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.

DCs are washed.

Day 3: change medium and start selection for stably transduced cells

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 ...

Staining of the cells in PEalphaDC80 in PBS + 2 % FCS

Staining of D2SC1/Flt3-DC and staining of the cells to be taken up by Mini67-1KT or Mini26-1KT (PKH67/PKH26 green/red fluorescent cell linker mini kit for general cell membrane labelling) from Sigma

Staining of GM-DC/Raw with anti-CD11c antibodies

This step is repeated several times to do several stimulations.

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.

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

Patients will undergo surgical removal of a metastatic lesion.

Patients with stage III or stage IV melanoma will undergo surgical resection of a melanotic lesion.

Transfection of CD40L-encoding RNA along with the tumor RNA payload.

Day 2: transfection

Transfection of immature DCs with different constructs encoding for the oncogene Her-2/neu and as control PSA using the technology of Amaxa biosystems or an adeno virus Her-2 full length construct.

DCs are treated with zymolyase.

Trypsinize cells and fix in 4 % PFA (or formalin)

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.