RF catabolites selectively inhibit ex vivo human MAIT cell activity. (A) Bar graph showing the proportion of CTV^low MR1-5-OP-RU tetramer⁺ MAIT cells as a proportion of total MAIT cells in PBMC after 7-day culture in the presence of 5-OP-RU (10 µM), RF (100 µM), lumiflavin (100 µM), lumichrome (100 µM), or alloxazine (100 µM) on day 7. (B–D) Bar graphs showing (B) MFI CD69, (C) proportion of CD69⁺, or (D) proportion of TNF⁺ MAIT cells from PBMCs coincubated with titrated doses of RF or RF catabolites in the presence of 5-OP-RU. (E and F) Bar graphs showing (E) proportion of CD69⁺ or (F) proportion of TNF⁺ MAIT cells from PBMCs coincubated with titrated doses of RF or RF catabolites in the presence of 5-A-RU. (G–J) Bar graphs showing (G) the proportion of CD69⁺ MAIT cells, (H) the proportion of CD69⁺ TRAV1-2^−ve MR1-5-OP-RU tetramer^−ve cells, (I) the proportion of TNF⁺ MAIT cells, or (J) the proportion of TNF⁺ TRAV1-2^−ve MR1-5-OP-RU tetramer^−ve cells from PBMCs preincubated with titrated doses of RF or RF catabolites followed by stimulation with PMA and ionomycin. (K and L) Bar graphs showing the proportion of CD69⁺ (K) MAIT cells or (L) TRAV1-2^−ve MR1-5-OP-RU tetramer^−ve cells, from PBMCs preincubated with titrated doses of RF or RF catabolites followed by stimulation with αCD3/αCD28-coated beads. Data from all graphs represent the average of three independent experiments performed in duplicate on PBMCs from three different donors. The error bars represent the standard error of the mean (SEM). One-way ANOVA statistical analysis was performed for all samples with Dunnett’s multiple comparisons performed using vehicle, 5-OP-RU, or 5-A-RU as controls (ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).