Nasal Immunophenotyping by Flow Cytometry

Immunophenotyping of nasal cells obtained by curettes was performed as previously described^{52 (link)}. In brief, cells were dislodged from curettes and stained with LIVE/DEAD Fixable Violet Dead Cell Stain (Thermo Fisher Scientific) and an antibody cocktail containing Epcam-PE (9C4; BioLegend), HLADR-PECy7 (L243; BioLegend), CD16-APC (3G8; BioLegend), CD66b-FITC (G10F5, BioLegend), CD3-APCH7 (SK7; BD Biosciences), CD14-PercpCy5.5 (MφP9, BD Biosciences) and CD45-PACOrange (HI30, Thermo Fisher Scientific). Whole blood was stained for 15 min at room temperature with TIGIT-PECy7 (A15153G, BioLegend) and CD16-APC, followed by 2 × 10 min incubation steps with FACSLysis buffer (BD Biosciences) to remove erythrocytes. Samples were acquired on an LSR II flow cytometer (BD Biosciences) and analyzed using Flowjo X (Treestar). Fluorescent minus one controls for each of the included antibodies were used to validate results. For the LAIV and control cohorts, but not the additional validation cohort (Supplementary Fig. 5c), 84 of 553 samples (15.2%) had less than 500 immune cells or 250 epithelial cells and were excluded from further analysis.