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

Take supernatant and freeze at -80°C for analysis of DC-stimulation

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.

Before injection all cell products will be thoroughly tested to exclude contamination and presence of viable tumor cells.

After thawing cells showed good viability (>90%) and the recovery was approximately 80%.

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.

The supernatants are transferred to a new tube and 2 ml of acid phenol is added to repeat the extraction.

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.

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