To assess the importance of ROS production in DC killing ability, DPI (10 microM) is added 30 minutes before stimulation.

Immature DCs are allowed to adhere onto fibronectin and subsequently incubated with FITC-labeled yeast or spores for 5, 15, 30 minutes at 37°C. At the end of the incubation period, the samples are fixed in 4% PFA, permeabilized in Methanol, and labeled for DC-SIGN and/or MR using specific mAb and isotype-specific fluorescent secondary Abs. Samples are analyzed using a Zeiss LSM 510 confocal microscope. Alexa647-conjugated goat-anti-mouse IgG, and Alexa568 goat-anti-mouse IgG2b from Molecular Probes.

Yeasts/spores are biotinylated using 10 mg/ml sulfo-NHS-LC-biotin (Sigma-Aldrich) in 50 mM NaHCO3 pH 8.5 for 2 hours at 4°C. The remaining reactive biotin molecules are inactivated by incubation in 100 mM Tris-HCl pH 8.0 for 40 minutes at 4°C. DCs are then treated with biotinylated spores/yeasts. After 1 hour, cells are permeabilized and labeled with aHLA-DR-FITC. After zymolyase treatment, intracellular yeasts/spores are detected using APC-labeled streptavidin and analyzed by flow cytometry.

Absorbance is measured using a microplate reader at 620 nm.

Blue formazan particles are dissolved using 2M KOH and DMSO.

DCs are stimulated with spores or yeasts, washed, supplemented with 0.1 mg/ml of NBT.

In order to assess the importance of IL-12p70 in balancing Th1/Th17 response two different experiments are performed. In one case, DCs are stimulated for 8 hours with live spores of S. cerevisiae and C. albicans hyphae at a stimuli:DC ratio of 4:1 in the presence of different concentration of human recombinant IL12p70 (0, 1, 10 and 100 ng/ml). In the second experiment, DCs are pre-incubated with different concentrations (0, 0.1, 1, 10, 100 microg/ml) of a monoclonal anti-human IL-12 antibody for 2 hours, then stimulated for 8 hours with live S. cerevisiae cel...

DCs are exposed to cytochalasin D (10 microg/ml, TebuBio) for 30 minutes at 4°C. After washing with cold PBS, DCs are stimulated with S. cerevisiae yeast cells or spores at a DC:stimuli ratio of 4:1 for 24 hours. IL-12p70 production is assessed by ELISA.

When evaluating survival after exposure, the possible effect of DMSO and DPI on the stimuli is taken into account.

Survival of yeast cells, spores or hyphae after uptake is reported as percentage of colony forming units after 3 days relative to the total number of cells growing in the absence of DCs exposure.

Cells, lysated with a hypotonic solution (KCl 0.05%), are plated on YPD.

DCs are washed.

DCs are washed.

DCs are treated with zymolyase.

DCs are collected and washed 3 times with PBS.

DCs are stimulated over 6 hours.

Serotype A C. albicans strain SC5314 is cultured overnight in Saboraud medium at 28°C and then shifted to RPMI medium for 18 hours at 37°C to allow hyphal growth. The culture is treated as for yeast cells. After microscopical inspection of the purity of hyphal culture, this is treated as for yeast cells. [It should be noted that in all the experiments performed in this study live microrganisms were used.]

In order to test homogeneous yeast populations, pure spore cultures are obtained by using this strain whose sporulation efficiency is 100%. To prepare spores, cells are grown on YPD plates, replicated on SPOIV medium (2% potassium acetate, 0.25% yeast extract, 0,1% glucose) and sporulation is assessed by optical microscopy. Zymolyase (2 mg/ml) is used to digest ascum and liberate spores. [We initially performed experiments to evaluate zymoliase sensitivity of asci for this specific strain and we assessed that 15 minutes incubation was the minimum time needed ...

Saccharomyces cerevisiae strain SK1 (MATa/alpha HO gal2 cupS can1R BIO, Kane SM and Roth J. 1974 Bacteriol. 118: 8-14) is cultured in complete medium (YPD, 2% yeast extract, 1% peptone, 2% glucose) for 18 hours, then collected, washed twice with sterile water and resuspended at 108 cells/ml. S. cerevisiae strains BY4741 (genotype, Mata his3delta1 leu2delta0 met15delta0 ura3delta0) and BY4741 och1 (Mata his3delta1 leu2delta0 met15delta0 ura3delta0 OCH1::kanMX4) are cultured in complete medium till exponentially phase and treated as before.

PBMC are stimulated in limiting dilution conditions with a pool of overlapping peptides (15 amino acids) covering the protein.