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

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

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

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.

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

In order to assess the importance of IL-12p70 in balancing Th1/Th17 response two different experiments are performed. In one case, DCs are stimulated for 8 hours with live spores of S. cerevisiae and C. albicans hyphae at a stimuli:DC ratio of 4:1 in the presence of different concentration of human recombinant IL12p70 (0, 1, 10 and 100 ng/ml). In the second experiment, DCs are pre-incubated with different concentrations (0, 0.1, 1, 10, 100 microg/ml) of a monoclonal anti-human IL-12 antibody for 2 hours, then stimulated for 8 hours with live S. cerevisiae cel...

The majority of monocytes (1 x 10e9) and all lymphocytes (6,4 x 10e9) are frozen in 90% A-plasma and 10% GMP-grade DMSO.

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.

DCs are washed.

refolded CD1 molecules are used to generate CD1 tetramers

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

Incubation at 65 °C for 12–15 h.

Day 4: harvesting (virus-containing)

On day 7 of culture 35/40 x 10e6 mature peptide pulsed DC are frozen at 5 x 10e6 cells per vial in freezing medium as above.

Fc-block for 30 min on ice

Cytokine accumulation is evaluated in the supernatants at 24h by ELISA, according to a standard protocol and it is measured at 450nm.

The CD8 T cells become evaluated on their cytokine secretion, cytotoxicity and % of antigen specific T cells.

Survival of yeast cells, spores or hyphae after uptake is reported as percentage of colony forming units after 3 days relative to the total number of cells growing in the absence of DCs exposure.

When evaluating survival after exposure, the possible effect of DMSO and DPI on the stimuli is taken into account.