ESAT-6 protein induces αβ but not γδ T cells' response in active TB patients and TST-positive LTBI subjects. Peripheral blood mononuclear cells (PBMCs) from a representative active TB patient (A) and a representative TST-positive LTBI subject (B-D) were stimulated with ESAT-6 protein (5 μg/mL; Lionex, Braunschweig, Germany) in presence of anti-CD28 monoclonal antibody (1 μg/mL; BD Biosciences, San Jose, CA). To detect intracellular expression of IFN-γ, brefeldin-A (Sigma-Aldrich, St Louis, MO) at 10 μg/mL was used. After overnight stimulation, cells were stained with CD4-peridinin chlorophyll protein (PerCP), Vδ2-phycoerythrin (PE), Vδ1-fluorescein isothiocyanate (FITC), IFN-γ allophycocyanin (APC) anti–human conjugated monoclonal antibodies (BD Biosciences). For all staining procedures, an isotype-matched negative control was used. Data acquisition and analysis were performed using a FASCalibur flow cytometer and CellQuest software (both BD Biosciences).