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 vector has been already successfully used to generate self-inactivating lentiviral vectors previously tested in DCs for transfection efficiency and absence of effects on their phenotype.

In our phase I clinical trial, we highlight the capacity of Dex based-vaccines to restore the number and NKG2D-dependent function of NK cells in 7/14 cancer patients.

We have prepared two plasmids coding for LACK linked to either eGFP or mCherry fluorescent proteins, under the control of the beta-casein promoter. In order to identify the APCs that were responsible for the development of this state of immune tolerance, we aimed to generate a transgenic mouse that express a recombinant fluorescent protein in the mammary glands, an approach that should result in high levels of expression of the fluorescent protein in the milk. We thus should be able to detect the cells that capture, degrade and present this antigen to T cel...

We have completed and published our work on biodegradable poly (D, L-lactide-co-glycolide) (PLGS) micropsheres (MS) coencapsulating ligands for endosomally expressed TLRs plus exogeneous Antigen (Ag) to deliver its cargo to endosomes of murine Dendritic cells (DCs), thus initiating TLR mediated DC maturation as well as processing of MHC class I and class II restricted epitopes.

We examined whether plasmacytoid pDC are infected and/or activated by MTb, and whether they play a role in regulating bacterial clearance during acute MTb infection and if so how. We have compared these DC with myeloid DC and macrophages with respect to MTb infection.

The CpG was used for stimulating DC. It was shown in mice that both IL-10 as well as IL-12 production is dependent on the signalling adaptor molecules either MyD88 or TRIF, respectively in response to CpG, LPS or PolyIC.

PBS (cells in FC-block 1:50) + 2 % FCS was used in incubation of cells for FACS staining for activation markers.

The patients were vaccinated with a vaccine composed by autologous DCs pulsed with apoptotic allogenic prostate carcinoma cell line LNCap.

These ovarian carcinoma patients are treated with a vaccine composed by autologous DCs pulsed with apoptotic autologous ovarian carcinoma cells.

PE-alpha DC80 is used for staining of incubated cells for FACS analysis for activation markers.

50 U/ml of penicillin were added to the monocyte cell culture.

This vaccine consists of G4-DC loaded with 8 different peptides. It was tested in a trial in small cohorts of patients (3-9) who were vaccinated with differently composed DC-vaccines.

We set an in vitro system to study ex-vivo responses able to detect fona fide in vivo primed CD4+ T cells. We investigated spontaneous CD4+ T cell responses to these antigens in normal donors and in patients with high-grade cervical lesions, pancreas adenocarcinoma and advanced melanoma. The antigens were: i) the E6 and E7 proteins of human papilloma viruses, ii) the carcinoembryonic antigen (CEA) and iii) the tumour specific antigen MAGE-A3.

We set an in vitro system to study ex-vivo responses able to detect fona fide in vivo primed CD4+ T cells. We investigated spontaneous CD4+ T cell responses to these antigens in normal donors and in patients with high-grade cervical lesions, pancreas adenocarcinoma and advanced melanoma. The antigens were: i) the E6 and E7 proteins of human papilloma viruses, ii) the carcinoembryonic antigen (CEA) and iii) the tumour specific antigen MAGE-A3.

We set an in vitro system to study ex-vivo responses able to detect fona fide in vivo primed CD4+ T cells. We investigated spontaneous CD4+ T cell responses to these antigens in normal donors and in patients with high-grade cervical lesions, pancreas adenocarcinoma and advanced melanoma. The antigens were: i) the E6 and E7 proteins of human papilloma viruses, ii) the carcinoembryonic antigen (CEA) and iii) the tumour specific antigen MAGE-A3.

These patients were treated in a long peptide trial. Analysis of the local immune response demonstrated the presence of HPV16-specific Th1/Th2 cells infiltrating the vaccination site and the VIN lesion after vaccination. 5 out of 20 patients showed complete clearance of all VIN III disease and in 4 of these this was also associated with complete clearance of HPV16 virus.

Perforine expressing T cells were observed in human Hemato-Lymphoid System Rag2-/-gc-/- mice after being infected with EBV and mounting an immune response. They infiltrated in B cell infected areas in lymphoid organs in situ.

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 similar to those elicited by ex vivo transduced DC.

We have analyzed the uptake, conservation and cross-presentation of the model antigen OVA after Fc receptor-mediated uptake by DC. 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.

Dendritic cell (DC) derived-exosomes (Dex) were described as nanovesicles harbouring functional MHC molecules stimulating T cell responses. 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 in an anti-metastatic effect. In humans, Dex bear functional IL-15R? which allow proliferation and IFNg secretion by NK cells. In contrast to immature DC, human Dex harbor NKG2D ligands on their surface leading to a direct engagement of NKG2D and NK cell activation ...