Some melanoma patients were vaccinated with the MAGE-A3, gp100, NA17 and tyrosinase peptides presented by HLA-A2 plus the montanide adjuvant, while others were vaccinated with the peptides without the adjuvant or with ALVAC canarypox virus (containing a MAGE-A3 minigene) viral vectors. There was a correlation between tumor regression and detection of anti-MAGE-3.A1 CTL responses.

The effect of environmental factors on development of high affinity CTL was investigated using a system whereby OT-1 cells were primed in vitro by engineered APC. After a 20 h priming phase, CTL were transferred to recipient mice that were either naïve, or had been injected with activated dendritic cells one day earlier, thus creating a reactive lymph node.

Subcutaneous injection of hypoxic DCs into the footpads of mice results in defective DC homing to draining lymph nodes, but enhanced leukocyte recruitment at the site of injection.

We infected mice with CD8+ T-cells primed with or without the influence of a reactive lymph node with a flu-virus encoding the specific antigen. After several days we determined the viral load in the lungs with RT-PCR. The results show that in mice with CD8+ T-cells primed with a reactive lymph node the viral load in the lungs was significantly smaller then in a control mice.

These patients were treated with monocyte-derived DCs matured with inflammatory cytokines and subsequently electroporated with mRNA encoding 6 tumor-associated antigens.

In these patients, dendritic cells were tracked for monitoring of cellular therapy by MRI. MRI cell tracking using iron oxides appears clinically safe and well suited to monitor cellular therapy in humans.

Melanoma patients were vaccinated with peptide-pulsed DC (MAGE-A3, gp100, NA17 and tyrosinase), while other patients were vaccinated with peptides +/s adjuvant or viral vectors (ALVAC canarypox virus containing a MAGE-A3 minigene). There was a correlation between tumor regression and detection of anti-MAGE-3.A1 CTL responses.

Melanoma patients were administered ALVAC canarypox virus containing a MAGE-A3 minigene, while others were vaccinated with MAGE-A3 peptide presented by HLA-A1, administered as peptide, and peptide-pulsed DCs. There was a correlation between tumor regression and detection of anti-MAGE-3.A1 CTL responses.

Anti-tumor responses were followed in melanoma patients that were vaccinated with autologous DCs loaded with a combination of tumor Ag peptides.

A significant number of patients was enrolled in the DC-based immunotherapy trial. After vaccination, vaccine-induced depigmentation and vitiligo were observed.

Small cohorts of patients (3-9) were vaccinated with differently composed DC-vaccines: DC loaded with a single MAA-derived peptide (MAGE-3.A1 or -A2 or Na17.A2) or pulsed with 2 to 3 MAA-derived peptides (combinations of MAGE-3.A2, MAGE-C2.A2 or Na17.A2) or G4-DC loaded with 8 different peptides. An important conclusion that can be made from these pilot-observations is that the potential therapeutic effect of our DC-based MAA-vaccination approach does not occur instantly but is delayed by 8-12w following the first vaccination (i.e.12-16w after the isolation o...

Patients with stage III or stage IV melanoma who undergo surgical resection of a melanotic lesion. These patients are vaccinated with pulsed and thouroughly tested dendritic cell vaccine.

DC-based vaccines that were given to these patients were combined with low dose IFN-alfa (3 x 10e6 U per wk). In a number of patients we observed vaccine-induced depigmentation and vitiligo.

The patients were vaccinated with 4 peptides (MAGE-A3, gp100, NA17 and tyrosinase) presented by HLA-A2. We measured responses to these peptides injected with Montanide, but did not know whether the Montanide adjuvant was important for these T cell responses. Indeed, melanoma patients may mount spontaneous responses to these antigenic peptides. We therefore injected the same peptides without adjuvant, and no response was observed. We conclude that Montanide had a clear adjuvant effect for the CD8 T cell responses measured against these four peptides.

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. With this approach we monitored the immune responses of 8 vaccinated patients and of more than 15 other stage IV melanoma patients.

This HSV vector expressing full length tyrosinase, gp100 and MART-1 was used to transduce dendritic cells from melanoma patients which were then to be used for vaccination.

This patient experienced an outstanding response to immunotherapy. The patient received several adoptive T cell transfers and regressed from stage IV melanoma to a disease free state and has remained tumor free for several years.

A recombinant Leishmania major parasite expressing a fluorescent tracer was used for the infection of BALB/c (H2-d) mice. This was done to investigate whether infected DC process pathogen-derived antigen and stimulate antigen-specific CD4+ T cells in vivo.

By immunizing mice with ovalbumin-conjugated anti-Siglec-H Ab in the presence of CpG, we demonstrated generation of antigen-specific CD8 T cells in vivo.

Antigen-specific CD4+ T cells were followed in these immunized individuals using generated peptide-MHC class II multimers.