Pos1012 single cell rna sequencing reveals distinct b cell characteristics in anti-ccp positive at-risk individuals with imminent rheumatoid arthritis

Background Anti-cyclic citrullinated peptide (CCP) antibody positive individuals with musculoskeletal (MSK) symptoms but no clinical synovitis are at elevated risk of developing rheumatoid arthritis (RA) [1]. Whilst risk stratification is possible, specific immunological changes underpinning the transition from pre-RA to clinical arthritis remain unclear. Objectives We aimed to define markers of rapid progression across immune-cell subsets to enable early differentiation of those who will progress to arthritis in ‘high-risk’ anti-CCP positive individuals. Methods Peripheral blood mononuclear cells (PBMCs) were collected from anti-CCP positive individuals with MSK symptoms but no clinical synovitis (CCP+ at-risk). We performed single cell RNA sequencing (scRNAseq) of PBMCs from 10 CCP+ at-risk individuals (6 CCP+ progressors, 4 CCP+ non-progressors) collected at baseline and follow-up time points (i.e. progression to arthritis in the CCP+ progressors). All subjects had high positive anti-CCP levels (>3x ULN) and were HLA-DR shared epitope positive with median time to progression in progressors being 4 months. Analysis was performed in R using SingleR for subset annotation and Seurat for differential gene expression analysis between progressors and non-progressors and across time points. Results Examining all subsets, we noted a generalised increased Type 1 interferon response in non-progressors, with B cells (n=8,659) showing the most prominent divergence between groups. Within B cells we observed 334 differentially expressed genes (DEG) in CCP+ progressors versus non-progressors at baseline and 143 DEG at follow up (padj<0.05), with 77 DEG consistent across timepoints. At baseline, CCP+ progressors demonstrated upregulation of genes associated with B cell antigen-presentation, including Class II MHC molecules, CD83 and CD74. Conversely, interferon stimulated genes (ISG) were enriched in the CCP+ non-progressor DEG. Notably, this ISG signature ceased at follow-up (median 14 months). Examining the DEG per B cell subset, we noted that upregulation of genes associated with B cell antigen-presentation is found across naïve, unswitched memory and switched memory B cells in CCP+ progressors at baseline. In contrast, ISG upregulation is specific to a separate subset of interferon-responsive B cells. At baseline, these cells form a significantly greater proportion of all B cells in the CCP+ non-progressor group versus the progressor group (17.8% versus 2.6%, p<10-5). At follow-up, the proportions are comparable between the two groups (2.5% in non-progressor versus 3.4% in progressor, p=0.08), reflecting the transient nature of ISG upregulation. Conclusion CCP+ at risk individuals with imminent arthritis demonstrate a distinct B cell gene expression profile. Imminent progression is associated with markers of B cell mediated antigen presentation across multiple subsets whereas non-progressors demonstrate transient over-expression of ISGs driven by expansion of a subset of interferon-responsive B cells. These data reveal mechanistic insights into the early divergence between these groups and the development of RA. Reference [1] van Venrooij, W., van Beers, J. & Pruijn, G. Anti-CCP antibodies: the past, the present and the future. Nat Rev Rheumatol 7, 391–398 (2011). Acknowledgements BF and KM are joint senior authors. Disclosure of Interests Weiyu Ye: None declared, Orion Tong: None declared, Diane Corscadden: None declared, Katie Mbara: None declared, Paul Emery Consultant of: PE has provided expert advice to Abbvie, Astra-Zeneca, BMS, Boehringer Ingelheim, Galapagos, Gilead, Janssen, MSD, Lilly, Novartis, Pfizer, Roche, and Samsung., Grant/research support from: PE has been involved in clinical trials for Abbvie, BMS, Lilly, Novartis, Pfizer, Roche and Samsung., Benjamin Fairfax: None declared, Kulveer Mankia Consultant of: KM has provided expert advice to Lilly, UCB, Galapagos and Abbvie., Grant/research support from: KM has received funding from Lilly, BMS and Gilead.