Mtb-specific T cell Responses Ex vivo

The freshly collected BAL cells were stimulated ex vivo with Mtb-specific antigens, ESAT-6/CFP-10 and Mtb Cell Wall Fraction (BEI Resources, 10 μg/mL) for a total of 16 h. Brefeldin A (0.5 μg/mL, SIGMA) was added 2 h after the onset of stimulation. After stimulation, the cells were stained with LIVE/DEAD fixable Near-IR stain (ThermoFisher) and stained subsequently with the surface antibodies: CD4-PerCP-Cy5.5 (L200, BD Biosciences), CD8-APC (RPA, T8, BD Biosciences), CD3-AlexaFlour 700 (SP34 2, BD Biosciences), CD95-BV421 (DX2, BD Biosciences), CD28-PECy7 (CD28.2, BD Biosciences) and CD45-BUV395 (D058 1283, BD Biosciences). Cells were then fixed, permeabilized and stained with intracellular antibodies: IFNγ-APC-Cy7 (B27, Biolegend), IL-17-BV605 (BL168, Biolegend) and TNF-α-BV650 (MAb11, Biolegend). Cells were washed, suspended in BD stabilizing fixative buffer and acquired on BD Symphony flow cytometer. Analysis was performed using FlowJo (v10.6.1) using previously published gating strategy (18 (link)–20 (link)) (Figures S1–S3).