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