Rankl pe

PB mononuclear cells (PBMCs) and SF mononuclear cells (SFMCs) were isolated by density gradient centrifugation. To detect the proportion of CD19⁺CD24^hiCD27⁺ B cells and the expression of RANKL in CD19⁺CD24^hiCD27⁺ B cells of PBMCs or SFMCs, the cells were isolated by density gradient centrifugation and then were stained with mouse monoclonal antibodies as follow: CD19-APC/Cy7 (BioLegend, San Diego, CA, USA), CD24-FITC (eBioscience, San Diego, CA, USA), CD27-APC (eBioscience), RANKL-PE (BioLegend). FMO controls were included. Data was acquired on a FACS Arial II flow cytometer (Becton Dickinson, NJ, USA) and analysed using FlowJo software.
To isolate CD19⁺CD24^hiCD27⁺ B cells, PBMCs were stained with mouse anti-CD19-APC/Cy7, CD24-PE (eBioscience), CD27-APC, then the aimed cell populations were sorted by flow cytometry. Sorted CD19⁺CD24^hiCD27⁺ B cells had a purity of > 95%. These sorted cells were subsequently subjected to reverse transcription-polymerase chain reaction (RT-PCR) or osteoclast differentiation assay.

Fibroblast-like synoviocytes (FLS) were generated from cRA SFMCs (33 (link)). FLS were evaluated at passages 3–4. Cultures were stimulated with interferon gamma (IFNγ) 10 ng/ml or TNFα 10 ng/ml (both from Peprotech) and subsequently with rhPD-1 (1 μg/ml) or left untreated as a control (34 (link)). Flow cytometry staining was performed after 48 h of stimulation, using anti-human antibodies, staining for CD90 FITC (BD, JJ, USA), PD-L1 PeCy7 (BD, JJ, USA), L/D nIR (Thermo Fisher, MA, USA), and RANKL PE (Biolegend). Gating was done on live cells, single cells, and CD90⁺ cells (Supplementary Figure S1). The flow cytometry was performed on the LSR Fortessa (BD), and data analysis in FlowJo. MCP-1 was measured by ELISA in the supernatant (Thermo Fisher, MA, USA).