Memory CD8 T cells, harvested from mice 35–45 days post-infection, were negatively selected from bronchoalveolar lavage (BAL) using Miltenyi CD8α T Cell Isolation Kit II. Influenza NP366–374/Db+ tetramer quantification allowed for equal numbers of antigen-specific cells to be i.t. transferred from donor mice to naïve recipient mice. No more than 1.5×105 antigen-specific airway CD8 TRM cells were transferred per recipient to approximate physiological numbers of airway TRM cells. Antibodies used for flow cytometry and cell sorting were BioLegend CD62L [MEL-14], CD8α [53–6.7], CXCR3 [CXCR-173]; eBioscience CD11a [M17/4], CD44 [IM7]; and BD Biosciences CD3ε [145-2C11], CD45.2 [104], CD90.2 [53–2.1], IFN-γ [XMG1.2]. Intravital staining was performed immediately before mouse euthanasia and tissue harvest as previously described (15 (link)). Briefly, to identify T cells resident in various tissues, including the lung parenchyma, 1.5µg of fluorophore-conjugated α-CD3ε antibody in 200λ 1× PBS was intravenously injected into the tail vein of mice; five minutes post-injection, mice were euthanized with Avertin (2,2,2-Tribromoethanol - Sigma) and exsanguinated prior to harvest of BAL and other tissues. Staining for intracellular cytokines was performed as previously described following stimulation in the presence of Brefeldin A for the indicated periods of time (25 (link)). To study cell proliferation, mice were given an intraperitoneal bolus of BrdU (0.8mg) at the time of infection and maintained on BrdU drinking water (0.8mg/mL) until harvest. BrdU incorporation was measured using the BrdU Flow kit (BD Biosciences) following tetramer and antibody staining. Samples were run on a BD Biosciences Canto II or LSR II flow cytometer and analyzed with FlowJo software. Sorting was performed on an Influx or Aria II cell sorter (BD Biosciences).