Sources for tumor antigens as well as monocytes and T cells will be isolated from the leukapheresis product by elutriation.

Leukapheresis products from cancer patients is used as a starting material for monocyte enrichment by elutriation technology with ELUTRA® (Gambro).

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

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

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.

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

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

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.

The DC with blocking receptors are exposed to fungi.

Fc-block for 30 min on ice

DCs are washed.

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.

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.

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.

Day 4: harvesting (virus-containing)

Incubation at 65 °C for 12–15 h.

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...