Take supernatant and freeze at -80°C for analysis of DC-stimulation

DCs are stimulated over 6 hours.

This step is repeated several times to do several stimulations.

Patients will undergo surgical removal of a metastatic lesion.

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

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

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

CD1 molecules are refolded in vitro.

All the individual microcultures are then screened for recognition of autologous EBV-B cells transduced with a retrovirus encoding the protein. Non transduced EBV-B cells are used as control stimulator cells.

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.

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 ...

DCs are washed.

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...

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.

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

Immature DCs are allowed to adhere onto fibronectin and subsequently incubated with FITC-labeled yeast or spores for 5, 15, 30 minutes at 37°C. At the end of the incubation period, the samples are fixed in 4% PFA, permeabilized in Methanol, and labeled for DC-SIGN and/or MR using specific mAb and isotype-specific fluorescent secondary Abs. Samples are analyzed using a Zeiss LSM 510 confocal microscope. Alexa647-conjugated goat-anti-mouse IgG, and Alexa568 goat-anti-mouse IgG2b from Molecular Probes.

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).