Analyzing Lung-Resident Memory CD8+ T Cells

Lung-resident memory CD8⁺ T cells were analyzed as previously described (34 (link)). Mice were intranasally infected with H1N1 or H5N1 (2:6) virus. On day 30 postinfection, mice were intravenously injected with 1 μg anti-CD8β antibody 5 minutes before tissue harvest. Lung tissues were perfused with PBS and single-cell suspensions were prepared after digestion with collagenase as described before. Cells were blocked first with FcRγIII/II antibody and stained with H-2D^b restricted tetramer conjugated to fluorophore R-phycoerythrin (PE) (NP_366-374 ASNENMETM). Tetramer-labeled cells were washed and stained with anti-CD4 (2 μg/ml; clone RM4-4; BioLegend), anti-CD3 (2 μg/ml; clone 145-2C11; eBiosciences), anti-CD8a (1 μg/ml; clone 53-6.7; eBiosciences), anti-CD44 (clone IM7; BioLegend), anti-CD103 (2 μg/ml; clone 2E7; eBiosciences), and anti-CD69 (clone H1.2F3; BioLegend). Dead cells were stained with the Live/Dead fixable near-IR staining kit (Life Technologies) in PBS for 15 minutes on ice. Surface-stained samples were fixed with FACS buffer containing 0.1% formaldehyde and analyzed using the BD LSR-II flow cytometer. Data analysis was performed using FlowJo software (Treestar Corp.).