The DC vaccine will consist of monocyte derived DC loaded with tumor lysate generated from the excised lesion.

Patients will undergo leukapheresis.

Such prepared DC will be used also to expand T cells (isolated during the elutriation) for subsequent adoptive transfer.

DCs will be generated from monocytes.

Leukapheresis will be performed on the following day.

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

Elutriation of the leukapheresis product.

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.

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

DCs are generated from adherent peripheral blood monocytes.

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

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

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.

HLA-A1 and/or HLA-A2 positive patients with stage III/IV melanoma undergo leukapheresis.

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

The pellets are washed with 70% Ethanol two times.

After the spin (10’ at 3,000 rpm), the supernatants are precipitated in RNase-free centrifuge tube by adding 1/10 volume 3 M NaAcetate (pH 5.3) and 2.5 volumes of cold ETOH 100% overnight at -20°C. Centrifugation (30’ at 12000 rpm).

After the centrifugation (10’ at 5000 rpm) the supernatant is transferred into a pre-spun (5’ at 1500 rpm) 50 ml Phase Lock Gel tube (Eppendorf 0032 005.330) and 4 ml of chloroform (Fischer BP1145-1) are added into the falcon.

Indirect labeling method. Briefly, the reactive amine derivative of dUTP, 5-(3- aminoallyl)-2?-deoxyuridine 5?-triphosphate (Sigma) is incorporated into cDNA using the Superscript II reverse transcriptase (Invitrogen) and oligo dT (Invitrogen) and random examers (Roche). After synthesis of cDNA (2–3 h at 42 °C), RNA is hydrolyzed by addition of sodium hydroxide and EDTA to a final concentration of 100 mM and 10 mM, respectively and incubated at 65 °C for 10 min. The hydrolysis reaction is neutralized with 1 M HEPES. After removing free nucleotides by purif...