Activation of human dendritic cells byA fumigatus. Immature (im) and mature (m) dendritic cells (DCs) DC1 and DC2 were obtained from CD11c⁺ blood mononuclear cells. (A) DCs were exposed to unopsonized conidia or hyphae for the assessment of phagocytosis after 60 minutes. After a Diff Quik staining, aliquots of cells were spun down on slides on a cytocentrifuge and examined for conidia internalization by light microscopy. For each experiment, at least 5 fields in each slide were counted, and at least 200 DCs were analyzed in each well. All conditions were tested in triplicate. The data are the means of 3 independent experiments and expressed as percent internalization. (B) Cytokine production by DCs pulsed with conidia or hyphae. DCs were exposed to the different stimuli for 2 hours at 37°C before the addition of amphotericin B to prevent fungal overgrowth, and left for an additional 22 hours. The levels of cytokines were determined in the culture supernatants by cytokine-specific ELISA. ^*P < .05 (cytokine production by conidia-pulsed versus hypha-pulsed DCs). Cytokine levels in unexposed cells were below the detection limits of the assays. Error bars indicate SE. (C) Proliferative response of peripheral blood T cells unstimulated (black histograms) or stimulated by autologous imDC1 and imDC2, either unpulsed (red lines) or pulsed with conidia (green lines) or hyphae (blue lines), for 6 days at 37°C. CFSE staining was done as described in “Materials and methods.” Shown are the results from one donor. Similar results were obtained with 5 different donors. Numbers with arrows to the median fluorescence intensity.