Figure 3

Preformed MR1 bound to 6-FP can present exogenously added antigens. (A) Left: A549 MR1^−/−: TET-MR1GFP were used as APCs in an IFN-γ ELISPOT assay with Msmeg supernatant as the antigen. The data are pooled from 2 independent experiments. An unpaired two-tailed t-test was used to calculate statistical significance (P = 0.0493). Right: A549 MR1^−/−: TET-MR1GFP were used as APCs in an IFN-γ ELISPOT assay with Mtb infection or with Msmeg supernatant comparing preformed MR1 to ongoing MR1 synthesis. Mean and SEM from technical replicates are plotted. Data are representative of 2 independent experiments. (B) A549 MR1^−/−: TET-MR1GFP with preformed MR1 were treated with 6-FP or control and used as APCs in an IFN-γ ELISPOT assay with Msmeg supernatant. Left) plot of IFN-γ cytokine activity. Data are pooled from 3 independent experiments. P = 0.0048 for 20 µL condition and P = 0.0256 for 10 µL condition (unpaired two-tailed t-test). Right) pooled data from 5 experiments using 10 µL Msmeg supernatant with IFN-γ SFU plotted. An unpaired two-tailed t-test was used to calculate statistical significance (P = 0.0072). (C) Flow cytometry of BEAS-2B:TET-MR1GFP with preformed MR1 treated overnight with 6-FP versus control. Left) flow cytometry of a representative experiment. Wild type (WT) BEAS-2B serves as the unstained control. Right) data from 4 independent experiments showing the percent of GFP positive cells. P = 0.0011 (unpaired two-tailed t-test).