We obtained data on the T-cell stimulatory capacity of human monocyte-derived DCs co-electroporated with different combinations of CD40L, CD70, and constitutively active toll-like receptor 4 (caTLR4) encoding mRNA. The effectiveness of the dendritic cell (DC) vaccination protocols that are currently in use could be improved by providing the DCs with a more potent maturation signal. We therefore investigated whether the T-cell stimulatory capacity of human monocyte-derived DCs could be increased by co-electroporation with different combinations of CD40L, CD70, and...

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...

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

Data is available from an evaluation of different stimulation conditions for the cytometric assessment of antigen-specific Th-cells based on CD154 expression induced during short-term in vitro activation, a method established by us during previous DC-THERA activities. This is important and essential for implementation of the technology into clinical immune monitoring in e.g. vaccination trials. Moreover, the affect of stimulation length was determined. Differences with respect to the induced frequencies of antigen-specific, activated CD154+ Th-cells were minor. I...

Data were obtained from an ex-vivo analysis of different peptide specific T cell functions like degranulation and production of cytokines such as IFN? and TNF? by 8-color flow cytometry. In general, pre-existing immune-responses were rather mono-functional with dominating “IFN?-only”, “TNF?-only” and “degranulation-only” cells in the CD8+ compartment. Vaccination not only increased some of these monofunctional subpopulations but also induced occurrence of tumorantigen specific polyfunctional subsets. The further course of our melanoma patients will sho...

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...

Data has been obtained on TriMix DCs co-electroporated with whole tumorantigen encoding mRNA. A critical factor determining the effectiveness of currently used dendritic cell (DC)-based vaccines, is the DC’s activation or maturation status. We have shown recently that the T cell stimulatory capacity of DCs pulsed with tumorantigen-derived peptides can be considerably increased by activating the DCs through electroporation with CD40L, CD70 and constitutively active TLR4 encoding mRNA (TriMix DCs). Here, we investigate whether TriMix DCs can be co-electroporate...

We have explored fundamental aspects of the anti-tumor responses. We obtained data examining the cell populations which infiltrate progressive versus regressing P815 mastocytoma in order to identify cells which display immunosuppressive properties. We found changes in regulatory T cells populations as well as in the “myeloid suppressor cells”. Our preliminary data suggest that two populations of regulatory T cells coexist (natural and induced) in progressing tumors, and that a population of Gr1+ cells may affect tumor resistance in vivo.

We obtained data from our activity to establish the identification of versatile central memory CD8+ T-cells capable of high IL-2 secretion according to new markers. Versatile human CD8+ T-cells represent up to 30% of the CD8+ central memory pool in healthy adults and are characterised by unique functional capabilities (ability to secrete IL-2 after restimulation in concordance with low effector cytokine (e.g. IFN) and lack of cytotoxic mediator (e.g. perforin) expression (Frentsch et al., unpublished)). They might present an attractive candidate memory/effector C...

We have obtained data on the immunogenicity induced by chemotherapeutics.

We obtained data on the immunotherapy potential of DC, pulsed with tumor antigens, through its evaluation in EG7-OVA and P815 tumor models. Depletion of natural regulatory T cells in tumor bearing mice resulted in rejection of P815 cells, but not EG7-OVA, suggesting that regulatory T cells have a stronger impact on immune responses against weakly immunogenic or auto-antigen (P1A). Treg are detected in growing P815 tumor and there is some evidence that 2 regulatory T cells develop sequentially. T cell activation was monitored by tetramer staining and in vivo CTL as...

Data will be available from in-vitro pre-clinical tests on the pharmacodynamic activity of the fusion vaccines. This test will verify the capacity of the fusion vaccine to present certain tumour-antigen derived peptides corresponding to antigens present in the fused cancer cells. BruCells has met with the Dutch MEB for a Scientific Advice Meeting on January 10, 2008 in The Hague. The Dutch authorities consider that the proposed in-vitro pre-clinical tests are adequate to check the pharmacodynamic activity of the fusion vaccines and that no animal model tests would be necessary.

This test will verify whether the fusion vaccine possesses the properties of both parental cell types for inducing primary CD8+ T-cell responses (data will become available). Detection of circulating tumour-specific CD8+ T-cells. BruCells has met with the Dutch MEB for a Scientific Advice Meeting on January 10, 2008 in The Hague. The Dutch authorities consider that the proposed in-vitro pre-clinical tests are adequate to check the pharmacodynamic activity of the fusion vaccines and that no animal model tests would be necessary.

This test, described for glioblastoma, performed with PBMC from newly diagnosed glioblastoma patients will verify the ability of tumour-specific T-cells to recognize the fusion vaccine. Data will be made available. BruCells has met with the Dutch MEB for a Scientific Advice Meeting on January 10, 2008 in The Hague. The Dutch authorities consider that the proposed in-vitro pre-clinical tests are adequate to check the pharmacodynamic activity of the fusion vaccines and that no animal model tests would be necessary.

We have data available on the inadequate nature of the antitumor CTL response, generated in a syngeneic mouse tumor model expressing an Ag derived from the early region 1A of human adenovirus type 5. We demonstrated using this syngeneic mouse tumor model that the inadequate nature of the antitumor CTL response is not due to direct Ag presentation by the tumor cells, but results from presentation of tumor-derived Ag by nonactivated CD11c(+) APC. Although this event results in division of naive CTL in tumor draining lymph nodes, it does not establish a productive i...

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 ...

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 has immunological data available on human Hemato-Lymphoid System Rag2-/-gc-/- mice infected with EBV. Both Epstein Bar Virus (EBV) and Human Immunodeficiency Virus (HIV) are human specific lymphotropic viruses. Human Hemato-Lymphoid System Rag2-/-gc-/- 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 directl...