CEA specific CD4+ T cells were found both in normal donors and in patients with high-grade cervical lesions, pancreas adenocarcinoma and advanced melanoma but CD4+ T cells from the patients compared to normal donors showed impaired IFN-gamma production.

The cell populations which infiltrate progressive versus regressing P815 mastocytoma were examined in order to identify cells which display immunosuppressive properties. We found changes in regulatory T cells populations as well as in the “myeloid suppressor cells”.

These cells are to be stained and are the input for FACS staining for activation markers.

A comprehensive protein inventory of clinical grade immature and cytokine cocktail matured (Il-6, IL-1 beta, TNF alpha, PGE2; 48 hours) monocyte derived human dendritic cells (DC) from a healthy donor has been established by using high accuracy, high sensitivity protein identification technology. We have identified 2794 proteins in DCs by liquid chromatography tandem mass spectrometry. Prior to MS analysis, DC were lysed and divided into a soluble fraction containing cytosolic proteins and into an insoluble fraction enriched for membrane containing proteins. Hi...

Our current efforts in a study of functional homogeinity involve a transition to 8 colours surface staining, and the evaluation of antigen-specific cell sets identified with MHC tetramers. For the later process, we initiated data collection of EBV, CMV and HIV specific T cell sets.

These DC were co-electroporated with constitutively active TLRs and TAA encoding mRNA. They were used to compare different maturation methods.

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.

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

The DC were matured with inflammatory cytokines followed by electroporation with TAA encoding mRNA. They were used to compare different maturation methods.

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

We decided to analyze the protein changes that occur at the cell surface of a maturing DC, induced by a prototypical maturation stimulus such as LPS. Results obtained from the LC-MS/MS analysis of the integral plasma membrane proteome of D1 cells stimulated for 24 hours with LPS have been compared to the results obtained from immature D1 cells.

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.

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

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

These cells were stained by Mini67-1KT or Mini26-1KT (PKH67/PKH26 green/red fluorescent cell linker mini kit for general cell membrane labelling, from Sigma) in order to analyse their uptake of dead cells.

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

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.

We have electroporated these DC with melanoma antigen RNA for a cross-presentation study and showed that they express the proteins ex vivo and in vivo.

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.