The frozen MoDC are subsequently used for tumor peptide specific stimulation of autologous T lymphocytes at a ratio of 1 DC per 10 T cells. Co-cultures led to antigen specific, IFN-? secreting T cells after 1 restimulation at a DC:T cell ratio of 1:20, as evaluated by IFN- ? Elispot, showing that the generated DC have good antigen presenting and co-stimulatory capacity.

Advanced melanoma patients are vaccinated with the DC vaccine.

Vaccination with a vaccine composed by autologous DCs pulsed with apoptotic autologous ovarian carcinoma cells. Apoptosis is induced by UV.

Patients with advanced prostate carcinoma are vaccinated with the DC vaccine.

The patients will be vaccined with the DC vaccine that consists of monocyte derived DC loaded with tumor lysate generated from the excised lesion.

DCs are treated with zymolyase.

DCs are stimulated over 6 hours.

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

This step is repeated several times to do several stimulations.

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

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

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 ...

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.

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.

Patients receive V administrations with eventual additional ones depending on clinical outcome.

The DC with blocking receptors are exposed to fungi.

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

Leukapheresis products from cancer patients is used as a starting material for monocyte enrichment by elutriation technology with ELUTRA® (Gambro).