Recognition of fungal RNA is defective in Tlr3^−/− DCs. C57BL/6 or Tlr3^−/− lung DCs from naive mice were left unpulsed (None) or were pulsed with resting (RC) or swollen (SC) A fumigatus conidia, DOTAP alone, or fungal RNA + DOTAP for 2 hours. (A) Phosphorylation of IRF3 (p-IRF3) in DCs pulsed as above. Shown is a representative Western blot of 3 independent experiments. The IRF3 bands were quantified and the ratios of phosphorylated to total IRF3 are shown. (B) Relative expression of Ifna1 and Ifnb1 (RT-PCR) and (C) expression of CD80, CD86, and MHC class II (flow cytometry) on pulsed DCs (mean values ± SD of at least 3 independent experiments assessed in triplicate). Flow cytometry was performed 6 hours after pulsing and results are expressed as the percentage of positive cells. (D, F) T-cell proliferation induced by conidia- or RNA-pulsed DCs in CD4⁺ or CD8⁺ T cells purified from lungs of C57BL/6 mice (D) or Tlr3^−/− mice (F) 1 week after the intranasal infection and assessed for lymphoproliferation after 72 hours coculture with pulsed DCs from naive mice. DNA synthesis was measured by ³H-thymidine uptake (cpm, counts per minute). Ct indicates pulsed DCs alone. (E, G) Relative expression of Ifng and Prf1 by RT-PCR in CD4⁺ and CD8⁺ T cells exposed to conidia- or fungal RNA–pulsed DCs for 24 hours from C57BL/6 mice (E) or Tlr3^−/− mice (G). (H) Cytolytic activity of CD8⁺ T cells from C57BL/6 mice, obtained as in panel D, against conidia-pulsed DCs at different effector cell:target cell (E:T) ratios. Shown is the percentage of specific cytotoxic activity determined by a standard 4-hour ⁵¹Cr-release assay. (I) Conidiocidal activity of culture supernatants from CD8⁺ T cells exposed as in panel H. Live conidia were labeled with the fluorescent molecular stain FUN-1 and incubated overnight with culture medium from cultured CD8⁺ T cells before examination by fluorescence microscopy. Metabolically active conidia accumulate orange fluorescence in vacuoles, whereas dormant and dead conidia stain green. Shown are representative fluorescence microscopy images of 3 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). (J) CD8⁺ T-cell proliferation induced by RNA-pulsed C57BL/6 DCs in the presence or not (−) of inhibitors targeting class II (bafilomycin A1 and chloroquine) or class I (brefeldin A and lactacystin) Ag-presentation pathways. Ct, CD8⁺ T cells alone. (K) Adoptive transfer of C57BL/6 or Tlr3^−/− DCs pulsed with conidia or fungal RNA into C57BL/6 or Tlr3^−/− mice. DCs were adoptively transferred by IP injection twice a week before the infection. Fungal growth in the lungs (mean CFUs ± SE, representative of at least 3 independent experiments) was assessed 3 days after the intranasal infection. In vivo data are from 4 independent experiments assessed in triplicate. *P < .05; **P < .01; and ***P < .001 for Tlr3^−/− versus C57BL/6 mice (panels B-C), Ag-pulsed DC-primed versus unpulsed DC-primed (None) T cells (panels D-G), stimulated versus unstimulated CD8⁺ T cells (panel H), inhibitors versus no inhibitors (−; panel J), and DC-treated versus untreated (None) mice (panel K).