Tlr3^−/− mice fail to develop MHC class I–restricted CD8⁺ T-cell responses after vaccination. (A) C57BL/6 mice were treated with a total of 300 μg of anti-CD4 or anti-CD8 Ab the day before or 1 or 3 days after the intranasal infection with live A fumigatus conidia (6-8 mice/group). “None” indicates mice receiving isotype control Ab. Depletion of the corresponding T-cell subsets with this regimen was between 95% and 98% at 3 and 7 days postinfection (dpi). (B) Periodic acid-Schiff staining of the lungs from mice treated in panel A. Note the presence of fungi (Gomori methenamine silver staining in the inset) and the sustained inflammation in the lungs of CD4-depleted mice throughout the infection, as opposed to the increased inflammation and fungal growth only observed at 7 dpi in CD8-depleted mice. Images were visualized with the 40×/0.75 objective on the BX51 upright microscope (Olympus) and captured using a high-resolution Microscopy Olympus DP71 camera (Olympus) and digitally acquired using the Cell∧P software (Version 3.3 build 2108, Olympus). Bars indicate magnifications. Lung histology of untreated mice was similar to those shown in Figure 1. (C) Survival (%), (D) fungal growth (CFUs, mean ± SE), and (E) relative lung expression of Ifng, Il17a, Il4, and Il10 by RT-PCR in C57BL/6 or Tlr3^−/− mice vaccinated with A fumigatus conidia 14 days before (blue color) or with the recombinant fungal Ag Crf1p + CpG 14 7 and 3 days (red color) before subsequent intranasal infection with live conidia (6-8 mice/group). Mice were given cyclophosphamide 1 day before the infection. Fungal growth and gene expression were assessed at 3 dpi. CpG alone failed to induce vaccine-induced resistance, as reported previously.²⁴  Survival (F and H) and fungal growth (at 3 dpi; G and I) in mice treated with anti-CD4, anti-CD8, anti–MHC class I, or anti–MHC class II mAbs in concomitance with A fumigatus conidia vaccination (blue color) or Crf1p + CpG vaccination (red color). Treatment with anti-CD4 alone, but not anti-CD8, anti–MHC class I-A, or anti–MHC class II mAbs alone, further increased the susceptibility to the infection in cyclophosphamide-treated mice compared with control mice. Ct indicates infected unvaccinated mice; none, vaccinated mice treated with an isotype control Ab. Bars indicate magnifications. Data are representative of at least 3 independent experiments. *P < .05; **P < .01; and ***P < .001 for vaccinated versus unvaccinated mice (panels D-E) or treated versus untreated mice (panels A, G, and I).