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.

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

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.

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

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

Activated T cells are detected by their expression of CD137.

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

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

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.

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.

The DCs are treated with the receptor antagonist laminarin, with chitin and mannan.

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.

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

DCs activation is induced by lipopolisaccaride (LPS 1microgram/ml, Sigma, St. Louis, MO), by Curdlan (100 micrograms/ml, Wako), by R848 and yeast RNA and by yeast cells in different conditions of culture.