Lung airway-resident cells were harvested by bronchioalveolar lavage (BAL) with 3 consecutive washes of 1 mL PBS. To isolate cells from the lung parenchyma, lungs were perfused with ,25 mL PBS/heparin sodium solution, harvested, minced and incubated at 37uC for 30 minutes in 1.25 mM EDTA. The tissue was subsequently incubated in collagenase diluted in RPMI (6 mg/mL) at 37uC for 1 hour and passed through a 5 mm cell strainer. Isolated cells were subjected to separation via density gradient centrifugation by resuspending cells in 47% Percoll underlain with 67% Percoll. The gradients were then centrifuged at 2800 rpm for 20 minutes, and lymphocytes at the interface were collected. Spleen and lymph nodes were harvested from animals, homogenized, and then passed through a cell strainer. Spleen homogenate was depleted of erythrocytes by incubation in tris-buffered ammonium chloride.