We investigated and obtained data on 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. Co-cultures of IFN-DCs with zoledronate-stimulated gamma delta T lymphocytes were also performed. gamma delta T-cell activation, as demonstrated by their up-modulation of activation marker expression levels (e.g. CD25 and CD69), was observed. These studies demonstrated for the first time the existence of a bidirectional activatin...

We have microarray data on macrophages and DC from different genetic backgrounds of mice, stimulated with CpG, in the presence or absence of a MEK inhibitor, and further data on the role of certain MAP kinases in the regulation of IL-10 and IL-12, IFN-gamma production. Using pharmacological inhibitors, or macrophages and DC from mice carrying a null mutation in MAP kinase signalling molecule(s), we have evidence for a role of certain MAP kinases in the regulation of IL-10 and IL-12, IFN-gamma production. The work on the role of the MAP kinases, TPL-2 and ERK in u...

Our lab has obtained data on the mechanism of how IL-10 regulates the immune response to MTb infection in mice. Control and clearance of intracellular pathogens such as MTb is dependent on the production of TNF and the induction of the T-helper 1 (Th1) cytokine IFN-gamma by IL-12. Conversely, IL-10 is a suppressive cytokine essential for dampening the immune response to a number of intracellular pathogens to limit host immune pathology. IL-10 has been shown to exert its suppressive effect by directly acting on the antigen presenting cell (APC), therefore functi...

We have been following up on the effort to generate proteomics map data of lysosomes purified from non-activated and activated mouse DCs. The decision to focus on the most important organelle for DC activity has been driven from the need to describe in greater detail the proteins involved in the DC response both qualitatively and quantitatively. The partner has been trying to quantify the relative amount of 400 proteins. They have also indexed better this list and are currently comparing it to a list of proteins present in macrophages lysosomes (in collaboration ...

Our group has obtained proteomic data on the three known splenic subsets (CD8+, CD4+, DN DC). Despite the significant advances in mass spectrometry, which has enabled much of proteomics, due to various analytical challenges so far no eukaryotic total cell proteome has been sequenced completely. An alternative approach is to purify single organelles or DC components in order to generate the proteomic data. An additional problem is to obtain highly pure fractions of DC from crude population of untouched splenic cells with a purity of 70-80%. This includes several...

Our lab has generated data on receptors on dendritic cells necessary for capture of exosomes. In 2007, we have finalized analysis of the receptors on DCs responsible for capture of exosomes secreted by mature DCs. We have shown that expression of LFA-1, but not of Mac-1, integrin, on DCs is required for capture of ICAM-1-bearing exosomes. Furthermore, the CD8+ subpopulation of DCs in lymph nodes expresses LFA-1 at higher levels than the CD8- subpopulation, and is responsible for in vivo capture of injected exosomes. We have thus proposed a new role for LFA-1 on DCs...

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. High...

Our lab has proteomic data available on the interaction between RIG-I and NS1 in influenza A. We have used fluorescence microscopy to analyse the interaction between RIG-I, a viral sensing protein, and NS1, an inhibitor of interferon production that is encoded by influenza A virus. This approach enabled us to determine that the two proteins form a complex in influenza infected cells.

We have established an in-depth proteomic map of murine dendritic cell subsets. We have been working in the attempt to overcome the limits in sample availability, exploiting the recent significant improvements of proteomic technology. The results allowed to lower the required sample amount significantly, an essential feature to analyze limited cell populations like dendritic cells. 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...

We obtained data on 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. As expected, DC up-regulated the cell surface expression of all proteins known to be part of the so-called “immunological synapse”, namely MHC class II (region E and K), CD40, CD80, CD86 and ICAM. Interestingly, LPS stimul...

Proteomic data was obtained from a proteomic analysis of DC-derived organelles such as the exosomes. Following the extensive proteomic analysis of immature and mature DC-derived exosomes, ICAM-1 was identified as the major protein on mature DC-derived exosomes, required for their functional activity. In order to functionally validate the proteomic data, the antigen-presenting function has been investigated. Exosomes bearing functional MHC-peptide complexes have been shown to be presented at the surface of recipient DCs as intact "antigen-presenting microdomains", w...

