These T cells were isolated during elutriation of a leukapheresis product (patients with stage III or stage IV melanoma).

These T cells were obtained from isolation during elutriation and were used for subsequent adoptive transfer.

DCs were pulsed with allogenic melanoma peptides (SK-Mel 24, HLA-A1 and HLA-A2 positive) plus KLH as immunological tracer and subsequently used to produce a vaccine for melanoma.

These T cells are to be purified using MACS cell separation to contain only CD8+ T cells. The latter are then co-cultured with electroporated DC, stimulated and become evaluated on their cytokine secretion, cytotoxicity and % of antigen specific T cells.

Untouched naive CD4+ T cells are co-coltured with fungi -pulsed DCs to prime naive T cells.

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

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

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.

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.

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.

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

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.

We have determined mechanisms for DC-induced Th1 development and the molecular pathways for inducing Th1 cells producing IFN-gamma solely, or Th1 cells producing IFN-gamma and IL-10, which have important implications for regulation of the immune response to eradicate pathogens with minimum pathology.

We have determined mechanisms for DC-induced Th1 development and the molecular pathways for inducing Th1 cells producing IFN-gamma solely, or Th1 cells producing IFN-gamma and IL-10, which have important implications for regulation of the immune response to eradicate pathogens with minimum pathology.

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.

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.

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 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 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.

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