Flow Cytometry analysis
Freshly processed nasal lavage cells were divided into 0.2 to 1 x 106 cells per tube, resuspended in 50 µl of phosphate-buffered saline (PBS) (Lonza) and incubated in the presence of antibodies for 20 minutes on ice in the dark. Cells were then washed with PBS and resuspended for analysis. The following antibodies to human cell membrane proteins were used: CD45-PE Cy7, CD14-APC eFluor, CD16-FITC, CD56-APC, CD3-PerCPCy5.5 (all from eBioscience, San Diego, CA) and NKp46-PE (9E2) (BioLegend). Data was acquired on an ARIA flow cytometer (Becton Dickinson) with FACSDiva acquisition software (Becton Dickinson BioSciences, Mississauga, Ontario, Canada) and analyzed using FlowJo flow cytometric analysis software version 10.1 (Tree Star, Ashland, OR). Leukocyte populations were identified in nasal lavage samples as described previously for induced sputums (17). Briefly, doublets were excluded by gating on forward scatter (FSC)-H and FSC-A, followed by gating on CD45 positive cells to discriminate leukocytes from contaminating squamous-epithelial cells. Cellular debris were then gated out on the basis of FSC and side scatter (SSC). Specific populations were identified on the basis of marker expression as well as size and granularity. Respective gating strategy is outlined in Fig 2A. For nasal lavage samples, as many events as possible were collected. For compensation control, BD CompBeads (BD PharMingen) were used.