Migratory CCR7⁺ DCs are defective in Tlr3^−/− mice. C57BL/6 mice (A) or Tlr3^−/− mice (B) were infected intranasally with GFP-expressing A fumigatus conidia, and the numbers of GFP⁺ CD11c⁺ cells were assessed by flow cytometry on T- and B-cell–depleted lungs or draining lymph nodes at different days postinfection (dpi). Numbers (mean values ± SD, n = 3) refer to the percentage of positive cells on gated CD11c⁺ cells. Shown is one representative experiment. (C) Phenotype of CD11c⁺ DCs from naive C57BL/6 or Tlr3^−/− mice. Flow cytometry was performed on gated CD11c⁺ DCs enriched from lungs. Numbers (mean values ± SD, n = 3) refer to the percentage of CD103⁺ cells. Histograms refer to cells expressing CD8α or CD11b on gated CD103⁺ cells from one representative experiment. Isotype-matched rat IgG2a (CD8α) or IgG2b (CD11b) control staining is shown in the blue histograms. Phagocytic index (n = 3; D) and relative expression of Ccr7 (RT-PCR, mean values ± SD of at least 3 independent experiments assessed in triplicates; E) by purified lung DCs. Monolayers of DCs were added to live GFP-expressing conidia for 2 hours at 37°C. Cytochalasin D was added for 1 hour at 37°C before phagocytosis. After quenching the fluorescence of bound, uningested conidia with Trypan blue, phagocytosis was quantified via phase contrast and fluorescence microscopy using the 100× objective and the DCs were fixed in 1% paraformaldehyde (shown are representative microscopic images of 2 independent experiments visualized with the 100×/1.3 oil objective on the BX51 upright microscope (Olympus). All images were captured using a high-resolution Microscopy Olympus DP71 camera (Olympus) and digitally acquired using the Cell∧P software (Version 3.3 build 2108, Olympus)). Ct indicates control, unexposed cells.