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

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)

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

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.

After the spin (10’ at 3,000 rpm), the supernatants are precipitated in RNase-free centrifuge tube by adding 1/10 volume 3 M NaAcetate (pH 5.3) and 2.5 volumes of cold ETOH 100% overnight at -20°C. Centrifugation (30’ at 12000 rpm).

The samples are centrifugated at 5000 rpm in an tabletop centrifuge.

Day 1: seed cells

Day 3: change of medium

A portion of pulsed and unpulsed mature dendritic cells will be frozen and used to monitor immunological responses elicited by the DC vaccine and transferred T cells during the clinical trial.

Cells are cultured in RPMI 1640 medium (GIBCO BRL) supplemented with 2 mM L-glutamine (Sigma), 1% (vol/vol) non-essential amino acids, 100 mM sodium pyruvate, 50 U/ml of penicillin and 50 mg/ml of streptomycin (Gibco BRL) containing 10% (vol/vol) FCS (Hyclone).

Autologous DC are pulsed with the apoptotic allogenic prostate carcinoma cell line LNCap to produce the vaccine for patients with advanced prostate carcinoma..

Pulsing of autologous DCs with apoptotic autologous ovarian carcinoma cells.

Serotype A C. albicans strain SC5314 is cultured overnight in Saboraud medium at 28°C and then shifted to RPMI medium for 18 hours at 37°C to allow hyphal growth. The culture is treated as for yeast cells. After microscopical inspection of the purity of hyphal culture, this is treated as for yeast cells. [It should be noted that in all the experiments performed in this study live microrganisms were used.]

2 x 200 x 10e6 monocytes are transferred into culture bags by sterile welding and cultured for 5 days at a density of 2.5-5 x 10e6 cells/mL in serum free medium (CellGro, Cell Genix) in the presence of 100 ng/mL GM-CSF (Leukine, Berlex) and 20 ng/mL IL-4 (Cell Genix). After 2 days one volume fresh medium containing 2x concentrated cytokines is added.

The cells are collected and the pellets are resuspended in 3.6 ml AE Buffer (AE=50mM NaAcetate pH 5.2, 10mM EDTA) and then transferred in 15 ml tubes wit 200 µl 10% SDS (w/v). 2 ml of preheated acid phenol (pH 4.3) are added and the falcon is mixed by vortex.

Serial diluition of yeast cells and preparations are added to the MoDCs.

The yeast oligonucleotide array is constructed using the S. cerevisiae Genome Oligo Set ™ (Operon Technologies, CA, USA) composed of 6240 optimized oligonucleotides (70mers) each representing one yeast gene.

The polarization is assessed by flow cytofluorimetry and cytokine production.