Clone rm4 4

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.).

B6 mice were injected intraperitoneally with 500 μg neutralizing antibodies against CD4 (BioXCell BE0003-1, clone GK1.5) and CD8 (BioXCell BE0223, clone 53-5.8) on days 1 and 3. On day 3, depletion of CD4⁺ and CD8⁺ T cells was confirmed by flow cytometry using different anti-CD4 (BioLegend 116025, clone RM4-4) and anti-CD8 (BioLegend 126610, clone YTS156.7.7) antibodies. The control mouse received PBS injections. On day 4, mice were implanted with 0.5 million αKO cells in the head of the pancreas as described above. Following implantation, mice received weekly injections of neutralizing antibodies or PBS until the end of the study. Tumor growth was monitored by IVIS imaging.