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.

DCs are treated with zymolyase.

The DC with blocking receptors are exposed to fungi.

Autologous DC are pulsed with the apoptotic allogenic prostate carcinoma cell line LNCap to produce the vaccine for patients with advanced prostate carcinoma..

Pulsing of autologous DCs with apoptotic autologous ovarian carcinoma cells.

On day 5 of culture immature DC will be pulsed with irradiated tumor cells (apoptotic bodies) prepared from the removed lesion and matured for 48 hours in presence of TNF-alpha. Monocyte and DC viability as well as functionality will be continuously monitored.

prepared DC will be used as DC vaccine, but also to expand T cells (isolated during the elutriation) for subsequent adoptive transfer

At day 6 DC are pulsed with the peptides and KLH in the presence of TNF-alpha overnight.

When the pellets are dry, they are resuspended in RNase-free water.

Monocyte-derived DCs are added at a final concentration of 5x105 cells/ml into 96-well plates.

Serial diluition of yeast cells and preparations are added to the MoDCs.

The cells are collected and the pellets are resuspended in 3.6 ml AE Buffer (AE=50mM NaAcetate pH 5.2, 10mM EDTA) and then transferred in 15 ml tubes wit 200 µl 10% SDS (w/v). 2 ml of preheated acid phenol (pH 4.3) are added and the falcon is mixed by vortex.

On day 5 cells are counted and pulsed with tumor antigen derived peptides (one per culture) at a concentration of 5 µg/mL in approximately 5 mL and at a cell density of 20 x 10e6/mL for 1 h.

After peptide pulsation cells are diluted in medium containing 20 ng/mL TNF-?, plus IL-4 and GM-CSF as stated above.