Multiparameter Flow Cytometry Analysis

For immunofluorescence staining, morphology of cells was determined via FSC-A and SSC-A, doublets excluded via FSC-A and FSC-H and dead cells excluded with Fixable-Yellow live/dead stain (Invitrogen, Carlsbad, CA, USA). Human and mouse MR1 tetramers were produced^{13 (link), 53 (link)}. Biotinylated MR1-5-OP-RU and control MR1-Ac-6FP monomers were tetramerized with streptavidin conjugated to PE (BD Pharmigen; San Jose, CA, USA). For intracellular detection of IFN-γ and TNF, cells were cultured for 4 h with 5 ng/mL PMA and 1 µg/mL Ionomycin in the presence of Golgi Plug and Golgi Stop (BD Pharmingen; San Jose, CA, USA). Data were collected using a BD FACSymphony and analyzed using FacsDiva v8 and FlowJo v10.2 (Treestar, Ashland, OR). A list of antibodies used for flow cytometry is provided in Supplementary Table 3and gating strategies are shown in Supplementary Fig. 9.