Il 2 pe

Whole blood was incubated with 1 μg/mL αCD28, 1 μg/mL αCD49d (BD Biosciences) and stimulated with 20 μg/ml PPD (SSI, Denmark), 5 μg/ml staphylococcal enterotoxin B (Sigma Aldrich), or no antigen (unstimulated). Samples were incubated at 37°C in 5% CO₂ for 6 h, 3 μg/ml Brefeldin-A (Sigma Aldrich) was added, and samples were incubated for another 6 h in a 37°C water bath. Samples were then treated with 2 mM ethylenediaminetetraacetic acid (Gibco), and red blood cells were lysed using FACS Lysing solution (BD Biosciences) and samples were frozen in PBS with 10% DMSO for batched ICS staining. Frozen samples were thawed, permeabilised and incubated with antibodies against CD3 (AF700), IFN-γ (PE-Cy7 (eBioscience); CD4 (APC), CD14 (Pacific blue), TNF-α (PerCP-Cy5.5), and IL-17 (AF488) (BioLegend); CD8 (APC-H7) (Becton Dickinson) and IL-2 (PE) (Beckman Coulter). Samples were acquired on an LSR II flow cytometer (BD Biosciences) and responses analyzed using FlowJo version 8.8.7 (Tree Star Inc., Ashland, USA). Cytokines were measured as the frequency of singlet CD14– CD3+ T cells, CD4+ T cells, and CD8+ T cells. Data are presented as percentages of cytokine-positive cells minus responses in unstimulated cells; the gating strategy is shown in Supplementary Figure 1.

BAL cells and PBMCs were isolated and stimulated at 1 × 10⁶ cells/mL with Ag85A, ESAT-6/CFP-10 peptides (2 µg/mL each), and PPD (20 µg/mL); unstimulated cells and SEB-stimulated cells were used as negative and positive controls, respectively. Brefeldin A (Sigma) was added to the cells 2 h after stimulation, and cells were incubated overnight at 37 °C and 5% CO₂.
Harvested BAL cells and PBMCs were stained with the live/dead red viability marker (Thermo Fisher) for the exclusion of dead cells, followed by surface staining with CD4-Pacific Blue (Biolegend, San Diego, CA, USA), CD14, and CD19 on ECD (Beckman Coulter, Brea, CA, USA). Cells were then permeabilised and stained with CD3-AF700 (Ebioscience, San Diego, CA, USA), CD8-APC/H7 (BD), IFN-γ-PECY7 (Ebioscience) and TNF-α-AF-647 (Biolegend), IL-2 PE (Beckman Coulter), and IL-17-AF488 (Biolegend).
Cytokine-producing CD4+ and CD8+ T-cells were gated on CD3+, CD14−, CD19− single T cells. Data were analysed using Flowjo (BD) and are presented as background-subtracted antigen-specific responses.