DCs are exposed to cytochalasin D (10 microg/ml, TebuBio) for 30 minutes at 4°C. After washing with cold PBS, DCs are stimulated with S. cerevisiae yeast cells or spores at a DC:stimuli ratio of 4:1 for 24 hours. IL-12p70 production is assessed by ELISA.

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.

After 10’ of incubation in 65 °C water bath, the samples are incubated on ice.

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

Incubate cells in Fc-block (1:50 in PBS + 2 % FCS) 30 min on ice

Day 2: remove the medium from the cells. Mix the medium containing virus gently by pipetting and add to the cells (+ 6 migrograms/ml of polybrene). Incubate the cells at 37°C overnight.

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

PBMC are isolated from buffy coats by density gradient centrifugation using Biocoll (BIOCHROM).

After 2 rounds of in vitro stimulation, the microcultures are left without stimulation for 2 weeks.

A HLA-A2 positive healthy donor undergoes leukapheresis.

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

Leukapheresis will be performed on the following day.

Patients will undergo leukapheresis.

Patients will undergo lymphodepletion prior to vaccination and prior to adoptive transfer of autologous T cells.

CD8 T cells are purified using MACS cell separation.

Absorbance is measured using a microplate reader at 620 nm.

Monitoring of immunological responses elicited by the DC vaccine and transferred T cells during the clinical trial

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.

The monocytes are cultured for five days to immature DC (imDC´s).