Collagenase type 2

Organoids were dissociated for flow cytometry analysis using collagenase type II (Sigma-Aldrich, cat. #C6885) resuspended in HEPES buffer (Sigma-Aldrich, cat. #H0887) at a concentration of 20 mg/mL. Samples were collected by gravitation in a 15-mL Falcon tube and washed 2× in PBS and then resuspended in collagenase type II. For dissociation, samples were incubated at 37°C for 5 minutes before trituration and a further 5-minute incubation. The dissociation reaction was stopped through the addition of PBS supplemented with FBS. Ten organoids were dissociated per flow cytometry experiment. Analysis was performed using either a cyan flow cytometer (Beckman Coulter) or an Attune NxT. Single color stained controls and fluorescence-minus-one controls were used for all experiments using antibodies as listed in Supplementary Table S5.

One week after the last vaccine dose, we euthanized mice and removed the spleen and lungs to assess immune responses. Single-cell suspensions of splenocytes were prepared by gently pressing the cells out of the spleen sac; lysing red blood cells with PharmLyse (BD Pharmingen); washing the cells; and filtering through a 70 µm nylon cell strainer (Falcon). Single-cell suspensions of lung cells were prepared by cutting the lung into small pieces with a scalpel; incubating at 37°C for 1 h with shaking in 10 mL of digestion solution (300 U/mL collagenase type II [Worthington] and 0.15 mg/mL DNase I [Worthington] in PBS); filtering through a 40-µm nylon cell strainer (Falcon); lysing red blood cells with PharmLyse (BD Pharmingen); and washing the cells. Advanced RPMI-1640 (Invitrogen) supplemented with 2% heat-inactivated fetal bovine serum, 2 mM glutamine dipeptide (glutaGRO Supplement, Corning), 10 mM HEPES buffer, 50 µM β-mercaptoethanol, and penicillin (100 IU/mL)-streptomycin (100 µg/mL) was used as the medium.