We have obtained valuable data on chemically well-defined TLR ligand conjugates and their effectiveness to induce a strong immune response via the cross-presentation pathway. We have studied long peptides which were covalently conjugated to either the TLR2 ligand or TLR9 ligand, Pam3CysSK4 or CpG, respectively have been used to study the uptake, antigen presentation, and induction of specific T-cells. A pronounced enhancement in antigen presentation by DC in vitro was facilitated by the conjugated peptides compared to peptide alone, or peptide mixed with the fre...

Preclinical data is available on the power of preventing disease and invoking immune response of our lead product for the prevention of infectious disease, ImmunoVexHSV2, a vaccine for genital herpes. ImmunoVex HSV2 is a novel live-attenuated vaccine candidate that expresses approximately 80 HSV-2 proteins intended to stimulate a broad and powerful immune response. In preclinical studies, ImmunoVex HSV2 completely prevents disease and invokes a powerful immune response. We intend to initiate a Phase I clinical trial for this product early in 2008.

In our lab, data was obtained from a phase I trial using peptide-pulsed Dex. Dendritic cell (DC) derived-exosomes (Dex) are nanomeric vesicles harboring functional MHC/peptide complexes promoting T cell -dependent tumor rejection. In this Phase I trial using peptide-pulsed Dex, the observation of clinical regressions in the absence of T cell responses prompted the search for alternate effector mechanisms. Mouse studies unraveled the bioactivity of Dex onto NK cells, Dex promoting a IL-15Ra-dependent NK cell proliferation and a NKG2D-dependent activation resulting...

We have obtained data from a phase I/II clinical trial of patients with end stage cervical cancer and of patients with vulvar intraepithelial neoplasia (VIN) grade III. Preclinical animal studies have shown that long peptides of 25-35 amino acids in length upon injection are only processed efficiently for MHC class I by dendritic cells in vivo. Long peptide injection is followed by appropriate cognate interactions between such DC, CD4+ T helper cells and CD8+ CTL precursors. Three groups of end stage cervical cancer patients (in total N=35) were subcutaneously va...

Data has been generated by multiplex analysis on the response of human MoDC to yeast, spheroplasts, pseudohyphae and spores. Human blood contains myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells that differ in cytokine secretion pattern and responsiveness to microbial stimuli. 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 ran...

Our lab has obtained data on maturation stimuli for GMP-grade DC.

Our group has produced in-vivo multi-photon microscopy imaging data on the distribution and motility of NK cells by tracking migration of DC-primed NK cells in vivo. Since LPS-activated DC are potent inducers of NK cell priming and lymph nodes appear to be a key place in which DC and NK cell interactions occur, we have investigated the in vivo capacity of resting and DC-primed NK cells to reach the draining lymph nodes. DC –activated NK cells reach the draining lymph node within 24 hours after intravenous injection and persist there for at least 72 hours. NK cell...

Multi-photon imaging data on DC-CD8+ T cell interactions in intact lymph nodes (LNs) have been obtained. The initiation of cytotoxic immune responses requires the direct interaction between naïve CD8+ T lymphocytes and dendritic cells. Multi-photon imaging showed that during priming, naïve T cells and dendritic cells (DCs) establish sequentially brief (i.e. minutes) and long (hours) antigen-specific contacts. We observed that ICAM-1 expression by mature DCs is critical for long-lasting contacts with CD8+ T cells, but dispensable for short-lived antigen-specific i...

Data were obtained on the effect of enhanced CD83 expression on dendritic cells and T cells and its correlation with effective immune responses. Human CD83 is a marker molecule for mature dendritic cells (DC) and is also expressed on activated B and T cells. Although CD83 has been implicated in immune responses, its function on DC and T cells remains unclear. In this study, we wanted to assess the role of CD83 expressed on DC and T cells in the immune response. Down-regulation of CD83 expression on human DC through RNA interference (RNAi) results in a less potent...