MAIT cell activation

APCs were seeded at 8 × 10⁴/well in U‐bottomed (or flat‐bottomed for monocyte‐derived macrophages) 96‐well plates; viable cells were counted using Trypan Blue (Invitrogen) and a Neubauer counting chamber. APCs were incubated overnight for 20 h. Fixed bacteria (25 bacteria per cell unless otherwise stated), bacterial cell lysate, or bacterial culture supernatant (both 10 μL unless otherwise stated) were added at the start of culture or, in some experiments, for the final 7 h of culture. In some experiments pharmacological inhibitors, TLR agonists, interferon‐α or IFN‐γ, were added at the start of culture as indicated. CD8⁺ T cells were added at 1–2 × 10⁵/well either at the start of culture (total 20 h co‐culture) or for the final 5 h (total 5 h co‐culture); blocking antibodies were added at the same time as the CD8⁺ T cells. When CD8⁺ T cells were added for the final 5 h of culture, APCs were washed three times in R10 before the addition of the CD8⁺ T cells. Brefeldin A (3 μg/mL) was added for the final 4 h of culture. Cells were analyzed for IFN‐γ production by intracellular cytokine staining.