We have identified some genes differentially expressed in different stimulation conditions. In particular, the most relevant gene upregulated in presence of stimuli typically inducing Th1 responses (LPS, CpG, Poly I:C) but not in presence of the Th2 stimulus, Pam3Cys, was Stat-1.

10 mg/ml sulfo-NHS-LC-biotin (Sigma-Aldrich) in 50 mM NaHCO3 pH 8.5 were used to biotinylate yeasts / spores for 2 hours at 4°C. The remaining reactive biotin molecules were inactivated by incubation in 100 mM Tris-HCl pH 8.0 for 40 minutes at 4°C. DCs were then treated with biotinylated spores/yeasts.

These cells were stained with anti-CD11c antibodies for a process of dead cell uptake by DC.

The D2SC1/Flt3-DC were stained (Mini67-1KT or Mini26-1KT (PKH67/PKH26 green/red fluorescent cell linker mini kit for general cell membrane labelling) from Sigma) for a process of dead cell uptake.

Migration of DCs was studied in vivo in melanoma patients exploring MRI. From isolated lymphnodes obtained after surgery we could identify single DC after staining with Prussian blue for iron. We are in the process of analysing the T cell rosettes (activation stage) that surround these DC to get insight in the functional activity.

It was evaluated whether STAT-1 was phosphorylated in DC following activation with TLR-dependent Th1 and Th2 stimuli and we found that only in presence of TLR stimuli able to induce Th1 responses STAT-1 was activated.

This supernatant contains the infective MAGE-3 encoding viruses and the natural tumour peptides and is needed for metastatic melanoma vaccine production.

iNKT cells were stimulated in vivo with the synthetic CD1d ligand alpha-GalCer. This significantly enhanced immune responses to protein and peptide based vaccines, due to rapid iNKT dependent DC maturation.

These plasmacytoid pDC that were stimulated with both CpG and the TLR-7 ligand R848 were shown to produce very high levels of IL-12p70 (in the ng/ml range per 5 x 105 cells/ml) and IFN-gamma.

Cells stained in PE-alpha DC80 in PBS + 2 % FCS for FACS analysis for activation markers.

These inhibitors were used to test the role of Src kinases in DCs during synergic engagement of TLR8 with either TLR4 or TLR3.

These stage III/IV melanoma patients underwent surgical removal of a metastatic lesion and lymphodepletion prior to vaccination and adoptive transfer of autologous T cells.

These patients receive DC transfected with RNA encoding MelanA, Mage3 and Survivin +/- E/L-selectin by the i.v. route. 2nd phase of the DERMA-ER-DC 06 trial.

Src kinases were found to be required for the initiation of some maturation characteristics of human monocytes derived dendritic cells upon stimulation with several toll like receptor (TLR) agonists but not of others, since their inhibition was able to dissociate cytokines and chemokines production from the induction of surface maturation markers.

In order to test homogeneous yeast populations, pure spore cultures were obtained by using this strain whose sporulation efficiency is 100%. To prepare spores, cells were grown on YPD plates, replicated on SPOIV medium (2% potassium acetate, 0.25% yeast extract, 0,1% glucose). Zymolyase (2 mg/ml) was used to digest ascum and liberate spores. Zymoliase was inactivated by heat (65°C for 2 minutes) and washed away carefully together with the remainings of the empty ascus, by resuspending twice the spore culture in 500 µl of distilled water centrifuging and disc...

Studies were carried out demonstrating that DC and other cell types can be activated in vitro by transfection with single stranded viral or synthetic RNA containing 5’ phosphates.

This vaccine consists of DC loaded with a single MAA-derived peptide (MAGE-3.A1 or -A2 or Na17.A2). It was tested in a trial in small cohorts of patients (3-9) who were vaccinated with differently composed DC-vaccines.

Shuttle plasmids into which antigens are inserted are available from a discontinued clinical program where an HSV vector expressing full length tyrosinase, gp100 and MART-1 was used to transduce DC from melanoma patients which were then to be used for vaccination. These could be of use to the project if partners would like to use HSV-based technology for the delivery of antigens to DC.

We have chemically coupled the S-epimer of the Pam3CSK4 to long peptides containing a CTL epitope and used this structure to investigate the behaviour of the diastereomer.

We have investigated the in vivo capacity of resting (and of DC-primed) NK cells to reach the draining lymph nodes.