Hybridized slides are washed in a solution of water, 20× SSC, and 10% SDS, rinsed in water and 20× SSC, and dried via centrifugation for 2 min at 1000 rpm.

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 collected and washed 3 times with PBS.

The pellets are washed with 70% Ethanol two times.

DCs are treated with zymolyase.

Day 2: transfection

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

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

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.

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

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

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

Patients will undergo surgical removal of a metastatic lesion.