Fig. 3.

Phenotypical analysis of the DN DC presenting viral antigen after i.n. infection. (A) DC from C57BL/6 mice infected 3 d earlier with Flu.gB (10 mice per group) were enriched as described in Materials and Methods. Enriched DC were stained for CD11c, CD8α, and CD45RA in addition to one of the following: CD24, F4/80, CD11b, or CD205. Cells staining for CD8α and CD45RA were excluded, and remaining CD11c⁺ cells were sorted according to their level of expression of each additional marker (expression levels are indicated on flow cytometry histograms). Sorted DC were cultured with 5 × 10⁴ CFSE-labeled gBT-I cells, and proliferation was assessed by loss of CFSE staining. (B) Mice were infected with either Flu.gB (Upper) or HSV (Lower), and DC populations were enriched as above but were additionally depleted of CD8⁺ and CD45RA⁺ cells and then stained for CD11c, CD11b, and F4/80 expression before sorting for CD11b^–F4/80⁺, CD11b⁺F4/80⁺, and F4/80^– cells. CFSE-labeled gBT-I CD8⁺ T cells (5 × 10⁴) were cultured with these DC subsets, and proliferation was analyzed at 60 h of culture. The percentage of proliferating cells for each culture is indicated in each histogram. Data are representative of three separate experiments. (C) Analysis of expression of F4/80 and CD11b on purified DN DC subsets from various LNs after depletion of CD8α⁺ and CD45RA⁺ cells. Profiles are gated on CD11c⁺PI^– cells.