Fig. 10

BEI-inactivated 22W_KY induces pronounced TNF-α + T-cell responses, with stronger immune activation in the lungs than in the spleen. (a) To assess T-cell responses induced by different vaccine inactivation methods, six-week-old BALB/c mice (n = 4–5) were intranasally immunized with F/A-, BEI-, or BPL-inactivated 22W_KY. Three weeks after booster vaccination, the mice were challenged with 10 LD₅₀ of either the SNU50-5 or the PR8 virus. Five days after the challenge, splenocytes and immune cells isolated from the lungs were restimulated in vitro with the respective challenge viruses to evaluate antigen-specific cytokine-secreting T-cell populations. The frequencies of (b, d, j, l) IFN-γ-producing and (c, e, k, m) TNF-α-producing CD4 + T cells and the frequencies of (f, h, n, p) IFN-γ-producing and (g, i, o, q) TNF-α-producing CD8 + T cells were measured. Fluorescence minus one (FMO) controls were used to gate IFN-γ and TNF-α positive populations. All the data are presented as the mean ± SD, and statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. Significant differences are denoted by asterisks, with asterisks displayed directly above the violin plots indicating statistical significance compared with the negative control (challenge-only group). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant)