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 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.

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.

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.

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.

Soluble recombinant HLADR1101 MHC class II molecules receptive for loading with either pathogen or tumor-derived peptides were constructed for a study of CD4+ T cell responses against pathogens or tumors in humans.

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.

We used a MHC II presentation pathway targeting vector originally published by Wang et al. 1999 for the delivery of antigens in both murine and human DC.

Co-electroporation of immature DCs with both poly(I:C(12)U) and Melan-A/MART-1 encoding mRNA induced strong anti-Melan-A/MART-1 CD8(+) T-cell responses in vitro. Higher numbers of Melan-A/MART-1-specific CTLs were consistently obtained with poly(I:C(12)U)-activated DCs compared to DCs matured in the presence of an inflammatory cytokine cocktail.

The melanin-free total tumor RNA is obtained from melanoma metastases; it is suitable for in vitro amplification and DC transfection.

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.

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.

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

Mannan (500 micrograms/ml) was added to DCs in order to block their mannan receptors.

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.

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...

Laminarin (500 micrograms/ml) was added to DCs in order to block their beta-glucan receptors.

2 mM L-glutamine (Sigma) was added to the monocyte cell culture.

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.