A 10-COLOR FLOW CYTOMETRIC PANEL FOR IDENTIFYING SYNOVIAL FIBROBLAST SUBSETS IN RHEUMATOID ARTHRITIS

Background: Synovial fibroblasts are key players in the disease pathology of rheumatoid arthritis and specific fibroblast subsets have been linked to joint inflammation and joint destruction[1]. These synovial fibroblasts can be cultured from both the easily obtained synovial fluid or the more time-consuming synovial biopsy or resection. Recently, cultured synovial fibroblasts from synovial fluid where confirmed to possess a similar phenotype to that of the synovial fibroblast podoplanin+/CD90+/human leukocyt antigen(HLA)-DR+ subset from biopsies[2]. The present advancement in our knowledge of synovial fibroblasts have been spearheaded through bulk and single cell RNA transcriptomics. Expensive techniques that may require supplementary identification of phenotype clusters and cell function on protein level. Objectives: The objective of this work was to plan, optimise and test a multi-color flow cytometry panel for identification of known synovial fibroblast subsets linked to arthritis pathology. The primary focus was well established markers; podoplanin, CD90, CD55, HLA-DR, CD34 and fibroblast activation protein alpha (FAPa). Methods: Several cell types were used in optimizing the panel. The majority of antibody titration was conducted on immortalized synovial fibroblasts from a patient with rheumatoid arthritis. Exclusion markers such as CD45 (blood cells) and CD31 (endothelial and monocytes/macrophages) were titrated on synovial fluid mononuclear cells and peripheral blood mononuclear cells. Finally, full panel testing on synovial fibroblasts cultured from synovial fluid was compared with immortalized synovial fibroblasts. All tests were carried out on a commercially available flow cytometer with 4-lasers (405, 488, 561 and 637nm) and 27 detectors. Results: Several panel iterations were tested during the optimization process. Two markers, cadherin-11 and receptor activator of nuclear factor kappa-b ligand, were abandoned after testing different antibody clones for staining and several detaching solutions on fibroblasts prior to staining. Initial compensation issues were overcome by rearranging fluorochromes and using cells as CD90 compensation controls. The final flow cytometry panel contained 10 markers; podoplanin, CD90, CD55, CD34, HLA-DR, FAPa, CD45, CD31, complement C3a receptor and a viability marker. Final testing on synovial fibroblasts cultured from synovial fluid yielded an acceptable viability > 90%. Within the synovial fibroblasts cultured from synovial fluid, the panel was able to identify podoplanin+/CD90+/FAPa+ synovial fibroblasts previously linked with joint inflammation. The panel was, furthermore, able to identify a podoplanin+/CD90+/HLA-DR+ population in the immortalized synovial fibroblasts. Conclusion: The optimized flow cytometry panel presented here enables researchers to identify synovial fibroblasts subsets which have been linked with disease traits in rheumatoid arthritis. This panel could therefore both be useful as a supplement to transcriptomic data and as a primary interrogation tool when investigating fibroblast subsets and cell function on a protein level. REFERENCES: [1]Croft, A.P., et al., Distinct fibroblast subsets drive inflammation and damage in arthritis. Nature, 2019. 570 (7760): p. 246-251. [2]Køster, D., et al., Phenotypic and functional characterization of synovial fluid-derived fibroblast-like synoviocytes in rheumatoid arthritis. Sci Rep, 2021. 11 (1): p. 22168. Acknowledgements: We would like to acknowledge the FACS Core Facility at Aarhus University, Denmark, and thank them for sparring throughout the optimization process. Disclosure of Interests: Søren Lomholt: None declared, Tue Wenzel Kragstrup Shareholder of: iBio tech ApS., Speakers bureau: Speaking fees from Pfizer, Bristol-Myers Squibb, Eli Lilly, Novartis, UCB, and Abbvie., Consultant of: Consultancy fees from Bristol-Myers Squibb and Gilead., Grant/research support from: Gilead. Citation: , volume 81, supplement 1, year 2022, page 465Session: Rheumatoid arthritis - aetiology, pathogenesis and animal models (POSTERS only)