Coculture of DCs and cells to be taken up (1(-2)x10^5 + 1(-2)x10^5 cells in 12-well) for 4 h to 24 h at 37°C

Untouched naive CD4+ T cells are co-coltured with fungi -pulsed DCs

Purified CD8 T cells using MACS cell separation are co-cultured with electroporated DC.

The T cell transfer will consist of T cells expanded by co-culture with tumor lysate loaded DC.

Different strains of Saccharomyces cerevisiae are cultured and collected in different conditions: different growth phase (exponential and stationary phase) different growth media (standard and promoting pseudohyphal growth) different cell form (spheroplast, spore and whole cell)

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.

DCs are generated from adherent peripheral blood monocytes.

DCs will be generated from monocytes.

Generation of tumor lysate loaded DC.

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

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

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.

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

Activated T cells are detected by their expression of CD137.

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.

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

Dendritic cells are electroporated.

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