Fig. 6: T cell responses in vaccinated mouse lungs.

Mice were challenged with 10x LD₅₀ of mouse-adapted A/Hong Kong/415742Md virus at 14 days after i.d. vaccination. Bronchoalveolar lavage fluid (BAL), lung, spleen, and serum samples were collected at 2 and 4 dpi. a T cell composition in mouse lung single-cell suspension by flow cytometry assays. Effector memory (EM) T cells were identified as CD3⁺, CD4⁺/CD8⁺, CD44⁺, and CD62L⁻ cells, whereas central memory (CM) T cells were identified as CD3⁺, CD4⁺/CD8⁺, CD44⁺, and CD62L⁺ cells. n = 3–5 mice. Error bars indicate standard error of mean. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001 when compared with PBS group by two-way ANOVA. b CD4⁺ and CD8⁺ T cell distribution in lung sections at 2 dpi. Representative images of immunohistochemically stained NP, CD4, and CD8. In vaccinated mouse lung, some viral NP-expressing cells were observed in the bronchiolar epithelium (solid arrows), extensive CD4⁺ and CD8⁺ T cell infiltration around the same bronchiole (open arrows). In PBS control mouse lung, abundant viral antigen-expressing cells in the entire epithelium layer (solid arrows) while CD4⁺ and CD8⁺ T cell infiltration were much less frequent (open arrows). Scale bars = 100 µm. c Relative expression levels of IL-2, IL-4, and IFN-γ in lung at 2 dpi by real-time RT-PCR comparing to uninfected lung samples. Viral-specific interferon-γ producing CD4⁺ and CD8⁺ T cells in (d) BAL and spleen (e) taken at 4 dpi. In vitro T cell epitope stimulation with CD4 epitope and CD8 T epitope from influenza NP (CD4 T epitope peptide: RLIQNSITIERMVLS; CD8 epitope peptide: TYQRTRALV) were performed for 48 h. Right-hand side is the representative images from EISPOT assay. n = 3 per group. Error bars indicate standard error of mean. ^*p < 0.05; ^**p < 0.01 when compared with PBS group by two-way ANOVA. f Antibody-mediated T cell depletion in vaccinated mice did not compromise vaccine-induced protection against A/Hong Kong/415742Md challenge, no weight loss (left) or lethality (right) were observed after virus challenge. g Representative images of H&E (left) and immunohistochemistry stained viral NP in the T cell-depleted mouse lung at 4 dpi. Scale bars = 100 µm. h Immunohistochemistry stained CD4, CD8 T cells, respectively, in the T cell-depleted or not depleted mouse lungs at 4 dpi. Scale bars = 100 µm. i Serum antibody titer, HI (left), MN (right) tested at 4 dpi in T cell-depleted or not depleted mice, and control mice. n = 3–5 per group.