The leukapheresis product is from patients with advanced (stage III or IV) melanoma. It is used to provide sources for tumor antigens as well as monocytes and T cells, which are isolated from the leukapheresis product by elutriation.

The leukapheresis product is taken from a HLA-A2 positive healthy donor and is subsequently separated into monocytes and lymphocytes.

It has been shown that DCs express specific receptors for exosomes. Since ICAM-1 on exosomes is required for their ability to activate T cells via DCs in vitro, the role of Mac-1 and LFA-1 (the major ligands for ICAM-1) as potential receptors for exosomes has been investigated. A new role for LFA-1 on DCs, as a receptor for exosomes has been proposed.

By selecting for cytokine receptor expression relevant to DC development, we were able to identify highly cycling Lin–c-KitintFlt3+M-CSFR+ cells with a distinct gene-expression profile in mouse bone marrow that, on a clonal level in vitro and as a population both in vitro and in vivo, efficiently generated plasmacytoid and conventional DCs but no other lineages, which increased in number after in vivo injection of the cytokine Flt3 ligand.

In a study aiming at the identification of molecular signatures for alternatively activated DC it was found that AA-DC induced in the presence of LPS and IL-10 also produced high levels of the long pentraxin PTX3. DC produced conspicuous amounts of PTX3 when stimulated with LPS and this production is further increased in the presence of IL-10. However, PTX3 production was not increased in other alternative conditions of activation, such as in the presence of dexamethasone or calcitriol. PTX3 has properties similar to antibodies. Its production is induced by pa...

LPS (1microgram/ml, Sigma, St. Louis, MO) (along with R848, Curdlan, yeast RNA and yeast cells in different conditions of culture) was used to induce DC activation.

Since LPS-activated DC are potent inducers of NK cell priming and lymph nodes appear to be a key place in which DC and NK cell interactions occur, we have investigated the in vivo capacity of resting and DC-primed NK cells to reach the draining lymph nodes.

CyP was extracted from the freshwater cyanobacterium Oscillatoria Planktothrix FP1. We found that CyP acts as a potent and selective antagonist of bacterial LPS.

We found that LPS-stimulated mDCs are able to induce up-regulation of co-stimulatory molecules on co-cultured pDCs. Likewise, CpG-stimulated pDCs activate co-cultured mDCs. The cross talk between these two populations of DC is very efficient since it can be observed at mDC/pDC ratio ranging from 1/10 to 10/1.

These are lymph nodes from patients that undergo surgery due to clinical situations that do not affect the immune system (most frequently malformations). We have attempted to identify and characterize CD8T lymphocyte populations in humans. We compared the distribution of phenotypically distinct cell sets using seven color staining in the blood, lymph nodes and spleen. We found that the addition of other markers allow to subdivide N, CM, TEM and TEMRA populations in additional cell types.

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.

It was found that lymph nodes that drain sites of mature DC or adjuvant inoculation recruited L-selectin- CCR7- effector and memory CD8+ T cells. This cell recruitment required CXCR3 expression on T cells and occurred through high endothelial cells (HEV) in concert with HEV luminal expression of the CXCR3 ligand CXCL9.

Cells are separated into lymphocytes (and monocytes) by elutriation in a closed system (Elutra). [The fraction richest in lymphocytes contained > 90% lymphocytes and viability was > 95%.]

A proteomics map of lysosomes purified from non-activated and activated mouse DCs was established.

This vaccine is composed of DC pulsed with 2 to 3 MAA-derived peptides (combinations of MAGE-3.A2, MAGE-C2.A2 or Na17.A2). It was tested in a trial in small cohorts of patients (3-9) who were vaccinated with differently composed DC-vaccines.

These macrophages were used to perform a transcriptional profiling. Stimulation was done with different stimuli at different time points in the presence or absence of a kinase inhibitors.

Using pharmacological inhibitors, or macrophages and DC from mice carrying a null mutation in MAP kinase signalling molecule(s), we have evidence for a role of certain MAP kinases in the regulation of IL-10 and IL-12, IFN-gamma production.

Melanoma patients were vaccinated with 4 peptides (MAGE-A3, gp100, NA17 and tyrosinase) presented by HLA-A2 and monitored. We measured responses to these peptides injected with Montanide, not knowing whether the Montanide adjuvant was important for these T cell responses. 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.

We have optimized the MAGE-A3 peptides to load onto HLA-DR*1101 tetramers.

MAGE-A3 specific CD4+ T cells were found also in a high percentage of melanoma patients but CD4+ T cells showed an unpolarized or Th2 skewed phenotype. We have optimized the MAGE-A3 peptides to load onto HLA-DR*1101 tetramers.