Our lab obtained data on the kinetics of effector gene expression and association in TCR-Tg CD8 cells responding to the male antigen and to Listeria-OVA with regard to « in vivo » responses to Ags. Surprisingly, instead of a single program of differentiation leading to the coordinated expression of all effector genes; we found that individual effector genes had different kinetics of expression and associated randomly generating several new effector cell types. These results are published in J. Exp. Med. Since these results were obtained in TCR-Tg mice, and thus ...

Our lab gathered data on DC activation by pathogen-derived stimuli. We focused on the study of the response of MDDCs to the combined stimulation with TLR ligands and we observed that simultaneous activation of TLR4 and TLR8 signaling cascades results in a marked inhibition of the secretion of the proinflammatory chemokine CCL2 with respect to stimulation through a single TLR. This inhibition is specific for both CCL2 and TLR agonist combination and could represent a novel regulatory mechanism evolved to maintain immunological balance (Del Cornò et al.). Future st...

My laboratory obtained data from a continued analysis of the ability of invariant NKT (iNKT) cells to assist priming of antigen specific T and B cell responses. We have carried out three complementary lines of research: 1) We have demonstrated that activation of human DC by Toll like receptor ligands (TLR-L) modulates the lipid biosynthetic pathway, resulting in enhanced recognition of CD1d-associated lipids by iNKT cells. 2) We have clarified the mechanisms by which CD1d-restricted lymphocytes translate T cell receptor (TCR) recognition of lipids with similar g...

Our lab obtained data on the activation of immature monocyte-derived dendritic cells after transduction with high doses of lentiviral vectors. Dendritic cells (DCs) are an attractive tool for immunomodulation, targeting mature DCs (mDCs) for immunization or immature/semimature DCs (iDCs) for tolerization. Therefore, introducing antigens into DCs has become a prime topic in various immunological disciplines. Numerous studies have shown that lentiviruses are an efficient vehicle for this purpose. This study evaluates the effects of lentiviral transduction on iDC ...

We have obtained data from in vitro studies to demonstrate that DC can be activated via the dectin-1/Syk pathway to become APC capable of priming CD8+ T cells. In addition, we have shown that the same DC can interact with Tregs and convert them into cells that co-express ROR?t and Foxp3 and produce IL-17. We have shown that this pathway can serve as an alternative to TLR signalling for allowing DC to become effector cells capable of priming CD4+ T cells. We have used agonists of the dectin-1/Syk pathway as adjuvants in vivo to induce DC activation and CD4+ T cell ...

The use of gene guns in ballistically delivering DNA vaccine coated gold micro-particles to skin can potentially damage targeted cells, including Langerhans cells, therefore influencing transfection efficiencies. We have assessed and obtained data on cell death in the viable epidermis by non-invasive near infrared two-photon microscopy following micro-particle bombardment of murine skin. We showed that the ballistic delivery of micro-particles to the viable epidermis can result in localised cell death. Furthermore, experimental results show the degree of cell deat...

Data has been obtained from a Balb-neuT mice model to study new strategies for advanced breast cancer. A colony has been established of transgenic Balb-neuT mice (founders provided by Forni, Torino IT) that over-express the rat HER-2/neu oncogene under the MMTV promoter. These mice develop mammary carcinomas with later lung and liver metastases; the course of disease and the type of tumours that develop closely recapitulate many features of human breast cancer. A collaborative study has commenced between Austyn and Cerundolo to use this model to study new strategi...

We have obtained data from a study where we addressed a model antigen to exosomes secreted by tumor cells in vivo, to allow capture of antigen-bearing exosomes by DCs and cross-presentation of the antigen. For this purpose, the model antigen OVA was fused to the C1C2 exosome-binding domain of MFG-E8/lactadherin. Tumors secreting the exosome-bound antigen grow slower than tumors secreting soluble OVA, because they induce activation of OVA-specific CD8+ T cells into killer cells. In addition, inducing in vivo secretion of the C1C2-coupled antigen by DNA vaccination i...

We have data available from a comparison of the use of fresh peripheral blood mononuclear cells (PBMC) versus fresh heparinised whole blood. Only when peptide pools as an antigen source are used PBMC exhibited a slightly higher stimulation capacity compared to whole blood. However, slightly higher background was observed when using PBMC to stimulate with antigen. Lowest background was observed when either peptides or protein were used for stimulation enabling assessment also of rare antigen-specific Th-cells below frequencies of 0.05% of peripheral CD4+ Th-cell...

