The cells were harvested from BALF, lung, and spleen tissues of immunized mice at day 6 after challenge with A/Phil/H3N2 (8× LD50). To determine IFN-γ secreting cells, lung and spleen cells were stimulated with M2e peptide or virus antigen for 3 days and then the cell spots secreting cytokine were determined by ELISpot analysis. (A) M2e-specific IFN-γ secreting cell spots in the lungs. (B and C) Virus antigen-specific IFN-γ secreting cell spots in the lung and spleen. (D and E) Antigen-specific T cell responses were analyzed by intracellular cytokine staining flow cytometry after in vitro stimulation with M2e peptide. Statistical significance was determined by using one-way ANOVA and Dunnett’s multiple comparison test. Data are representative of individual animal out of two independent experiments. Error bars indicate the means ± SEM. ***; p<0.001, **; p<0.01, *; p<0.05.