Data has been obtained on the role of Src kinases in DCs during synergic engagement of TLR8 with either TLR4 or TLR3. Using specific inhibitors we have previously found that src-family tyrosine kinases are required for cytokines and chemokines production during maturation of human monocyte-derived dendritic cells (DCs) upon stimulation with several toll like receptor (TLR) agonists. We have now evaluated the role of Src kinases in DCs during synergic engagement of TLR8 with either TLR4 or TLR3. We tested if src-family tyrosine kinases inhibitors had any effect on...

Data were obtained from an ex-vivo analysis of different peptide specific T cell functions like degranulation and production of cytokines such as IFN? and TNF? by 8-color flow cytometry. In general, pre-existing immune-responses were rather mono-functional with dominating “IFN?-only”, “TNF?-only” and “degranulation-only” cells in the CD8+ compartment. Vaccination not only increased some of these monofunctional subpopulations but also induced occurrence of tumorantigen specific polyfunctional subsets. The further course of our melanoma patients will sho...

We will explore the immunogenicity of necrotic cells as a tool to dissect the molecular determinants involved in “danger” recognition and will obtain data on this.

We have further data available on the expression and function of FcgammaR in mice. We had previously shown that conventional DCs express the inhibitory type II FcgammaR and the activatory type I and type III FcgammaRs. Therefore, we next investigated the expression and function of these murine FcgammaR in CD11c+CD11b-B220+ plasmacytoid DCs (pDCs). 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 Fcg...

Data has been obtained on expression and regulations of Notch ligands on DC subsets.

Flow cytometric analysis data were obtained of translation activation in DC-activated T cells.

We have obtained data by fluorescence microscopy analysing DC distribution in spleen and how this is affected during inflammation driven by stromal cells or hematopoietic cells. One of our images of DC was selected as the cover for the June 2007 issue of Nature Immunology.

We obtained data on anti-tumor responses in melanoma patients that were vaccinated with autologous DCs loaded with a combination of tumor Ag peptides. This study is a collaboration with the lab of Didier Colau and Thierry Boon in Brussels. HLA-DP4/MAGE3 molecules have been prepared to stain ex vivo PBL from immunized melanoma patients.

Data was obtained on CD4+ T cell responses against pathogens or tumors in humans. Our goal was to generate peptide-MHC class II multimers to follow antigen-specific CD4+ T cells in immunized individuals. The study was a collaboration with Monica Moro in P. Dellabona’s lab. to study CD4+ T cell responses against pathogens or tumors in humans. Soluble recombinant HLADR1101 MHC class II molecules receptive for loading with either pathogen or tumor-derived peptides were constructed. Although all these molecules could stain CD4+ T cells that had been stimulated for f...

Work has continued to characterise the function of a novel tick-derived DC modulator that perturbs various signalling pathways of human DC but not others; transcriptional profiling and pathways-based analysis will be undertaken in the next phase and data will become available.

We obtained data from studies of CD8 T cell properties in man comparing HIV, CMV and EBV-specific cells. It is usually assumed that effector gene expression in CD8 T cells may be similar in all immune responses, CD8 T cells in vivo differentiating into cytotoxic T cells that also produce g-inf. In contrast we published in 2007 that CD8 follow several differentiation waves and that effector gene expression is heterogeneous, different effector genes having different kinetics of expression-down regulation and associating randomly, suggesting that different immune re...

In our lab, we have gene expression data in T cells available in responses to LCMV-GP33 protein and listeria-Ova. We studied TcR-Tg clones specific of each antigen (P14-GP33 specific, 0T-1-OVA specific), as well as endogenous polyclonal T cells, specific for the same antigens, identified by MHC pentamers loaded with specific peptides. In each individual T cell, we studied the expression of twenty different genes either mediating effector functions, or coding for different receptors involved in T cell differentiation and memory generation. We were surprised to obs...

We are currently performing gene silencing experiments in human monocyte-derived DCs (MDDCs) with the dual aim of generating relevant knowledge and data on (i) the transcriptional regulation of DC differentiation, and (ii) the safe and rationale in vitro manipulation of gene expression in DCs. One of the two different approaches that have been set up in parallel is: Lentiviral transduction of shRNAs: to this aim, shRNA specific for IRF4 and STAT3 are in the process to be cloned into the pLVTHM lentiviral vector (obtained from the repository Addgene). This v...

We are currently performing gene silencing experiments in human monocyte-derived DCs (MDDCs) with the dual aim of generating relevant knowledge and data on (i) the transcriptional regulation of DC differentiation, and (ii) the safe and rationale in vitro manipulation of gene expression in DCs. One of the two different approaches that have been set up in parallel is: Transient transfection of synthetic siRNA: to this aim, commercially available siRNA Smart Pool from Dharmacon targeted against the STAT3 transcription factor have been successfully transfected i...

The goal of this initiative is to obtain sufficient genomic data and a reference data set from retrospective studies of a large number of stored human DC samples to understand the genetic basis of inter-donor variability in responses to DC maturation stimuli. ‘Illumina’ microarrays rather than the ‘Affymetrix’ platform will be used for the necessary transcriptional profiling and subsequent data analysis. Jointly, P2 and P6 identified a large number of stored samples of DC from melanoma patients (who had been treated in clinical trials of DC-based therapy ...

High-throughput data is available on tumour-associated antigen (TAA) specific T-cells in breast cancer patients using the Becton Dickinson Lyoplate™ technology. Therefore we have established multiparameter assessments of antigen-specific CD4+ and CD8+ T-cells enabling a simultaneous quantitative and qualitative analysis.

Data has been obtained on the homing capacity of DC transfected with RNA encoding E/L-Selectin. We have efficiently transfected DC with RNA encoding a functional protein (E/L-Selectin) which allows entry of DC into LN from HEV. Mouse E/L-S transfected DC, when given i.v., homed to LN, whereas non transfected DC home only to the spleen. The novel adherence capacity of human E/L-S transfected DC was demonstrated via sticking to sialyl-LewisX coated slides using a parallel plate flow microscope. RNA transfected human DC could be frozen and thawed without loosing th...

First clinical vaccination data has been obtained using an HSV vector expressing full length tyrosinase, gp100 and MART-1 to transduce DC from melanoma patients. Biovex has developed HSV-based vectors optimised for antigen delivery to dendritic cells by the deletion of HSV genes which usually inhibit DC function. These deliver antigens to DC at very high efficiency and activate the transduced cells similarly to LPS. Based on this technology, Biovex began a clinical program where an HSV vector expressing full length tyrosinase, gp100 and MART-1 was used to transdu...

Data has been obtained on TriMix DCs co-electroporated with whole tumorantigen encoding mRNA. A critical factor determining the effectiveness of currently used dendritic cell (DC)-based vaccines, is the DC’s activation or maturation status. We have shown recently that the T cell stimulatory capacity of DCs pulsed with tumorantigen-derived peptides can be considerably increased by activating the DCs through electroporation with CD40L, CD70 and constitutively active TLR4 encoding mRNA (TriMix DCs). Here, we investigate whether TriMix DCs can be co-electroporate...

We have obtained data on CD8T lymphocyte populations in humans from our effort to identify and characterize these populations. We compared the distribution of phenotypically distinct cell sets using seven color staining in the blood, lymph nodes and spleen. Due to our location in a pediatric hospital, we have access to these organs removed from patients that undergo surgery due to clinical situations that do not affect the immune system (most frequently malformations). We found that the addition of other markers allow to subdivide N, CM, TEM and TEMRA populations ...