DCs are generated from adherent peripheral blood monocytes.

Generation of tumor lysate loaded DC.

This step of the new maturation protocol involves maturation with TNF-alpha, IFN-gamma, and PGE2.

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.

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

DCs are stimulated with spores or yeasts, washed, supplemented with 0.1 mg/ml of NBT.

After peptide pulsation cells are diluted in medium containing 20 ng/mL TNF-?, plus IL-4 and GM-CSF as stated above.

Blue formazan particles are dissolved using 2M KOH and DMSO.

Dendritic cells are electroporated.

Electroporation of autologous DCs electroporated with mRNA encoding CD40L, CD70, caTLR4 and one tumorantigen (gp100, Tyrosinase, Mage-C2 or Mage-A3) to produce the vaccine

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

Elutriation of the leukapheresis product.

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

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

The DC with blocking receptors are exposed to fungi.

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.

Day 4: harvesting (virus-containing)