OP0032 SINGLE CELL ANALYSIS OF SPONDYLOARTHRITIS REGULATORY T CELLS IDENTIFIES DISTINCT SYNOVIAL GENE EXPRESSION PATTERNS AND CLONAL FATES
Authors: D. Simone, F. Penkava, A. Ridley, S. Sansom, H. Al Mossawi, P. Bowness
Journal: Annals of the Rheumatic Diseases (2021)

Background: Regulatory T cells (Tregs) play an important role in controlling inflammation and limiting autoimmunity, but their phenotypes at inflammatory sites in human disease are poorly understood. Whilst the phenotype and transcriptional profile of Tregs have been studied in some immune mediated conditions, they have been little studied (especially at the single cell level) in synovial fluid in the course of inflammatory arthritis. In Spondyloarthritis (SpA), in particular, where pathogenesis and inflammation is driven by dysregulated effector immunity, the role of the regulatory arm of immunity is largely unknown. Objectives: We aimed to draw an atlas of Tregs in the context of SpA joint inflammation using single cell RNA sequencing of blood and SF Tregs of patients with Ankylosing Spondylitis (AS) and Psoriatic Arthritis (PsA). Functionally distinct specialised Treg subtypes, and specific changes in transcriptional profile occurring in synovial fluid Tregs, providing an insight on Treg adaptation during inflammation. Furthermore, by coupling gene expression analysis with TCR sequencing, we aimed to describe clonally expanded and likely antigen-driven Tregs in the SF. Methods: Fluorescent activated cell sorting (FACS) was used to isolate 13,400 memory CD3+ CD45RA-ve CD25 + 127low Tregs from the blood and synovial fluid (SF) of 2 patients with HLA-B27+ AS presenting with active knee arthritis. Single-cell RNA sequencing (scRNA-seq) using 5’ V(D)J 10x Genomics technology allowed both transcriptional definition of Tregs, and exploration of their immune TCR repertoire. Findings were compared to &gt;3,000 SF and blood Tregs from 3 patients with olygoarticular PsA 1 . Multicolor flow cytometry and in vitro cell-based assays using patient-derived cells were used to confirm and expand, at protein and functional level, the findings that emerged from the gene expression analysis. Results: We report a large scRNAseq dataset (approx. 17,000 cells) comparing Tregs from SpA blood and joints. We identify multiple Treg clusters with distinct transcriptomic profiles, including, among others, a regulatory CD8+ subset expressing cytotoxic markers/genes, and a Th17-like RORC + Treg subset characterized by IL-10 and LAG-3 expression. Synovial Tregs show upregulation of interferon signature and TNF receptor superfamily genes, and marked clonal expansion, consistent with tissue adaptation and antigen contact respectively. Individual synovial Treg clones map to different clusters indicating cell fate divergence. Finally, we demonstrate that LAG-3 directly inhibits IL-12/23 and TNF secretion by patient-derived monocytes, a mechanism with translational potential in SpA. Conclusion: Our detailed characterization of Tregs at an important inflammatory site illustrates the marked specialization of Treg subpopulations and identifies a broad transcriptional profile upregulated across all synovial regulatory cells. Our TCR analysis provides evidence of Treg clonal expansion, which may be driven by antigen, and confirms functional specialisation of individual clones. We also propose a new insight into a Treg functional mechanism through LAG-3 that suggests a novel therapeutic approach to immune-driven diseases. References: [1]Penkava et al., Nature Communications, 2020 Disclosure of Interests: Davide Simone: None declared, Frank Penkava: None declared, Anna Ridley: None declared, Stephen Sansom: None declared, Hussein Al Mossawi Employee of: UCB, Paul Bowness Grant/research support from: Regeneron, Celgene/BMS and GSK