Treatment induced T cell responses will be evaluated by flow cytometry based methods (T cell phenotype, proliferation, perforin degranulation) and cytokine detection (Elispot, Elisa). The patients will be monitored for safety and toxicity of the treatment and for clinical response to immunotherapy.

A portion of pulsed and unpulsed mature dendritic cells will be frozen and used to monitor immunological responses elicited by the DC vaccine and transferred T cells during the clinical trial.

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

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

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.

DCs will be generated from monocytes.

The patients are monitored for safety and toxicity of the treatment and for clinical response. T cell stimulatory activity of DC is monitored by induction of KLH specific T cells, tumor specific responses are evaluated by induction of SK-Mel 24 peptides specific T cells.

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

The next day the peptides and KLH are replaced and incubated for further 4h and then used for quality controls and intradermal administration.

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

DCs are generated from adherent peripheral blood monocytes.

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

Data are normalized to mean ratio intensity.

Intensity values are adjusted by subtracting surrounding background from spots. The median of spot intensities is corrected for background. To eliminate signals that are most prone to estimation error, any spot is excluded from analysis if both the Cy3 and Cy5 mean fluorescence signals are within two standard deviations of the mean background signals for that spot. This procedure avoids artificially inflated measurements of expression due to low signals. Additional to eliminating flagged spots, spots are also visually inspected and flawed ones discarded from the analysis.

The arrays are scanned immediately. Each comparison is performed in duplicate. Fluorescent cDNA bound to the microarray is detected with a GenePix 4000 microarray scanner (Axon Instruments, Foster City, CA), using the GenePix 4000 software package to quantify microarray fluorescence.

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.

Incubation at 65 °C for 12–15 h.

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...