γδ T cells, but not CD4, CD8, or NK cells, express IFN-γ in ESAT-6–stimulated PBMCs. (A) Identification of IFN-γ–producing cells in PBMCs induced by ESAT-6. PBMCs were cultured in the presence of ESAT-6. After stimulation for 5 hours, cells were harvested and FACS stainings were conducted as described in “Flow cytometric analysis of cell-surface markers and intracellular staining.” Subpopulations of CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells, CD3⁻CD56⁺ NK cells, and γδ T cells were analyzed by FACS for IFN-γ expression. (B) One representative result from 3 independent experiments showing the expression of TNF-α and IFN-γ. The numbers in each quadrant represent the percentages of positive cells in gated γδ T cells. (C) Statistical results of IFN-γ expression by subpopulations of PBMCs from 3 independent experiments were shown. ***P < .001.