value p-value<0,01). Lower antibody levels before third vaccination correlated with lower antibodies after third immunization. Conclusion Patients with autoimmune rheumatic diseases undergoing combination therapy may be more vulnerable to SARS-CoV-2 infection, due to reduced antibody levels 6 months following two doses of mRNA vaccine. Our data strongly recommends antibody measurements in patients receiving combination therapy and individualized earlier booster vaccination. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests Elisabeth Simader Speakers bureau: Lilly, Thomas Deimel: None declared, Felix Kartnig: None declared, Selma Tobudic: None declared, Helmuth Haslacher Grant/research support from: Glock Health, BlueSky Immunotherapies and Neutrolis, Thomas Maria Karonitsch: None declared, Daniel Mrak: None declared, Thomas Nothnagl: None declared, Thomas Perkmann: None declared, Helga Lechner-Radner: None declared, Judith Sautner: None declared, Florian Winkler: None declared, Heinz Burgmann Speakers bureau: speaker fees from Shionogi, Pfizer, MSD, Paid instructor for: advisory boards for Valneva, MSD, Gilead, Consultant of: consulting fees from MSD, Pfizer, Takeda, Gilead, Daniel Aletaha Speakers bureau: other from Abbvie, Amgen, Lilly, Merck, Novartis, Pfizer, Roche, Sandoz, Grant/research support from: grants from Abbvie, Amgen, Lilly, Novartis, Roche, SoBi, Sanofi, Stefan Winkler: None declared, Stephan Blüml Speakers bureau: personal fees from Abbvie, personal fees from Novartis, Peter Mandl Speakers bureau: reports speaker fees from AbbVie, Janssen and Novartis, Grant/research support from: research grants from AbbVie, BMS, Novartis, Janssen, MSD and UCB.Figure 1Anti-SARS-Cov-2 S antibody titers. A: Antibody titers measured 6 months after two doses of mRNA vaccination in patients with connective tissue disease, vasculitis and healthy controls. B, Antibody levels according to disease entity. AB: antibody; BAU: binding antibody unit; CTD: connective tissue disease; HC: healthy control; mono: disease modifying anti-rheumatic drug monotherapy; combination: combination therapy of disease modifying anti-rheumatic drugs; RBD: receptor binding domain;Table 1Demographic parameters and therapy of study participants.SARD (n=53)HC (n=73)Age, mean (standard deviation)53.55 (±14.04)51.27 (±14.07)Female45 (84.9%)47 (64.4%)Connective tissue disease42 (79%)Vasculitis11 (21%)csDMARD or b/tsDMARD monotherapy22 (41%)csDMARD and/or b/tsDMARD combination therapy13 (25%)No therapy18 (34%)Methotrexate8 (15%)Mycophenolate mofetil10 (19%)Hydroxychloroquine17 (32%)Azathioprine8 (15%)Belimumab3 (6%)Tocilizumab3 (6%)Glucocorticoid dose 1. vaccination, mean (standard deviation)2.8 (±10.8)Glucocorticoid dose 2. vaccination, mean (standard deviation)2.6 (±10.7)SARD: Systemic autoimmune rheumatic disease, HC: Healthy controls, csDMARD: conventional synthetic disease modifying antirheumatic drugs and b/tsDMARD: biological/ targeted drugs disease modifying antirheumatic drugs

Introduction Structural reorganisation of the synovium with expansion of fibroblast-like synoviocytes (FLS) and influx of immune cells is a hallmark of rheumatoid arthritis (RA). Activated FLS are increasingly recognised as a critical component driving synovial tissue remodelling by interacting with immune cells resulting in distinct synovial pathotypes of RA. Methods Automated high-content fluorescence microscopy of co-cultured cytokine-activated FLS and autologous peripheral CD4 + T cells from patients with RA was established to quantify cell–cell interactions. Phenotypic profiling of cytokine-treated FLS and co-cultured T cells was done by flow cytometry and RNA-Seq, which were integrated with publicly available transcriptomic data from patients with different histological synovial pathotypes. Computational prediction and knock-down experiments were performed in FLS to identify adhesion molecules for cell–cell interaction. Results Cytokine stimulation, especially with TNF-α, led to enhanced FLS-T cell interaction resulting in cell-cell contact-dependent activation, proliferation and differentiation of T cells. Signatures of cytokine-activated FLS were significantly enriched in RA synovial tissues defined as lymphoid-rich or leucocyte-rich pathotypes, with the most prominent effects for TNF-α. FLS cytokine signatures correlated with the number of infiltrating CD4 + T cells in synovial tissue of patients with RA. Ligand-receptor pair interaction analysis identified ICAM1 on FLS as an important mediator in TNF-mediated FLS-T cell interaction. Both, ICAM1 and its receptors were overexpressed in TNF-treated FLS and co-cultured T cells. Knock-down of ICAM1 in FLS resulted in reduced TNF-mediated FLS-T cell interaction. Conclusion Our study highlights the role of cytokine-activated FLS in orchestrating inflammation-associated synovial pathotypes providing novel insights into disease mechanisms of RA.

