Klrg1 percp cy5

Cells isolated from spleen, lungs or BAL were cultured with 10 µg/ml Purified Protein Derivative of M. tb (PPD-T) (Statens Serum Institut, Copenhagen, DK), 1 µg/ml anti-CD28 (BD Biosciences) and 10 µg/ml Brefeldin A (Sigma-Aldrich) for 16 h at 37 °C/5% CO₂. Cells were washed (300 g/5 min) and surface stained for 15 min/4 °C with pre-titrated antibodies: PD-1⁻FITC, CD44-BV785, CD8-AF700, KLRG1-PerCP-Cy5.5, CXCR3-BV421, live/dead-Zombie Aqua (all BioLegend, San Diego, CA, USA), CD90.2-eFluor 450 (eBioscience), and CD4-APC-H7 (BD Biosciences). Cells were then washed, treated with BD Biosciences Cytofix/Cytoperm as per the manufacturer’s instructions and stained intracellularly for 30 min/4 °C with IFN-γ-PE-Cy7, IL-2-APC (both eBioscience) and TNF-α-BV605 (BioLegend). Cells were washed again and acquired using an LSRFortessa™ analyser, utilising a 532 nm laser for PE and PE-conjugate excitation, with FACSDiva™ software (BD Biosciences). Final analysis was performed using FlowJo® software (Tree Star, Ashland, OR, USA) on a minimum of 100,000 live lymphocytes (50,000 for BAL).

The following antibodies were purchased from Biolegend: CD3 BV510, CD49b (DX5) APC-Cy7, NKp46 BV421, CD11b PE, Ly49D PE, Streptavidin BV650, CD27 biotin, KLRG1 BV605, KLRG1 PerCP/Cy5.5, CD69 PerCP/Cy5.5, and Ki67 APC. The following antibodies were purchased from eBioscience: CD107a PerCP-eFluor710, Ly49I PE, CD94 FITC, IFNγ APC, and Ly49H PE-Cyanine7. The following antibodies were purchased from BD Biosciences: NKG2A/C/E BV605. The following antibodies were purchased from Miltenyl Biotec: NKG2D (CD314) biotin, Ly49D biotin, and Ly49D PerCP-Vio700. To determine viability Invitrogen Fixable live/dead Aqua stain was used.