IM prime-derived, cross-reactive T cells are involved in the latter mucosal boost response in the lungs. (A) Schedule for the vaccination of donor CD45.1⁺ mice, transfer of donor cells, and subsequent vaccination of recipient CD45.2⁺ (CD45.1⁻) mice. Lungs were harvested 3 wk after Ad-o vaccination. (B) Total number (log₁₀) of donor⁺ cells (CD45.1⁺CD45^IV−) in the lungs of mice, as measured via cell staining and flow cytometry. The median response of the negative control mouse group that received PBS instead of cells, and were not vaccinated, is indicated as a dashed horizontal black line. (C) Proportion (median for the group) of donor⁺ (CD45.1⁺CD45^IV−) CD8⁺, CD4⁺, and CD19⁺ cells in the lungs. (D) Total number (log₁₀) of cross-reactive CD8⁺ T cells (MHC I tet⁺CD45^IV−) in the lungs of mice binding the conserved H-2K^b-restricted S_539-546 epitope, as measured via cell staining and flow cytometry. (E) Proportion of donor⁺ cells, and T_RM⁺ (CD69⁺CD103⁺CD62L⁻CD44⁺) and T_EM⁺ (CD62L⁻CD44⁺CD127⁺) cells, within the measured cross-reactive lung CD8⁺ T cell (MHC I tet⁺CD45^IV−) population. For all data, **P < 0.001, ***P < 0.001, ****P ≤ 0.0001. Parametric one-way ANOVA tests were completed when data were normally distributed; otherwise, a nonparametric Kruskal–Wallis test was performed (indicated with a + at the top left of the graph). For multiple comparison testing, the Ad-WT^IM+Ad-WT^IM>Ad-o^IN regimen was compared against all other regimens. On violin plots, the dashed black line represents the group median response and dots represent individual mice. Data are representative of two independent experiments, where experiment 1 (shown) was n = 6 for test groups WT^IM+WT^IM>o^IM, WT^IM+WT^IM>o^IN, and Unvaccinated>o^IN and n = 4, n = 3, and n = 2 for groups WT^IM+WT^IM>, Unvaccinated>, and PBS>, respectively. In experiment 2 (all n = 5), test groups WT^IM+WT^IM>o^IM, WT^IM+WT^IM>o^IN, and critical control group WT^IM+WT^IM> were exclusively repeated.