OBJECTIVES:TNF-induced activation of fibroblast-like synoviocytes (FLS) is a critical determinant for synovial inflammation and joint destruction in RA. The detrimental role of TNF-receptor 1 (TNFR1) has thoroughly been characterized. The contributions of TNFR2, however, are largely unknown. This study was performed to delineate the role of TNFR2 in human FLS activation. METHODS:TNFR2 expression in synovial tissue samples was determined by immunohistochemistry. Expression of TNFR2 was silenced using RNAi or CRISPR/Cas9 technologies. Global transcriptional changes were determined by RNA-seq. QPCR, ELISA and immunoblotting were used to validate RNA-seq results and to uncover pathways operating downstream of TNFR2 in FLS. RESULTS:TNFR2 expression was increased in RA when compared with OA synovial tissues. In particular, RA-FLS demonstrated higher levels of TNFR2 when compared with OA-FLS. TNFR2 expression in RA-FLS correlated with RA disease activity, synovial T- and B-cell infiltration. TNF and IL1β were identified as inflammatory mediators that upregulate TNFR2 in RA-FLS. Silencing of TNFR2 in RA-FLS markedly diminished the TNF-induced expression of inflammatory cytokines and chemokines, including CXCR3-binding chemokines and the B-cell activating factor TNFSF13B. Immunobiochemical analyses revealed that TNFR2-mediated expression of inflammatory mediators critically depends on STAT1. CONCLUSION:Our results define a critical role for TNFR2 in FLS-driven inflammation and unfold its participation in the unresolved course of synovial inflammation in RA.

BackgroundIdiopathic inflammatory myopathies (IIM) comprise a heterogenous group of autoimmune diseases characterised by inflammation of muscle and affection of other organs, such as lung or skin. Some cases of IIM non-responsive to conventional treatment with glucocorticoids and DMARDs require treatment escalation. There are only limited data on efficacy and safety of immune-apheresis (IA) in IIM patients.ObjectivesThis retrospective cohort study aims to determine whether IA is effective in treating therapy-refractory IIM.MethodsPatients with active IIM undergoing IA (either plasma-exchange or immunoadsorption) at the Medical University Vienna were included in this explorative study. Patient characteristics and clinical data including serum levels of creatine kinase and concomitant medication were extracted from electronic medical records. As a primary endpoint, efficacy of IA was evaluated four weeks after initiation of IA, calculating absolute and relative change of CK-values as well as changes in steroid dose. Secondary endpoints included absolute and relative changes of CK-values at week 8 and week 12.ResultsFrom 2000 to 2021 25 IIM patients treated with IA were identified, 24 could be used for further analyses. Patient characteristics at start of IA are displayed in Table 1. Subtypes of IIM included dermatomyositis (DM 54.2%), polymyositis (PM 8.3%), Overlap-Myositis (20.8%), mmune-mediated-necrotising-myositis (IMNM 8.3 %), and antisynthetase syndrome (ASS 8.3 %). The majority of patients received concomitant steroid therapy (87.5 %) and DMARD therapy (62.5 %).Table 1.Cohort descriptives:Population, n (n= female%)24 (n=19;79.2 %)Age at Baseline (years; mean [SD];)42.5 [11.5]Subtyp Myositis (%,n)-Dermatomyositis54.2 % (13)-Polymyositis8.3 % (2)-Overlap-Myositis20.8 % (5)-Immune mediated necrotising myositis8.3 % (2)-Anti-Synthetase-Syndrom8.3 % (2)Immune Apharesis within 4 weeks (number of cycles)mean: 9.54,median: 9.5,[IQR: 8; 11.5]Previous DMARDs (%,n)-Any DMARDs-Mean number of previous DMARDs amongst all 24 patients62.5% (15)-Azathioprin33.3% (8)-Methotrexat45.8% (11)-Rituximab16.7% (4)-Mycophenolat mofetil16.7% (4)-Cyclophosphamid8.3% (2)-Ciclosporin8.3% (2)-(Hydroxy)-Chloroquin20.8% (5)-Etanercept4.2% (1)-Infliximab4.2% (1)Concomitant DMARDs (%,n)-Any DMARDs62.5% (15)-Azathioprin20.8% (5)-Methotrexat20.8% (5)-Rituximab4.2% (1)-Cyclophosphamid4.2% (1)-Ciclosporin4.2% (1)-(Hydroxy)-Chloroquin8.3% (2)Concomitant Steroids % (n)87.5% (21)-Dose prednisone at Baseline (mg/day; median [IQR])25 [12.5; 50] (21)-Dose prednisone at week 4 (mg/day; median [IQR]12.5 [0; 37.5] (22)CK-level at baseline (U/ml; median [IQR])970.5 [157.5; 3795.5]Change in CK-values from baselineAbsolute changeRelative change-to week 4 (n=24)median [IQR] U/ml% [IQR]-to week 8 (n=16)304.5 [28.8;2051]49.1 [22.4; 79.3] %-to week 12 (n=15)648.5 [25.8; 3939.3]70.0 [13.6; 87.7] %559 [-7; 3988]63.8 [-5.8; 98.1] %Until week 4 significant decrease in CK-values was observed in 21/24 patients (p<0.01; Figure 1), from median 970.5 [157.5; 3795.5] to 347[63; 1010] U/ml. Median [IQR] dose reduction of steroids was 12.5 [0; 12.5] mg/day absolute and 25% [0%, 100%] relative. No differences were observed within patients of different myositis subtypes. One patient died after 4 weeks, in 15 patients IA was maintained until week 12. Significant reduction in CK-values was observed from baseline to week 8 and 12 respectively (141 [78; 460], 111 [57;338]. Median and relative changes from baseline until week 12 are displayed in Table 1.Figure 1.Individual response in CK-levels from baseline to week 12ConclusionImmune-apheresis seems an effective therapeutic option in refractory IIM, leading to decrease of CK-values and steroid dose.References[1]Lundberg IE. Expert Perspective: Management of Refractory Inflammatory Myopathy. Arthritis Rheumatol. 2021 Aug;73(8):1394-1407.Disclosure of InterestsNone declared