Studies have continued and data obtained on the effects of biolistic (Gene Gun) delivery of vectors engineered to express distinct chemokines at the local skin site of mice. In particular, attention has been focussed on the recruitment of distinct subsets of DC to skin. This work, which is being performed in collaboration with PowderMed, a former SME now owned by Pfizer, may lead to the development of more targeted strategies to induce distinct T cell responses using this technology. A publication has resulted from this work.

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...

We have obtained data from an analysis of the uptake, conservation and cross-presentation of the model antigen OVA after Fc receptor-mediated uptake by DC and from an analysis of the immunological consequences of the antigen depot. We found that MHC class I presentation is relatively short-lived in contrast to MHC class II. However, CD8 cross-priming capacity of OVA-loaded DC was functionally retained for many days, while peptide-pulsed DC had lost their priming capacity after 24 hours. Strikingly, OVA protein antigen was conserved intracellularly in DC for many ...

Our laboratory obtained data on the induction of immune responses after targeting antigens to DC via TLR agonists.

Our lab obtained data on the induction of antigen-specific CD8+ cytotoxic T cells by dendritic cells co-electroporated with a dsRNA analogue and tumor antigen mRNA. The maturation state of dendritic cells (DCs) is an important determinant for the initiation and regulation of adaptive immune responses. In this study, we wanted to assess whether functional activation of human monocyte-derived DCs can be achieved by electroporation of an activation signal in the form of double-stranded (ds) RNA and whether simultaneous electroporation of the dsRNA with tumor antigen...

We have data available on the induction of effective therapeutic antitumor immunity by direct in vivo administration of ovalbumin (OVA) encoding lentiviral vectors. Ex vivo lentivirally transduced dendritic cells (DC) have been described to induce CD8+ and CD4+ T-cell responses against various tumor-associated antigens (TAAs) in vitro and in vivo. We report here that direct administration of ovalbumin (OVA) encoding lentiviral vectors caused in vivo transduction of cells that were found in draining lymph nodes (LNs) and induced potent anti-OVA cytotoxic T cells ...

Our lab has gathered data in establishing a per-clinical model of breast cancer.

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

In 2007 we established routine monitoring of tumor-specific T-cell subsets by multicolor flow cytometry and obtained immunological data from this. For this purpose we used multimers kindly provided by Pierre Coulie (Brussels) together with surface markers (such as CD8, CD4, CD3, CD45RA, CCR7, CD25, CD27, CD137, CD127) in different 8-color panels on the BD FACSCanto II. Additionally we used functional markers such as CD107a and intracellular cytokine staining for the further analysis of individual T-cell populations and T-cell clones. Monitoring of the ongoing vacc...

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...

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 ...

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 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...

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...

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...

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 ...

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 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...