i.n. vaccination with recombinant MVA induces a distinct population of antigen-specific cytotoxic T cells in the lung. (A) Mice i.n. received 10⁷ IU MVA-Cy5, and 3 d later the phenotype of Cy5⁺ cells in the draining LNs was determined by flow cytometry. (B) Expression of CD86 on CD11c⁺MHCII⁺ brLN DCs before and 2 d after i.n. instillation of MVA (shaded area, isotype control). (C–G) 1 d after the i.v. transfer of 200 CD45.1⁺ CD8⁺ OT-I T cells into CD45.2⁺ recipients, mice were i.n. or i.m. infected with 10⁷ IU MVA-OVA. At the indicated time points (days after infection) the absolute number of OT-I cells was determined in the brLNs (C), BAL (D), lung (E), and spleen (F). (G) The percentage of OT-I cells expressing CD69 in the lung and spleen. (H) Expression of CD3 and CD69 in BALT at 12 d after infection. Bar, 50 µm. Data in A and B are representative of four mice analyzed in two independent experiments. Data shown in C–G are pooled from two independent experiments with two to three mice per time point (means + SD). *, P < 0.05; ***, P < 0.001.