The dead cells 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 by DC.

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

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.

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.

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.

50 mg/ml of streptomycin (Gibco BRL) containing 10% (vol/vol) FCS (Hyclone) were added to the monocyte cell culture.

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.

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

This supernatant was created during a co-culture of the stained cells and subsequently frozen at -80°C for analysis of DC-stimulation.

We demonstrated using a syngeneic mouse tumor model, expressing an Ag derived from the early region 1A of human adenovirus type 5, that the inadequate nature of the antitumor CTL response is not due to direct Ag presentation by the tumor cells, but results from presentation of tumor-derived Ag by nonactivated CD11c(+) APC.

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.

These T cells stem from an elutriation process from a metastatic lesion of stage III/IV melanoma patients. Subsequently, these T cells will be expanded by co-culture with tumor lysate loaded DC.

We have used “conventional” techniques to monitor the vaccine induced specific anti-tumor T cell responses (thymidine-incorporation, ELISA, LDA and ELISPOT assays). We have set the staining conditions for the HLA-DR*1101 tetramers loaded with tetanus toxoid and MAGE-3 peptides corresponding promiscuous CD4+ T cell epitopes. Antigen-specific CD4+ T cells are visualized after in vitro short-term expansion; we are currently optimizing the conditions for the ex-vivo staining.

We attempted to improve the adoptive transfer protocol for immunotherapy by stimulating T cells with monocyte derived DC pulsed with tumor lysate, instead of simply adding tumor lysate into PBMC cultures. T cells raised exhibited some tumor reactivity and outgrowth of a distinct T cell population could be observed.

It was found that these cells do not migrate to lymph nodes in the steady state.

By using TAP deficient cells, we found that TAP is also necessary for the process of presentation of the antigen in MHC class I. We further characterized the antigen depot by confocal microscopy and found that the antigen co-localized with the lysosomal marker LAMP1, but not with the endosomal marker EEA1, TAP, MHC class I or MHC class II. We conclude that the antigen depot is a storage compartment and not a loading compartment. We have started to examine depot formation by targeting antigen to other receptors. For example, we have found that presentation ...

These TcR-Tg clones are specific for the P14-GP33 antigen. In the individual T cells, we studied the expression of twenty different genes either mediating effector functions, or coding for different receptors involved in T cell differentiation and memory generation.

The TcR-Tg clones are specific for the 0T-1-OVA antigen. In the individual T cells, we studied the expression of twenty different genes either mediating effector functions, or coding for different receptors involved in T cell differentiation and memory generation.

Concerning « in vivo » responses to Ags, we studied the kinetics of effector genes expression and association in TCR-Tg CD8 cells responding to the male antigen and to Listeria-OVA.