Quantification of Airway Immune Cells

Endobronchial biopsies were processed and embedded into glycol methacrylate resin (Polyscience), as previously described [80 (link)]. Sections from biopsies (2 μm) were stained in duplicates with anti-CD8, HLA-DR, CD11c, and CD123 (all BD Biosciences) followed by the rabbit anti-mouse (Dako) biotinylated secondary antibody. The immunostaining was performed as previously described [13 (link)]. All sections were visualized with 3-amino-9-ethylcarbazole (AEC), and cell nuclei were counterstained with Mayer hematoxylin (Histo Lab). Finally, all sections were analyzed using a high-resolution digital scanner, NanoZoomer-XR (HAMAMATSU) to convert them into digital images. A blinded analysis was performed using the scanned sections and NanoZoomer Digitial Pathway View2 software (NDP View; HAMAMATSU). The number of positive cells was expressed as cells/mm and cells/mm² of epithelium and lamina propria, respectively. Quantification of HLA-DR molecules was carried out with a Leica DMR-X microscope (Leica Microsystems GmbH) coupled to computerized image analysis (Leica Qwin 5501W; Leica Imaging Systems) as described previously [81 (link)].