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

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.

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.

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.

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.

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 ...

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.]

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

The DC with blocking receptors are exposed to fungi.

CD8 T cells are purified using MACS cell separation.

Untouched naive CD4+ T cells are co-coltured with fungi -pulsed DCs

Purified CD8 T cells using MACS cell separation are co-cultured with electroporated DC.

This step is repeated several times to do several stimulations.

Dendritic cells are electroporated.