fumigatus infection, which suggests that IFN-β is a possible adjuvant to elicit an appropriate Th reactivity to A. fumigatus. Dendritic cells were prepared as previously described.9 CD14+ monocytes were cultured with 25 ng/ml granulocyte–macrophage colony-stimulating factor (GM-CSF; Schering-Plough, Levallois Perret, France) and 1000 U/ml IL-4 (R&D Systems, Minneapolis, MN) for 5 days. On day 5, about 90% of the cells express CD1a+ and 95% express

CD14−. The DCs were starved from IL-4 and GM-CSF for 20 hr before infection or treatments. Monoclonal antibodies specific for CD1a, CD14, CD38, CD40, CD83, CD86, HLA-DR, CD3 and CD4 as well as immunoglobulin G1 (IgG1), IgG2a HM781-36B cost and IgG2b (BD Bioscience PharMingen, San Diego, CA) were

used as direct conjugates to fluorescein isothiocyanate (FITC) or phycoerythrin. Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (Sigma-Aldrich, St Louis, MO) was used at a concentration of 100 ng/ml to stimulate DC maturation and IFN-β expression. The IFN-β (Avonex®; Biogen Inc., Cambridge, MA) was used at 200 pm. A wild-type clinical isolate of A. fumigatus (CBS 144 89) was grown on Sabouraud–chloramphenicol agar for 3 days, at 37°, as previously described.23 Preparations of A. fumigatus were analysed for LPS contamination by the Limulus lysate assay (Biowhittaker, Verviers, Belgium) and were found to contain less than PF 2341066 10 pg/ml LPS. In all experiments, DCs were infected with live A. fumigatus conidia at a 1 : 1 ratio. Amphotericin B (0·75 μg/ml; Sigma-Aldrich) was added to the cell

cultures to prevent fungal overgrowth 6 hr after infection when the internalization of A. fumigatus conidia was completed.9 For the adherence assay, A. fumigatus conidia were incubated with FITC at a final concentration of 3 mg/ml overnight at 4°, and then washed extensively with PBS. After a 6-hr incubation with FITC-labelled Adenosine triphosphate conidia (ratio 1 : 1), DCs were washed and the adherence was measured by flow cytometric analysis. The cells were incubated with purified monoclonal antibodies at 4° for 30 min. After washing, the cells were fixed with 2% paraformaldehyde before analysis on a FACScan using the cellquest software (BD Bioscience PharMingen). A total of 5000 cells were analysed per sample. RNA extraction, reverse transcription (RT) and real-time RT-polymerase chain reaction (PCR) assays were performed as previously described.24 Sequences of the primer pairs used for glyceraldehyde 3-phosphate dehydrogenase (GaPDH), IFN-β, IL-12p35, IL-23p19 and IL-27p28 quantification were previously described.24 Cytokine concentration in filtered supernatants was evaluated with the human inflammation cytometric bead array (CBA) [for IL-12p70, IL-10, tumour necrosis factor-α (TNF-α) and IL-6: BD Bioscience PharMingen] and enzyme-linked immunsorbent assay (ELISA; for IFN-β: PBL Biomedical Laboratories, Piscataway, NJ; for IL-23: eBioscience, San Diego, CA).