prepared DC will be used as DC vaccine, but also to expand T cells (isolated during the elutriation) for subsequent adoptive transfer

Staining of the cells in PEalphaDC80 in PBS + 2 % FCS

Day 3: change medium and start selection for stably transduced cells

In order to test homogeneous yeast populations, pure spore cultures are obtained by using this strain whose sporulation efficiency is 100%. To prepare spores, cells are grown on YPD plates, replicated on SPOIV medium (2% potassium acetate, 0.25% yeast extract, 0,1% glucose) and sporulation is assessed by optical microscopy. Zymolyase (2 mg/ml) is used to digest ascum and liberate spores. [We initially performed experiments to evaluate zymoliase sensitivity of asci for this specific strain and we assessed that 15 minutes incubation was the minimum time needed ...

Saccharomyces cerevisiae strain SK1 (MATa/alpha HO gal2 cupS can1R BIO, Kane SM and Roth J. 1974 Bacteriol. 118: 8-14) is cultured in complete medium (YPD, 2% yeast extract, 1% peptone, 2% glucose) for 18 hours, then collected, washed twice with sterile water and resuspended at 108 cells/ml. S. cerevisiae strains BY4741 (genotype, Mata his3delta1 leu2delta0 met15delta0 ura3delta0) and BY4741 och1 (Mata his3delta1 leu2delta0 met15delta0 ura3delta0 OCH1::kanMX4) are cultured in complete medium till exponentially phase and treated as before.

When the pellets are dry, they are resuspended in RNase-free water.

DCs are washed.

CD1 molecules are refolded in vitro.

Day 1: 4 x 10^6 293 FT in 10 ml DMEM (+10 % FCS (Tet-frei), +P/S) in a 10cm plate

On day 5 cells are counted and pulsed with tumor antigen derived peptides (one per culture) at a concentration of 5 µg/mL in approximately 5 mL and at a cell density of 20 x 10e6/mL for 1 h.

Indirect labeling method. Briefly, the reactive amine derivative of dUTP, 5-(3- aminoallyl)-2?-deoxyuridine 5?-triphosphate (Sigma) is incorporated into cDNA using the Superscript II reverse transcriptase (Invitrogen) and oligo dT (Invitrogen) and random examers (Roche). After synthesis of cDNA (2–3 h at 42 °C), RNA is hydrolyzed by addition of sodium hydroxide and EDTA to a final concentration of 100 mM and 10 mM, respectively and incubated at 65 °C for 10 min. The hydrolysis reaction is neutralized with 1 M HEPES. After removing free nucleotides by purif...

Cells, lysated with a hypotonic solution (KCl 0.05%), are plated on YPD.

Cells are subsequently separated into monocytes and lymphocytes by elutriation in a closed system (Elutra). The two most monocyte-rich fractions collected contained > 80% monocytes, the fraction richest in lymphocytes contained > 90% lymphocytes and viability in both fractions was > 95%.

Monocytes are isolated from PBMC using MACS anti-CD14 microbeads and a Midi-MACS® magnetic cell sorting device (both from Miltenyi Biotec, Bergisch- Gladbach,Germany).

After the centrifugation (10’ at 5000 rpm) the supernatant is transferred into a pre-spun (5’ at 1500 rpm) 50 ml Phase Lock Gel tube (Eppendorf 0032 005.330) and 4 ml of chloroform (Fischer BP1145-1) are added into the falcon.

The monocytes are cultured for five days to immature DC (imDC´s).

Yeasts/spores are biotinylated using 10 mg/ml sulfo-NHS-LC-biotin (Sigma-Aldrich) in 50 mM NaHCO3 pH 8.5 for 2 hours at 4°C. The remaining reactive biotin molecules are inactivated by incubation in 100 mM Tris-HCl pH 8.0 for 40 minutes at 4°C. DCs are then treated with biotinylated spores/yeasts. After 1 hour, cells are permeabilized and labeled with aHLA-DR-FITC. After zymolyase treatment, intracellular yeasts/spores are detected using APC-labeled streptavidin and analyzed by flow cytometry.

Differentiation of monocytes into dendritic cells is promoted by addition of granulocyte-macrophage colony-stimulating factor (GMCSF 1000U/ml, Chemicon) and recombinant IL-4 (1000U/ml, R&D Systems) for 5 days.

Leukapheresis will be performed on the following day.

HLA-A1 and/or HLA-A2 positive patients with stage III/IV melanoma undergo leukapheresis.