Flow cytometry, sorting, and intracellular transcription factor staining

Mouse and human cells were stained with viability dye 7-aminoactinomycin D (Sigma-Aldrich) and cell surface antibodies concurrently with MR1-tetramers, at room temperature (RT) for 30 min. A list of antibodies used is detailed in table S4. Transcription factors were assessed by staining with antibodies after the cells were surface-stained and permeabilized with the eBioscience Foxp3 Fixation/Permeabilization kit, according to the manufacturer’s instructions. Apart from intracellular analysis, mouse cells were analyzed unfixed after staining, whereas human cells were fixed with 1% paraformaldehyde. Analysis was carried out using a BD LSRFortessa equipped with a 561-nm yellow-green laser, and data were processed using FlowJo software (TreeStar). Mouse cells are gated on B220⁻ lymphocytes and human cells on CD14⁻CD19⁻ lymphocytes after dead cell and doublet exclusion. Flow cytometric sorting for live, unfixed bulk (100 cells) or single cells was performed using a BD FACSAria III cell sorter. Flow cytometry gating strategy is shown in fig. S10.