Patients with resected HPV16-positive cervical cancer were vaccinated with an overlapping set of long peptides comprising the sequences of the HPV16 E6 and E7 oncoproteins emulsified in Montanide ISA-51.

HSV vector expressing full length tyrosinase, gp100 and MART-1optimised 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. Biovex provides the possibility of using these vectors for a variety of pre-clinical, and ultimately clinical studies.

Hu-SCID mice were used to study responses to EBV and HIV.

This is blood from patients that undergo surgery due to clinical situations that do not affect the immune system (most frequently malformations). We have attempted to identify and characterize CD8T lymphocyte populations in humans. We compared the distribution of phenotypically distinct cell sets using seven color staining in the blood, lymph nodes and spleen. We found that the addition of other markers allow to subdivide N, CM, TEM and TEMRA populations in additional cell types.

Human DC exposed to TLR ligands and activated iNKT cells in vitro. They had enhanced expression of maturation markers, suggesting that a cooperative action of TLR ligands and iNKT cells on DC function is a generalizable phenomenon across species. These studies highlight the potential for manipulating the interactions between TLR ligands and iNKT cell activation in the design of effective vaccine adjuvants.

The exosomes were used to immunize mice, hybridomas were generated that produce antibodies recognizing at least the human exosomes.

We have efficiently transfected DC with RNA encoding a functional protein (E/L-Selectin) which allows entry of DC into LN from HEV. 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 their functionality.

The mice were infected with EBV and mount an immune response (i.e. cytotoxic T cell proliferation, some control of EBV driven B cell proliferation, perforine and granzyme expressing T cell infiltration in B cell infected areas in lymphoid organs in situ), however, specific T cells could not be detected directly ex vivo by looking at the most common EBV derived/presented epitopes (tetramer staining). Some animals developed EBV induced B cell lymphoproliferative disease.

These mice were infected with both CXCR4 as well as CCR5 tropic HIV-1 strains. HIV causes a disseminated infection and spreads in all newly generated lymphoid tissues, thus closely resembling HIV infection in humans. We are now aiming to improve the recipient mouse background by co-transplanting human mesenchymal stroma cells, and by adding human cytokines as well as human MHC. Furthermore, we use the mice to evaluate targeted therapies directed at human immune system cells as T cells, B cells, and dendritic cells.

We established “human hemato-lymphoid-system mice” by transplanting human CD34+ cord blood cells into irradiated newborn Rag2-/-gc-/- mice, leading to de novo development of human B, T, and dendritic cells.

Commercially available siRNA Smart Pool from Dharmacon targeted against the STAT3 transcription factor have been successfully transfected in these human MDDCs.

We studied the response of human MoDC to yeast, spheroplasts, pseudohyphae and spores. Analysis of IL-6, IL-8, TNF?, IL-1?, IFN?, IL-10, IL-12p70, IL-12p40 secretion was performed with a multiplex assay.

We have stimulated human Monocyte derived Dendritic Cells (hMoDC) with yeast, spheroplasts, pseudohyphae, spores, purified RNA and not purified RNA, LPS.

We used specific inhibitors to study the role of src-family tyrosine kinases in the maturation of human monocyte derived dendritic cells upon stimulation with several toll like receptor (TLR) agonists. The effect of these kinase inhibitors on the capacity of DC to be activated by a TLR2 (PAM3CSK4), TLR3 (Poly I:C), TLR5 (Flagellin), and TLR8 (Poly U) agonist was evaluated.

These DC were exposed to different maturation cocktails and various activators or inhibitors and profiled.

On these cells, transcriptional profiling was performed and a comparative pathway-based analysis was done.

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 encoding mRNA can lead to the induction of a cytotoxic T-lymphocyte (CTL) response.

We are currently performing gene silencing experiments in human monocyte-derived DCs (MDDCs) with the dual aim of generating relevant knowledge on (i) the transcriptional regulation of DC differentiation, and (ii) the safe and rationale in vitro manipulation of gene expression in DCs.

These dimers were injected into TCR transgenic mice which exhibited an increased frequency of LACK-specific T cells with the aim of producing a mAb reacting to the immunodominant LACK156-173 peptide of leishmania bound to I-Ad MHC class II molecules.

These dendritic cells were generated with interferon-beta and interleukin-3. They were used in a clinical trial in melanoma patients who were randomized to receive immunizations either with these I3 DC or with G4 DC (DC generated with GM-CSF and IL-4).