For studying in vivo responses to antigens, we initiated a long program comparing the behaviour of TCR-Tg cells specific for GP-33 peptide.

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

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.

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.

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 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 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.

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

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.

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

These Treg were induced after treatment of primed mice with intact anti-CTLA-4 antibodies. These regulatory T cells inhibit Th1 responses, in vitro and in vivo, and repress experimental intestinal inflammation, by a mechanism involving IL-10 and IDO.

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

RCC product that was tested in an RCC phase 2 trial. The product does indeed lead to restoration of both IL-2 and IFN responses and leads to a gradual decline of the immune effector T cells in the peripheral circulation (i.e., weaker signal after the fifth dose). Using the PME-CD40L DC product we have observed tumor regressions in a number of patients in the current study. At present, 6 of 12 patients that have been restaged using RECIST criteria have less tumor burden that when they enrolled in the study (15 weeks earlier).

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.

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.

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.

Purified CD8 T cells using MACS cell separation were co-cultured with electroporated DC and subequently became evaluated on their cytokine secretion, cytotoxicity and % of antigen specific T cells.