These DC were matured with inflammatory cytokines and subsequently electroporated with mRNA encoding 6 tumor-associated antigens. They were used for DC-based immmunotherapy of melanoma patients.

These monocytes have been obtained from leukapheresis of tissue from patients with stage III or stage IV melanoma (tissue taken from a surgical resection of a melanotic lesion). They will subsequently be used to generate dendritic cells.

Patients underwent surgical removal of a metastatic lesion and leukapheresis at the beginning of the trial to provide sources for tumor antigens as well as monocytes and T cells, which will be isolated from the leukapheresis product by elutriation.

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.

A quantitative microarray approach that detects 723 human mature miR (miRBase 10.1, Dec 2007), was used to measure miR expression profile in these cells.

MoDC were stimulated with yeast.

Monocytes were cultured in serum free medium in the presence of IL-4 and GM-CSF, and concentrated cytokines were added.

We showed that, in the absence of adjuvants, DEX mediate potent antigen dependent-antitumor effects against preestablished tumors in mice pretreated with immunopotentiating dosing of cyclophosphamide (CTX).

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.

We have immunized mice with human DC derived-exosomes and generated hybridomas that produce antibodies recognizing at least the human exosomes.

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.

An mAb reacting to the immunodominant LACK156-173 peptide of leishmania bound to I-Ad MHC class II molecules was prepared. To this aim, we injected I-Ad/LACK recombinant dimers to TCR transgenic mice which exhibited an increased frequency of LACK-specific T cells. Four out of 600 supernatants, i.e. 2C44, 2F74, 2E60 and 2X8, readily stained LACK156-173-pulsed fibroblasts, but neither unpulsed fibroblasts nor OVA323-339-pulsed cells. BIAcore measurements yielded equilibrium dissociation constants ranging from 1.1 nM for 2C44 mAb to 120 nM for 2F74 mAb.

4 metastatic melanoma patients were actively vaccinated and additional injections were performed in disease free patients, in patients with stable disease or with slowly progressive disease.

These patients are involved in a phase II clinical trial for melanoma and are treated with OncoVexGMCSF. Data so far indicates that OncoVex can destroy both injected and un-injected melanoma deposits. The trial is still in progress.

Monocyte-derived and cocktail-matured DC electroporated with a combination of RNAs encoding tumor antigens (MelanA, Mage3, Survivin) and E/L-selectin are currently used in a clinical trial.

It was found that lymph nodes that drain sites of mature DC or adjuvant inoculation recruited memory CD8+ T cells.

These DC were used in an experiment for identification of antigens recognised by T cells from melanoma patients responding to immunotherapy.

We have generated a monoclonal antibody, 2C44, that reacted to a peptide derived from the Leishmania LACK protein bound to I-Ad MHC class II molecules. We have successfully used the 2C44 mAb to visualize LACK-presenting DCs in mice infected with the L. major.