The mature peptide pulsed DC were frozen at 5 x 10e6 cells per vial in freezing medium for subsequent stimulation of autologous T cells.

The tumor antigen derived peptide pulsed monocyte-derived DC were diluted in medium containing 20 ng/mL TNF-?, plus IL-4 and GM-CSF.

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.

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

We have used “conventional” techniques to monitor the vaccine induced specific anti-tumor T cell responses (thymidine-incorporation, ELISA, LDA and ELISPOT assays). We have set the staining conditions for the HLA-DR*1101 tetramers loaded with tetanus toxoid and MAGE-3 peptides corresponding promiscuous CD4+ T cell epitopes. Antigen-specific CD4+ T cells are visualized after in vitro short-term expansion; we are currently optimizing the conditions for the ex-vivo staining.

We have demonstrated that the target of FcgammaRs with the antigen induces the internalization of the immune complex, leading to the maturation of CD11c+CD11b+ conventional DCs. These DCs become thus efficient to present antigenic peptides to helper CD4 T cells and cytotoxic CD8 T cells via cross-presentation pathway.

We used specific inhibitors to study the role of src-family tyrosine kinases in the maturation of human monocyte derived dendritic cells upon stimulation with several toll like receptor (TLR) agonists. The effect of these kinase inhibitors on the capacity of DC to be activated by a TLR2 (PAM3CSK4), TLR3 (Poly I:C), TLR5 (Flagellin), and TLR8 (Poly U) agonist was evaluated.

We studied the correlation between CD8 multiple cell surface markers and functional profiles studied at single cell level. For that purpose, we subdivided peripheral CD8 T cells into eleven different cell subtypes based on the association of multiple cell surface markers. In each subtype, we isolated single-cells. In each single cell, we quantified the expression of multiple genes. Moreover, we isolated and studied cells from different normal donors.

We investigated the interactions between human monocyte derived DCs (MDDC), generated in the presence of GM-CSF and IL-4 (IL-4 DC), and antigen-stimulated circulating gamma delta T lymphocytes, bearing the Vgamma2 TCR. The studies demonstrated for the first time the existence of a bidirectional activating interaction between DCs and gamma delta T lymphocytes activated by aminobiphosphonates. These data suggest a potential adjuvant role of this early cross-talk in the therapeutic activity of aminobiphosphonate drug that are currently used as anti-tumor drugs.

We examined whether plasmacytoid pDC are infected and/or activated by MTb, and whether they play a role in regulating bacterial clearance during acute MTb infection and if so how. We have compared these DC with myeloid DC and macrophages with respect to MTb infection.

The crude population of DC is divided into the three known splenic subsets (CD8+, CD4+, DN DC) using Fluorescence activated cell sorting (BD FACS Aria). This procedure allowed to obtain highly pure fractions of each subset. However, because DC are a very rare population, this method is very material- and cost-intensive. Furthermore, it is not surprising that the obtained cell numbers of each subset after isolation are below 107. However, these cell numbers should be sufficient for proteomic investigations. The proteomes of all three subsets using mass spectrometry is in progress.

The crude population of DC is divided into the three known splenic subsets (CD8+, CD4+, DN DC) using Fluorescence activated cell sorting (BD FACS Aria). This procedure allowed to obtain highly pure fractions of each subset. However, because DC are a very rare population, this method is very material- and cost-intensive. Furthermore, it is not surprising that the obtained cell numbers of each subset after isolation are below 107. However, these cell numbers should be sufficient for proteomic investigations. The proteomes of all three subsets using mass spectrometry is in progress.

The crude population of DC was divided into the three known splenic subsets (CD8+, CD4+, DN DC) using Fluorescence activated cell sorting (BD FACS Aria). This procedure allowed to obtain highly pure fractions of each subset. However, because DC are a very rare population, this method is very material- and cost-intensive. Furthermore, it is not surprising that the obtained cell numbers of each subset after isolation are below 107. However, these cell numbers should be sufficient for proteomic investigations. The proteomes of all three subsets using mass spectrometry is in progress.

The group completed the measurements of murine dendritic cell subsets and established an in-depth proteomic map of them. These preliminary results show more than 4500 identified proteins in each subset. All of the known markers like CD11c, CD11b, DEC-205, Langerin, etc. were found according to their differential expression pattern.

DCs pre-loaded in vitro with ICs are at least 1000-fold more potent than ICs injected directly into mice or DCs loaded with the same amount of non-complexed protein. FcgammaRs on DCs were required for efficient priming of Ag-specific CD8+ cells in vivo and induction of tumor protection.

We studied the response of human MoDC to yeast, spheroplasts, pseudohyphae and spores. Analysis of IL-6, IL-8, TNF?, IL-1?, IFN?, IL-10, IL-12p70, IL-12p40 secretion was performed with a multiplex assay.

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.

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.

The capacity of splenic DC, pulsed in vitro with protein antigens or peptides, to induce immunity was monitored after transfer into syngeneic animals. To improve existing strategies, the amplitude and polarisation of T cell responses was monitored in the presence or absence of regulatory T cells.

Down-regulation of CD83 expression on human DC through RNA interference (RNAi) results in a less potent induction of allogeneic T cell proliferation, reduced IFN-gamma secretion by established T cells and decreased capacity in the priming of functional tumor antigen-specific CD8+ T lymphocytes. In addition, CD83 mRNA-electroporated DC are stronger T cell stimulators. However, CD83 overexpression on Melan-A/MART-1-specific tumor-infiltrating lymphocytes (TIL) circumvents the need for CD83 expression on DC. Co-culture of immature DC with TIL or K562 cells overex...