OP0074 DISTINCT CIRCULATING LYMPHOCYTE SUBSETS DISTINGUISH FLARE FROM DRUG-FREE REMISSION IN RHEUMATOID ARTHRITIS

Background Rheumatoid arthritis (RA) is characterised by relapsing joint and systemic inflammation, yet the immunopathological basis of these disease flares their clinical prediction remain uncertain. Objectives Using mass cytometry single cell RNA sequencing, we aimed to identify circulating lymphocyte subsets associated with RA flare, potential cellular biomarkers predict flare versus drug-free remission (DFR). Methods We analysed peripheral blood mononuclear cells (PBMCs) from patients recruited BioRRA study (Figure 1), a prospective trial conventional synthetic disease-modifying anti-rheumatic drug (csDMARD) cessation.[1] Patients in (DAS28-CRP < 2.4) ultrasound (absence power Doppler signal 7 joints) stopped csDMARDs, defined as DAS28-CRP ≥ 2.4 during 6 month follow-up. A 44-marker panel was used profile PBMCs 36 (20 16 DFR) at two time points each (baseline, onset / DFR). In subset (n = 12: 8 4 DFR), fluorescence-activated sorting T B followed sequencing 81,923 cells) incorporating 320 immune genes, 34 oligo-tagged surface protein antibodies, TCR/BCR CDR3 sequence. Clones were ≥2 identical nucleotide sequence, clonal expansion significant increase proportion baseline final visit. Statistical significance assessed after Benjamini-Hochberg multiple test correction (adj p 0.05). Figure 1. Results Mass revealed 31 distinct clusters: notably, greater proportions memory (CD45RO+/PD1hi) CD4+ CD8+ cells, (CD27+/CD21-) observed (Table 1). Table 20 + Contrast Cluster Median % Adj. (GLMM) Flare vs baseline: CD4+/CD45RO+/PD1+ 2.14 0.24 <0.001 CD8+/CD45RO+/PD1+ 6.64 0.07 CD19+/CD27+/CD21- 2.39 0.03 Single RNAseq (Wilcoxon) IgA+ plasma 0.37 0.21 0.020 DFR patients: Baseline CD4+/CD25+/Foxp3+ Treg 0.55 1.27 0.022 To better characterise flare-associated subsets, CD45RO+/PD1hi CD19+ performed identified 21 clusters. (Fisher exact, adj. 0.05) within five unique clones (4 patients), one clone (1 patent), no clones. Overall, there significantly baseline. contrast, lower CD25+/FoxP3+ regulatory present csDMARD cessation (baseline) subsequent suggesting biomarker potential. further assess predictive performance Tregs for DFR, an independent cohort 50 (25 25 stopping csDMARDs ongoing BIO-FLARE study.[2] By flow cytometry, confirmed CD4+/CD25hi (median 4.74 6.37%, Wilcoxon 0.037; AUC: 0.67). this cohort, only elevated (> 6.11% total CD4) would have prevented 18/25 (72%) patients; 9/25 (36%) continued unnecessarily. Conclusion detailed longitudinal characterisation phenotype, gene expression, DFR. Furthermore our data, across cohorts, suggests role promoting meriting investigation, future development. References [1]Baker et al; J Autoimmunity; 105:102298 [2]Rayner BMC Rheumatology; 5:22 Acknowledgements This work funded research grants Wellcome Trust [102595/Z/13/A KFB], Newcastle NIHR Biomedical Research Centre [BH136167/PD0045 British Society Rheumatology [KFB], Academy Medical Sciences [SGL022\1074 University Translational Partnership Hospitals Charity [8033 National Institute Health Clinical Lectureship [CL-2017-01-004 KFB]. Our supported into Inflammatory Arthritis Versus (RACE) (grant number 20298), Rheuma Tolerance Cure (European Union Innovative Medicines Initiative 2, grant 777357). AGP JDI are named inventors on patent application (“Prediction Drug-Free Remission Arthritis”; International Patent Application Number PCT/GB2019/050902). The views expressed those authors not necessarily NHS, NIHR, or Department Social Care. Disclosure Interests Kenneth F Baker Consultant of: Modern Biosciences Ltd, Grant/research support from: Pfizer, Genentech, Fiona Rayner: None declared, Henrique Lemos: David McDonald: Gillian Hulme: Rafiqul Hussain: Jonathan Coxhead Speakers bureau: Tesaro, Arthur Pratt Gilead, Amy E. Anderson: Andrew Filby Becton Dickinson, John Isaacs Abbvie, Roche, UCB, GSK, Janssen, Pfizer.