Data was obtained on the DC-activating, TLR-4-mediated stimulus, LPS, on rat intestinal and hepatic DC in vivo. DC present peripheral Ags to T cells in lymph nodes, but also influence their differentiation (tolerance/immunity, Th1/Th2). To investigate how peripheral conditions affect DC properties and might subsequently regulate T cell differentiation, we examined the effects of a potent DC-activating, TLR-4-mediated stimulus, LPS, on rat intestinal and hepatic DC in vivo. Our results suggest that any explanation of switching between tolerance and immunity as wel...

Central to all T cell responses is the interaction between the T cell receptor and peptide antigen presented by the MHC molecules. This interaction selects the dominant T cell clones with the most favourable (not necessarily highest) affinity and determines the extent of cross-reaction with epitope variants. I am currently collaborating with the structural biology group of Professor Yvonne Jones in the Nuffield Department of Medicine (NDM) at the Wellcome Trust Centre for Human Genetics. This collaboration has led to structural data on the CD1b in complex with s...

We obtained data from a trial in small cohorts of patients (3-9) who were vaccinated with differently composed DC-vaccines: DC loaded with a single MAA-derived peptide (MAGE-3.A1 or -A2 or Na17.A2) or pulsed with 2 to 3 MAA-derived peptides (combinations of MAGE-3.A2, MAGE-C2.A2 or Na17.A2) or G4-DC loaded with 8 different peptides. Our experiences can be summarized as follows: (1) preparation of an autologous dendritic cell based vaccine was feasible for every patient enrolled in our studies; (2) we did not observe vaccination related toxicities beside mild...

Our laboratory has data on autologous dendritic cells loaded with apoptotic leukemic cells from patients with B-CLL used as vaccine in patients. In preclinical experiments DC-loaded with apoptotic leukemic cells were shown to induce a strong preferentially Th1 T-cell response. Apoptotic tumor cells were more effective than lysate, RNA and cell hybrids. A clinical trial has been initiated where autologous dendritic cells are loaded with apoptotic leukemic cells from patients with B-CLL. So far “vaccines” have been produced for six patients. Four patients are...

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 ...

We obtained data from a study of 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. These results were published in Blood. We showed that - this strategy allowed the identifi...

We obtained data assessing the entire CD4+ Th-cells specific for defined antigens also for murine cells (based on the strategies developed in work performed in 2005 (Frentsch et al., Nature Medicine 2005)). For intracellular assessment of CD154 induced in the course of short-term activation target cells are stimulated with antigen for 6h in the presence of the secretion inhibitor BrefA (for 4h). For extracellular analysis of live antigen-specific Th-cells cells are stimulated in the presence of an anti-CD40 blocking antibody and biotinylated anti-CD154 antibody. W...

We have obtained data monitoring the vaccine induced specific anti-tumor T cell responses (thymidine-incorporation, ELISA, LDA and ELISPOT assays) using “conventional” techniques. 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 identified new MHC class II restricted epitopes on tumor associate...

We have obtained data from monitoring vaccine induced T cell responses for two clinical trials. We found CD8+ and CD4+ T cell responses against the immunological tracers KLH and TK, and against the NTPs and MAGE-3, respectively. Assays used have been 3H-thymidine-incorporation, ELISA, CBA, LDA and 51Cr-release assays. Autologous tumour specific CD8+ and CD4+ T cell clones have also been obtained from the blood and tumour infiltrating lymphocytes. We investigated the role of spontaneous CD4+ T cell responses against the HPV-18 and 16 E6 and E7 proteins in women wi...

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...

We obtained data on antigen delivery by a vector with ovalbumin and CMVpp65 in mice or human, respectively. We used a MHC II presentation pathway targeting vector originally published by Wang et al. 1999. Proof of principle studies for antigen delivery have been performed with ovalbumin (murine studies) and CMVpp65 (human studies).

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.

Our group has obtained imaging data on the role of DALIS and related aggregates in antigen presentation and their interaction with the regulation of translation in response to pathogens. We have implemented in collaboration with lead coordinator Carl Figdor, the organization of the imaging core-facility of the DC-THERA NoE. Experimentally, we have been applying confocal microscopy to the study of DALIS and related aggregates in order to understand their role in antigen presentation and their interaction with the regulation of translation in response to pathogens....

We have data available on a novel M-CSF dependent DC developmental pathway that is independent of Flt3L. These DC have unique characteristics and precursor origin. The cells can be found in vivo in Flt3L gene deleted (-/-) mice injected with recombinant M-CSF.

Our group has MRI data available on the migration of DCs in vivo in melanoma patients. From isolated lymphnodes obtained after surgery we could identify single DC after staining with Prussian blue for iron. We are in the process of analysing the T cell rosettes (activation stage) that surround these DC to get insight in the functional activity. It was also demonstrated that DCs transfected with RNA produce derived proteins in vivo in the lymph nodes. Also efforts were spent to get further information on the dynamics of immature and mature DCs. We discovered (van...

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...

We have generated data on the kinetic of the transcriptional profiling of D1 cells treated with either DMSO or LPS.

Our laboratory has obtained data on the kinetic of the transcriptional profiling of purified mouse DC treated with IL10.

Data has been obtained from an analysis of immature DC and mature DC from two strains of mice: the MyD88 deficient and the wild-type mice.