BALFs were harvested from the lung airways by infusion with PBS via trachea using a 25-gauge catheter (26 (link)). Lung tissues were homogenized, passed through a cell strainer, and spun on 44/67% Percoll gradients to collect lung cells. Cellular phenotypes were determined by flow cytometric analysis using cell surface marker Abs specific for CD3, CD4, CD8, CD11b, CD11c, CD45, F4/80, Siglec F, DX5, and Ly6c (eBioscience, San Diego, CA, USA or BD Pharmingen, San Jose, CA, USA) as previously described (17 (link)27 (link)28 (link)). Cells from BALF and lungs were stimulated with the synthetic F85-93 (KYKNAVTEL) peptide (29 (link)) for CD8 T cell activation. Intracellular cytokine-producing cells stained with monoclonal IFN-γ and TNF-α and cell phenotypic marker Abs were acquired by the Becton-Dickinson LSR-II/Fortessa flow cytometer and data analyzed by Flowjo software (Tree Star Inc., Ashland, OR, USA). In the determination of cellular phenotypes, the fraction (%) of each cell phenotypes obtained by flow cytometry was multiplied by the total cell numbers counted in the cell preparations from the lung tissues and BALF.