Mice were euthanized by cervical dislocation, and spleen, inguinal lymph nodes, liver, and lungs were harvested. Lungs, inguinal lymph nodes, and liver were finely chopped and digested with 1.5 mg/ml collagenase D (Sigma-Aldrich), 0.1 mg/ml DNase I (ITW Reagents), 0.6 mg/ml NADase (Sigma-Aldrich), and 5% FBS (Thermo Fisher Scientific) in PBS for 60 min at 37°C with gentle shaking. Cells were then passed through a cell strainer (40–70 μm) and washed with sterile PBS. For flow cytometric analysis, cell suspensions were depleted from red blood cells using the hypotonic buffer Tris-based amino-chloride (5 min, room temperature) prior to staining with the antibodies depicted below.

Flow cytometry staining was performed in FACS buffer (1% FBS, 1% BSA, 0.02% sodium azide) using the following antibodies from Biolegend unless specified otherwise: Anti-mouse antibodies: B220 (RA3-6B2), CD11b (M1/70), CD11c (N418), CD27 (LG.3A10), CD28 (37.51), CD69 (H1.2F3), CD25 (PC61), CD44 (IM7), CD137 (17B5), ICOS (7E.17G9; Invitrogen), PD-1 (29F.1A12), TCRβ (H57-597), TCR γ/δ (clone GL3), IL-17A (TC11-18H10.1), IFNγ (XMG1.2), Granzyme B (QA16A02), and pSTAT1(Ser727; A15158B). Anti-human antibodies were as follows: CD14 (HCD14), CD19 (HIB19), CD3ε (HIT3a), CD8α (HIT8a), CD161 (HP-3G10), Vα7.2 (3C10), CD69 (FN50), and Granzyme B (QA16A02). Dead cells were excluded from the analyses using Zombie fixable viability dye (Biolegend). For intracellular staining, cells were fixed and permeabilized using BD Cytofix/Cytoperm Solution kit (BD Biosciences) according to the manufacturer’s instructions. MR1(5-OP-RU), MR1(6-formyl pterin, Ac-6-FP), and CD1d(PBS-57)-loaded tetramers were provided by the National Institutes of Health (NIH) Tetramer Facility. The MR1 tetramer technology was developed jointly by James McCluskey, Jamie Rossjohn, and David Fairlie, and the material was produced by the NIH Tetramer Core Facility as permitted to be distributed by the University of Melbourne. Data were recorded using LSR-II or LSR Fortessa cytometers (BD Biosciences) and analyzed with FlowJo software (TreeStar).

For cell sorting of pulmonary murine MAIT cells, T cells were previously enriched in lung single-cell suspensions using αCD3ε microbeads (Miltenyi Biotec). Murine MAIT cells were sorted as Zombie−B220−CD11b−CD11c−TCRβ+MR1(5-OP-RU) tetramer+ cells.

Human MAIT cells were sorted from PBMCs as Zombie−CD14−CD19−CD3ε+CD8α+CD161+Vα7.2+ cells.