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.

Yeast cells in different conditions of culture (along with R848, Curdland, yeast RNA and LPS) were used to induce DC activation.

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.

We established tumor cell lines from renal cell cancer in our laboratory.

We established tumor cell lines from pancreatic cancer in our laboratory.

We established tumor cell lines from melanoma in our laboratory.

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.

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

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.

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 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 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.

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 ...