Fig. 3: Histological analyses of lung tissue post immunization or H1N1 infection.

A Lungs from rAd-immunized or H1N1-infected mice were obtained 18 days post-treatment, and HE-stained lung sections were scanned at 40x magnification. Airways were generally intact and solely H1N1 infection caused lymphocytic infiltration reflected by areas of lymphocyte accumulations, without neutrophils, monocytes, and macrophages (arrows). One representative example of four mice per group is shown. Scale bars in whole slide lung images are 1000 µm and scale bars in magnified images are 100 µm. B At the indicated time points, lungs of naïve, immunized, and infected mice were removed, embedded into O.C.T. compound, and finally stained with anti-CD4, anti-CD8, and anti-B220. Nuclei are shown in blue (Hoechst33342), CD4⁺ T cells are shown in yellow, CD8⁺ T cells are shown in green, and B cells are shown in red. C To identify the localization of antigen-specific CD8⁺ T cells in the lung parenchyma, lung sections from day 18 p.i. were primary stained with anti-CD8, followed by MHC-I pentamer staining. The signal intensity of bound APC-labeled pentamers was amplified with anti-APC-AF647, and anti-mouse IgG2b-AF647. Nuclei are shown in blue (Hoechst33342), CD8⁺ T cells are shown in green, and MHC-I pentamer-specific T cells are shown in red. Images were acquired on a Leica SP5X laser scanning confocal microscope using a 40x oil objective and are representatives from a collection of different slices of four mice per group (n = 4). Scale bars in each image are 50 µm.