Foxp3 fixation permeabilisation concentrate and diluent

PBMCs were rapidly thawed in medium heated to 37°C and kept overnight at 4°C. Cells were then immunostained during 30 min at 4°C with the appropriate monoclonal antibodies detailed in Supplementary file 1. Intracellular staining of transcription factors and cytotoxic molecules was performed with Foxp3 Fixation/Permeabilisation concentrate and diluent (eBioscience). Intracellular staining of cytokines and chemokines was performed with Cytofix/Cytoperm (BD Biosciences). Intracellular staining of phosphorylated proteins was performed with Lyse/Fix and Perm III buffers (BD Biosciences). Phosphorylated proteins were then stained during 40 min at RT. Kynurenine uptake was measured as previously described (Sinclair et al., 2018 (link)). Briefly, PBMCs were stained for surface markers for NK cell identification and resuspended in phosphate-buffered saline (PBS). Baseline fluorescence in the BV421 channel was recorded for 30 s, and kynurenine (200 µM final concentration) was added before a further 4 min acquisition. The assay was run at 37°C. Flow cytometric analysis was performed on LSR Fortessa 5L (Becton-Dickinson). Fluorescence Minus One controls were used to set the gates, and data were analysed with FlowJo 10.5.0 software (Tree Star). Gating strategy is presented is Figure 1—figure supplement 2.

PBMCs from HD were stimulated for 1 hr with ionomycin at the indicated concentration. They were then stained with a fixable viability dye (ThermoFisher Scientific) and stained for surface markers allowing NK cell identification (CD3, CD19, CD14, and CD56). The samples were then fixed and permeabilised using the Foxp3 Fixation/Permeabilisation concentrate and diluent (eBioscience) and stained with an anti-NFAT1 (Cell Signaling Technology). Sample acquisition was made on an ImageStream X Mark II (Amnis-EMD Millipore, Darmstadt, Germany) with ×40 magnification and analyzed with IDEAS software (v6.0).