Mtb-infected human AEC are efficiently recognized by Mtb-specific CD8⁺ T cells.

<p>A–B) Mtb-infected DC, BEAS-2B cells, or LAEC (MOI:10) were used as APCs in an IFN-γ ELISPOT assay. A) IFN-γ release by the HLA-B45-restricted CD8⁺ T cell clone D466H4 was assessed by ELISPOT assay. 10,000 T cells were used with a titration of APCs from 10,000 - 1,250 cells per well. Results are representative of all experiments (N = 3). Error bars represent the mean and standard error from duplicate wells. B) IFN-γ release by the MR1-restricted (D426B1), HLA-E-restricted (D160 1-23), and HLA-B45-restricted (D466H4) CD8⁺ T cell clones was assessed for Mtb-infected DC, BEAS-2B, or primary LAEC (except D466H4). The fold-difference in IFN-γ release was normalized to the T cell response to Mtb-infected DC (5,000 DC/well) for each APC type and T cell clone. Results are representative of all experiments (N = 3 for DC and BEAS-2B cells and N = 1 for LAEC). C–F) Following fixation in 4% PFA, uninfected or infected BEAS-2B cells, DC, or LAEC were surface stained with either the pan-HLA-I antibody W6/32 (C-F) or the anti-MR1 antibody 26.5 (G) and analyzed by flow cytometry. C-E: shaded histogram, UI; black line, Mtb. F-G: shaded histogram, isotype staining for each cell type. Results are representative of all experiments (N = 5 for W6/32 and N = 4 for 26.5). H) DC or BEAS-2B cells were pulsed with CFP10_2-9 peptide at the indicated concentrations for 1 hour and assessed for their ability to stimulate IFN-γ production by the HLA-B45 restricted, CFP10_2-9 specific, D466H4 T-cell clone. APCs were added at 10,000 cells/well, while T cells were kept constant at 500 T cells per well. Results are representative of all experiments (N = 3). Error bars represent the mean and standard error from duplicate wells.