For a transcriptional profiling of dendritic cells, DC were stimulated with PolyI:C, pretreated or not with PP2, and microarray analysis was performed.

These DC were generated from monocytes after elutriation of leukapheresis product (patients with stage III or stage IV melanoma) and pulsed with irradiated tumor cells (apoptotic bodies) prepared from the removed lesion and matured for 48 hours in presence of TNF-alpha, and subsequently frozen.

A portion of the DCs is pulsed with irradiated tumor cells (apoptotic bodies) prepared from the removed lesion and matured for 48 hours in presence of TNF-alpha.

The kinetic of the transcriptional profiling of purified mouse DC treated with IL10 was analysed.

We have studied the immuno-physiology of rat and mouse intestinal dendritic cells. We have used a known adjuvant, E. coli heat-labile toxin (Etx) and a potential adjuvant R-848, a small TLR7/8 ligand.

A vaccination trial with RNA transfected autologous DC was done with overlapping peptides covering the whole protein sequence of the tumor antigens MageA3, MelanA and Survivin.

The capacity of splenic DC, pulsed in vitro with protein antigens or peptides, to induce immunity was monitored after transfer into syngeneic animals. To improve existing strategies, the amplitude and polarisation of T cell responses was monitored in the presence or absence of regulatory T cells.

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.

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

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.

The immature monocyte-derived dendritic cells where transduced with high doses of lentiviral vectors to monitor activation of the immature DC.

The DC are transfected with RNA encoding MelanA, Mage3 and Survivin +/- E/L-selectin. They are used in the DERMA-ER-DC 06 trial.

Immature DC (imDC´s) are transfected with different constructs encoding for the oncogene Her-2/neu and as control PSA. The technology of Amaxa biosystems or an adeno virus Her-2 full length construct are used.

We have efficiently transfected DC with RNA encoding a functional protein (E/L-selectin), which allows entry of DC into LN from HEV. These DC rolled in vitro on sialyl-LewisX-coated slides, and in vivo, mouse E/L-selectin-transfected DC homed to LN after i.v. application.

These monocyte-derived dendritic cells are loaded with tumor lysate generated from the excised metastatic lesion of stage III/IV melanoma patients. They will be used for vaccine generation.