Mice were injected i.v. with 1 µg anti-CD8β 3 min before tissue harvest. Cells in the lung airways were recovered by lavage with PBS (5 × 1 ml), followed by plastic adherence (1 h at 37°C). Lung tissues were digested by collagenase D (Roche Diagnostics) for 30 min at 37°C, and enriched by centrifugation over a 40/80% Percoll gradient. Spleen cells were obtained by straining through nylon mesh and enriched by panning on anti-mouse IgG-coated flasks, followed by RBC lysis in buffered ammonium chloride. Cells were blocked first with mAbs to FcRγIII/II and then stained with APC-conjugated influenza NP366-374/Db tetramer. MHC class I tetramers were generated by the Molecular Biology Core Facility at the Trudeau Institute as described previously (Takamura et al., 2010 (link)). Tetramer-labeled cells were washed and stained with fluorescent-conjugated reagents purchased from BD Bioscience (CD45.1, CD90.1, Siglec F, and CD31), BioLegend (CD8α, CD8β, CD44, CD69, CD49a, CD103, CD11a, EpCAM, CCR3, CCR5, CCR6, and CCR9), TONBO Bioscience (CD90.2, CD11c, and F4/80), or R&D Systems (CXCR3, CXCR4, and CXCR6). Samples were run on LSRFortessa flow cytometers (BD Bioscience), and data were analyzed using FlowJo (Tree Star).