aDC depletion at 48 h p.i. does not alter IAV-specific CD8 T cell proliferation in the lungs. (A and B) Groups of BALB/c mice were infected with a sublethal dose of IAV. Half of the mice were subsequently aDC depleted at 48 h p.i. (aDC depleted), whereas the other half remained nondepleted (control). On days 4 and 5 p.i., mice were administered CFSE i.n, followed 2 h later by BrdU i.n. 4 h after BrdU administration, the proliferation, as measured by BrdU incorporation, of lung-resident CFSE⁺tetramer⁺ CD8 T cells was assessed by flow cytometry using the gating strategy outlined in A. Data in A and B are representative of two to three separate experiments (n = 5–8 mice/group). (C and D) Influenza-specific, CD90.2⁺ CL-4 T cells were adoptively transferred to groups of CD90.1⁺ BALB/c mice as in Fig. 1 (D and E). 24 h later, mice were infected with a sublethal dose of IAV with or without aDC depletion. On days 4 and 5 p.i., mice were treated with i.n. CFSE and BrdU. 4 h after BrdU administration, mice were sacrificed and their lungs were analyzed by flow cytometry for the frequency of BrdU⁺ cells among CFSE⁺CD8⁺CD90.2⁺ T cells using the representative gating strategy shown in C. Data in C and D are representative of one experiment (n = 3–5 mice/group). (E) Mice were infected and aDC depleted as in A and B; however, groups of aDC-depleted mice were subsequently reconstituted with purified pulmonary pDCs (light gray bars) or CD8α⁺ DCs (dark gray bars) on day 3 p.i. (i.e., 24 h after depletion). On day 5 p.i., virus-specific CD8 T cell proliferation was determined as in A and B. Data in E are representative of two to three separate experiments (n = 5–8 mice/group). Means ± SEM are shown. No statistical difference was observed between the analyzed groups. SSC, side scatter.