Infected macrophages present antigen in DC-ablated mice. (A) VV-NP-S-eGFP–infected cells (green) just under the collagenous capsule of the node (second harmonic generation (blue)). Numbers of infected cells in nonablated (untreated) and DC-ablated (DTx-treated) mice (right). (B) Confocal microscopy of a frozen section of a DC-ablated LN 10 hpi with VV-NP-S-mCherry (red) showing only the red and green channels (left), only the red and grey channels (middle), or all channels (right). FITC-dextran (green) and anti-CD11b staining (white) identify macrophages. (C) Same as shown in B, except staining with F4/80 (grey) to identify medullary macrophages. (D) Numbers of infected cells of each cell type in the node. Cells were gated into CD11c⁺CD169⁻ (DCs) or CD11c^dimCD169⁺ (CD169⁺ macrophages), then CD169⁺ macrophages were further gated based on F4/80 expression. Background (blue) is caused by low cell recovery and macrophage autofluorescence. (E) Flow cytometric analysis of LN single-cell suspensions from untreated or DTx-treated animals 6 hpi with VV-venus-ubiquitin-SIINFEKL. Cells were stained with 25D-1.16 recognizing K^b-SIINFEKL complexes. Histograms are gated on infected cells (Venus eYFP⁺). Infected with virus lacking SIINFEKL (grey shaded histograms), infected without 25D-1.16 stain (black lines), infected untreated mice (blue lines), and infected DTx-treated mice (red lines). Scale bars are shown in micrometers. Results are shown from one experiment of two (A–C) or four (D and E).