Expansion and Characterization of Lung MAIT Cells

Lung MAITs were expanded ex vivo using a previously published method for the expansion of human MAIT cells (Liu et al., 2020 (link)). Briefly, lung homogenates from healthy WT mice were cultured for 14 days in the presence of mouse recombinant IL-2 and sulfate latex beads of 5-OP-RU/MR1 artificial antigen-presenting cells. To isolate monocytes, lung cells from healthy WT mice were surface stained with anti-CD45 AF700 (BioLegend), anti-CD11b-FITC (BioLegend), anti-Siglec-F- BV711 (BD), and anti- Ly6C (PE dazzle 594) for 30 min at 4°C. We obtained CD45⁺, Siglec-F^-, Ly6C⁺, CD11b⁺ monocytes by flow sorting on a FACS Aria II (BD, Franklin Lakes, NJ). We incubated 20,000 MAITs with 10,000 monocytes for 18 hr, in a total volume of 200 µL in 96-well U-bottom plates. MAIT-monocyte co-culture was stimulated with 200 nM 5-A-RU and 50 µM MeG (Li et al., 2018 (link)) in RPMI 1640 with 10% FBS with 1% penicillin/streptomycin and 1% HEPES. MAIT cells that were stimulated/unstimulated and monocytes that were stimulated/unstimulated were kept as controls. After 18 hr, the cell supernatant was collected and stored at −20°C. We measured IFN-γ and GM-CSF levels in supernatants using ELISA as per manufacturer’s instruction in BioLegend ELISA MAX standard sets for mouse IFN-γ and mouse GM-CSF.