These constructs were used to transfect immature DCs.

DCs were exposed to cytochalasin D (10 microg/ml, TebuBio) for 30 minutes at 4°C.

We have prepared several new constructs for the expression of (modified) adhesion receptors in DC.

This cytokine cocktail was used to mature human monocyte-derived dendritic cells, on which subsequently, transcriptional profiling was performed.

The cultured cells were subsequently pulsed with tumor antigen derived peptides (one per culture) at a concentration of 5 µg/mL in approximately 5 mL and at a cell density of 20 x 10e6/mL for 1 h.

D1 cells will be stimulated with LPS and harvested at different time-points for GeneChip analysis and for tandem mass spectrometry-based analysis of the integral plasma membrane proteome.

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.

Cytotoxic T cells proliferation was observed in human Hemato-Lymphoid System Rag2-/-gc-/- mice after being infected with EBV and mounting an immune response.

DC pulsed with tumor antigens were evaluated for their immunotherapy potential in EG7-OVA and P815 tumor models. Depletion of natural regulatory T cells in tumor bearing mice resulted in rejection of P815 cells, but not EG7-OVA, suggesting that regulatory T cells have a stronger impact on immune responses against weakly immunogenic or auto-antigen (P1A).

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

The cDNA’s encoding these early expressed HIV antigens have been human codon optimized and modified with lysosomal targeting sequences.

These DC still have functional receptors and are exposed to the receptor antagonist laminarin in order to block them.

100 micrograms/ml curdlan (Wako) was used (along with R848, LPS, yeast RNA and yeast cells in different conditions of culture) to induce DC activation.

Monocytes were cultured in RPMI 1640 medium (GIBCO BRL) supplemented with 2 mM L-glutamine (Sigma), 1% (vol/vol) non-essential amino acids, 100 mM sodium pyruvate, 50 U/ml of penicillin and 50 mg/ml of streptomycin (Gibco BRL) containing 10% (vol/vol) FCS (Hyclone).

The kinetic of the transcriptional profiling of D1 cells treated with either DMSO or LPS was analysed.

CX3CR1 GFP mice, in which bmDC are green fluorescent (GFP), were analyzed according to their bmDC localization. It was found that the cells were organized in unique clusters, mainly located in the endosteum of the BM. These DC co-localize with B cells and these clusters are occupying architecturally definable niches and are reminiscent to the niches that were found in the cranial BM parenchyma.

DC exosomes along with various TLR ligands or cytokines such as IL-2 or alpha IFN were used to promote Mart-1 specific T cell priming and/or NK cell triggering in HHD2 mice. We demonstrated that only TLR3 and 9 ligation is able to induce CTL priming in vivo. However, exosomes in the absence of adjuvants can induce NK cell activation.

The kinetic of the transcriptional profiling of D1 cells treated with either DMSO or LPS was analysed.

These HIV strains were used to infect Human Hemato-Lymphoid System Rag2gc-/- mice to closely resemble HIV infection in humans.

These animals were used to test the combined effects of these cytokines on DC development in vivo.