Fig. 2.

Legionella activates tetramer⁺ mouse MAIT cells in vitro via MR1. a Splenocytes (2 × 10⁵) from Vα19iCα^−/−MR1⁺.Ly5.1 mice were prepared and co-cultured with iBMDM.MR1 cells (10⁵) overnight with or without lysate (20 μl or 10 μl) of L. longbeachae. 5-OP-RU was used as a positive control at a final concentration of 1 nM. Anti-mouse MR1 monclonal antibody 8F2 or its IgG1 isotype control (in-house) were added at 20 μg ml⁻¹ to examine MR1-specific Ag presentation. CD69 upregulation (MFI) on MAIT cells was measured by flow cytometry for MAIT cell activation after staining together with other T-cell antibodies and MR1-5-OP-RU tetramers. Two way ANOVA with ****P < 0.0001. b Immunofluorescence micrographs of murine lungs showing TCRβ⁺, MR1-5-OP-RU-tetramer⁺ MAIT cells (white boxes) in C57BL/6 mice infected intranasally with 2 × 10⁴ CFU L. longbeachae 3 days prior. Images from uninfected or MR1^−/− mice or stained with control tetramer MR1-6-FP are presented in Supplementary Fig. 3. Red, MR1-5-OP-RU tetramer; yellow, TCRβ; cyan, L. longbeachae; blue, nuclei (Hoechst)