Data has been obtained on the effect of exposure of DCs or gamma-delta T lymphocytes to HIV-1 and on the miR profile expressed in HIV-exposed DCs. Based on our previous findings demonstrating a bidirectional activating interaction between immature MDDCs and gamma-delta T lymphocytes (Conti et al., JI. 2005), as well as on the demonstration that HIV-1 exposed MDDCs exhibit an impaired functional maturation (Fantuzzi et al., J Virol 2004), we are currently investigating whether exposure of DCs or gamma-delta T lymphocytes to HIV-1 can directly modulate their functi...

Protein expression data is available for our pre-clinical work aiming at developing DC based vaccines expressing the tumor antigens PSA or Her2/neu. Monocyte rich fractions were produced by elutriation under GMP conditions and cultured for 5 days with GM-CSF and IL-4. Conditions for transfection with electroporation (Amaxa) or with Adenovirus constructs have been established. Protein expression (Her2/neu, PSA) following transfection with a panel of different full length or truncated Her2/neu or PSA constructs was assessed using Facs analysis (Her2/neu) or assessme...

We have data available on dendritic cell development from hematopoietic stem and progenitor cells in steady-state and in inflammatory conditions, and, in particular, gene-expression data of Lin–c-KitintFlt3+M-CSFR+ cells in mouse bone marrow. Lymphoid tissue plasmacytoid and conventional dendritic cells (DCs) are continuously regenerated from hematopoietic stem cells. The cytokine dependence and biology of plasmacytoid and conventional DCs suggest that regeneration might proceed through common DC-restricted developmental intermediates. By selecting for cytokin...

We have data available on the role of chemokine receptors in regulating migration of mature DC and subsets of effector and memory T cells in secondary lymphoid organs. CCR7 is a chemokine receptor that is expressed by mature DCs and central memory T cells (TCM) and regulate migration of these cells in secondary lymphoid organs in the steady state. T lymphocytes lacking the lymph node homing receptors L-selectin and CCR7 do not migrate to lymph nodes in the steady state. In contrast it was found that lymph nodes that drain sites of mature DC or adjuvant inoculatio...

BruCells is collaborating with Professor De Witte from ULB for the preclinical study in which circulating tumour-specific CD8+ T-cells will be detected in the blood of newly diagnosed glioblastoma patients and from which data will be obtained. In order to obtain the blood samples and tumour biopsies from the glioblastoma patients, a file has been submitted to the Ethics Committee of the Erasme Hospital and has been recently approved.

We have generated data on the developmental control of translation during DC activation. Our work (Lelouard et al., JCB) demonstrates that DCs down-regulate and change the quality of translation in order to increase their survival, however during this process MHC I antigen processing is affected. This has important consequences for the way antigens should be introduced in DCs and how long after stimulation they should be used during vaccination application. We have also initiated studies on the impact of dsRNA on the regulation of translation and the functional re...

Data were produced in our lab on a broad spectrum of priming events and their impact on the effector and memory phases of the CTL response. Activation of a cytotoxic T lymphocyte (CTL) response in an antigen-exposed lymph node involves a great diversity of encounters between naive CTLs and APCs that differ in both duration and quality. This broad spectrum of priming events instigates a complex blend of CTL developmental pathways. Using an experimental system that allows tight control over CTL priming, we have singled out defined priming events and analyzed the ...

Although many bacteria and protozoa can invade dendritic cells (DC), it is not clear whether infected DC process pathogen-derived antigen and stimulate antigen-specific CD4+ T cells in vivo. To address this issue and obtain data on this question, we have infected susceptible BALB/c (H2-d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. We have directly visualized antigen-presenting cells using a mAb reacting to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad MHC class II molecule. I-Ad/LACK complexes wer...

We have obtained data addressing the effectiveness of TLR ligand-long peptide conjugates in vivo by either direct vaccination or ex vivo DC loading prior to vaccination. Both approaches show similar effectiveness in CTL priming suggesting that TLRL-peptide conjugates efficiently target DC in vivo.

Knowledge and gene expression data were gained on differentiation patterns of T cells.

We investigated the effects of tumour hypoxia on DC differentiation and maturation and gathered data in mice. This is a an important topic as sites of inflammation are often characterized by both low oxygen tension and an extensive infiltration of inflammatory leukocytes, including DC, which need to move against oxygen gradients. We showed that oxygen availability is necessary to promote full monocyte-derived DC differentiation and maturation. Low oxygen tension (hypoxia) inhibits expression of several differentiation and maturation markers (CD1a, CD40, CD80, CD8...