The moDCs are used in the DERMA-ER-DC 05 trial. They are multipeptide loaded cytokine matured moDC + prior Treg elimination by 3x ONTAK treatment [5µg/kg]. 8 patients are enrolled and receive this substance.

In the process of total RNA extraction from yeast cells, the supernatants were precipitated in RNase-free centrifuge tube by adding 1/10 volume 3 M NaAcetate (pH 5.3) and 2.5 volumes of cold ETOH 100%.

The role of IL-10 in the immune response to MTb was studied. We have identified that IL-10 plays an early and transient role in the control of the immune response to MTb and now show that this involves enhanced migration of IFNgamma producing CD4+ and CD8+ T cells into the lung rather than enhanced mycobacterial killing. The effects are accompanied by enhanced G-CSF, GM-CSF, TNF, IL-6 and IL-17 levels although the enhanced clearance of bacilli in IL-10 deficient mice we now show is independent of IL-17.

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, but did not know 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.

The expression and function of these murine FcgammaR in CD11c+CD11b-B220+ plasmacytoid DCs (pDCs) was investigated. pDCs express mostly FcgammaRIIB while the expression of FcgammaRI and FcgammaRIII is only detected by RT-PCR at low but significant level. Moreover, the ITAM-containing intracellular chain associated to FcgammaRI and FcgammaRIII is strongly expressed in pDCs as detected by biochemical assay.

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.

When we tested in therapeutic tumor experiments with OVA+ melanoma cells, direct administration of lentiviral vectors slowed down tumor growth to a comparable extent with the highest dose of ex vivo transduced DC.

1% (vol/vol) non-essential amino acids were added to the monocyte cell culture.

This is mRNA encoding CD40L, CD70, caTLR4 and one tumorantigen (gp100, Tyrosinase, Mage-C2 or Mage-A3). It was used to electroporate autologous dendritic cells in order to produce a vaccine for advanced melanoma patients.

We set an in vitro system to study ex-vivo responses able to detect fona fide in vivo primed CD4+ T cells. We investigated spontaneous CD4+ T cell responses to these antigens in normal donors and in patients with high-grade cervical lesions, pancreas adenocarcinoma and advanced melanoma. The antigens used were: i) the E6 and E7 proteins of human papilloma viruses, ii) the carcinoembryonic antigen (CEA) and iii) the tumour specific antigen MAGE-A3.

We found that BM-myeloid and splenic CD8alpha+ DC produced at least fivefold less IL-12p70 than plasmacytoid pDC responding to both CpG and the TLR-7 ligand R848.

We found that CD8alpha- DC produced undetectable levels of IL-12p70 upon stimulation with CpG.

These multimers were provided by P. Coulie and used to establish routine monitoring of tumor-specific T-cell subsets by multicolor flow cytometry.

We have efficiently transfected DC with RNA encoding a functional protein (E/L-Selectin). The mouse E/L-S transfected DC, when given i.v., homed to the lymph nodes, whereas non transfected DC home only to the spleen.

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.

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.

The animal(s) were immunized in vivo with in vitro matured DC and / or treated using a maturation signal.

Cells from healthy blood donors are separated into monocytes (and lymphocytes) by elutriation in a closed system (Elutra). [The two most monocyte-rich fractions collected contained > 80% monocytes, viability was > 95%.]

These monocyte come from an elutriation process from a metastatic lesion from stage III/IV melanoma patients and will be used for the generation of a vaccine and generation of tumour-lysated loaded DC.

Melanoma patients were vaccinated with peptides (MAGE-A3, gp100, NA17 and tyrosinase), with and without montanide as adjuvant. No response was observed when vaccinating without the adjuvant. We conclude that Montanide had a clear adjuvant effect for the CD8 T cell responses measured against these four peptides.