CD8–MR1 interactions enhance antigen-dependent MAIT cell responses. (A) MAIT cells identified using surrogate markers CD161 and TRAV1-2 (left plots), and analysis of coreceptor usage and cytokine production (TNF and IFNγ) of unstimulated and 5-OP-RU stimulated MAIT cells (middle and right plots). (B and C) Percentage of TNF- or IFNγ-producing MAIT cells in response to 10 nM 5-OP-RU in the absence of C1R cells, or in the presence of MR1 deficient (C1R.MR1^null) or WT MR1 expressing (C1R.MR1^null+MR1) C1R cells. Mean and SD are displayed. (D and E) Percentage of TNF- or IFNγ-producing MAIT cell coreceptor subsets in response to WT MR1 expressing C1R cells (C1R.MR1^null+MR1) pulsed with titrating doses of 5-OP-RU. Mean, SD, and nonlinear regression line (least squares) are displayed. (F and G) Percentage of TNF- or IFNγ-producing MAIT cells by individual donors in response to WT MR1 expressing C1R cells (C1R.MR1^null+MR1) pulsed with 100 pM 5-OP-RU (∼EC₅₀ dose). (H and I) Percentage of TNF- or IFNγ-producing MAIT cells, comparing the response in individual donors to WT or CD8-null MR1 expressing C1R cells (C1R.MR1^null+MR1 or C1R.MR1^null+MR1 CD8-null) pulsed with titrating doses of 5-OP-RU. (J and K) As above, comparing the percentage of TNF- or IFNγ-producing DN or CD8 SP MAIT cells. (B–J) Data are from 12 healthy blood donors from three independent experiments. Statistical significance was determined using a Friedman test with Dunn’s multiple comparison (F and G) or a two-way ANOVA with Sidak’s multiple comparisons test (H–K).