Intracellular Cytokine Staining of Lung T Cells

For intracellular cytokine staining, 1 million lung single cells were Fc-blocked with anti-CD16/32 and LIVE/DEAD-stained with Ghost Dye BV510 (Tonbo Biosciences) and surface-stained as described above. The cells were fixed, permeabilized with CytoFix/CytoPerm, and stained with anti-mouse GzmB-conjugated BV421 and APC-conjugated perforin. The perforin and GzmB expression represent the response from bulk CD8⁺ T cells. For IFN-γ detection (IFN-γ), lung single cells prepared from mock- and IAV-infected mice were stimulated with 10 μM IAV peptide NP_366–374 for 5 hours with brefeldin A, in RPMI medium containing 10% fetal bovine serum (FBS) and supplemented with antibiotics, before intracellular staining with anti-mouse IFN-γ–PE–Dazzle 594 (26 (link)). A BD FACSymphony or SONY MA900 flow cytometer was used to acquire 100,000 events, and data were analyzed using FlowJo (Tree Star Inc.). The list of the antibodies used in this study including their clone and catalog number is provided in the Supplementary Materials (table S1).