Pos0138 rheumatoid synovial fibroblasts display imprinted memory of their synovial endotype which can be plastically modulated by b-cells crosstalk

BackgroundDespite advances in the treatment of Rheumatoid Arthritis (RA), synthetics and biologicals drugs are ineffective in ~40% of patients. The origin of this refractoriness is unclear, but several clues point at the synovial microenvironment (SE) and the relative cellular heterogeneity between patients. We previously described the existence of different RA endotypes such as the lympho-myeloid, LM, which is B-cell rich and the fibroid-paucimmune, FPI, which is devoid of B-cells. While there is clear evidence that the crosstalk between stromal and immune cells in rheumatoid joints is critical for the perpetuation of chronic inflammation and autoimmunity, it is currently unknown whether transcriptional signatures identified in synovial fibroblasts (SFs) derived from different RA endotypes are driven by “imprinted” properties of the SFs or are shaped by the interaction with infiltrating immune cells in the RA joints.ObjectivesI) to identify “imprinted” vs “inducible” RASFs signatures trough the comparison of freshly isolated SFs and primary established SFs cultures obtained from LM vs FPI RA synovial biopsies and ii) to investigate the identified RASF signature as predictive biomarkers of disease evolution and of response to conventional and biological DMARDs.MethodsWe performed flowcytometry and single cell RNA sequencing (sc-RNAseq) on SFs obtained from LM and FPI biopsies, in isolation or in co-culture with RA B cells. Next, supernatant has been screened trough Multiplex and ELISA. Furthermore, we compared our results to publicly available sc-RNAseq datasets on freshly isolated SFs and to our bulk-RNAseq data from clinical trials patients.ResultsHierarchical clustering from sc-RNAseq transcriptional profiling of LM vs FPI RASF - after several cell passages - identified profoundly different gene signatures: whereby LM-RASF were characterised by genes involved in inflammation, proteoglycan formation and integrin binding, FPI-RASF were defined by genes related to collagen biosynthesis. Comparing the above signatures with those of freshly isolated RASF we identified both imprinted (i.e. maintained through several in vitro passages) and inducible (i.e. loss after long term culture) gene signatures. Notably, RA B-cells co-cultured with FPI-RASF profoundly altered the FPI-RASF transcriptional profile including the ex-novo expression of gene signatures typical of LM-RASF. Consensus gene modules constructed on LM vs FPI RASF imprinted gene signatures could be tracked in longitudinal whole tissue bulk RNA-seq data obtained from both early arthritis and established RA and were associated with synovial pathotype-specific histological and clinical features. Finally, modulation of FPI-RASF related genes following B-cell depletion identified poor responders to Rituximab in the R4RA randomised clinical trial.ConclusionOur work demonstrates that RASFs from different endotypes display imprinted memory of their original synovial tissue when maintained in culture over several months. We also demonstrated that imprinted memory typical of RASF isolated from B-cell rich LM synovial tissues can be dynamically modulated in FPI RASF following crosstalk with RA B cells. Finally, consensus gene modules based on FPI vs LM RASF-gene signatures were able inform on response/resistance to targeted biologic therapies.References[1]Lewis, M. J. et al. Molecular Portraits of Early Rheumatoid Arthritis Identify Clinical and Treatment Response Phenotypes. Cell Rep (2019)[2]Humby, F. et al. Synovial cellular and molecular signatures stratify clinical response to csDMARD therapy and predict radiographic progression in early rheumatoid arthritis patients. Ann Rheum Dis (2019)[3]Zhang, F. et al. Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry. Nat Immunol (2019)[4]Humby, F. et al. Rituximab versus tocilizumab in anti-TNF inadequate responder patients with rheumatoid arthritis (R4RA). Lancet (2021)Disclosure of InterestsNone declared