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

Abstract

BackgroundRheumatoid arthritis (RA) is characterised by relapsing joint and systemic inflammation, yet the immunopathological basis of these disease flares and their clinical prediction remain uncertain.ObjectivesUsing 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).MethodsWe 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.ResultsMass 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)ContrastClusterMedian %Adj. p (GLMM)Flare onset vs baseline: Flare patientsCD4+/CD45RO+/PD1+ memory T cells2.14 vs 0.24<0.001CD8+/CD45RO+/PD1+ memory T cells6.64 vs 0.07<0.001CD19+/CD27+/CD21- memory B cells2.39 vs 0.03<0.001Single cell RNAseq (n = 8 flare + 4 DFR)ContrastClusterMedian %Adj. p (Wilcoxon)Flare onset vs baseline: Flare patientsIgA+ plasma cells0.37 vs 0.210.020Flare vs DFR patients: BaselineCD4+/CD25+/Foxp3+ Treg cells0.55 vs 1.270.022To 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.ConclusionWe 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.