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 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...

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...

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...

(This trial is expected for 2009 and data will become available.)

We have obtained data from a phase I long peptide vaccination trial with p53 peptides in Montanide adjuvant in patients with colorectal cancer or ovarium cancer. This vaccine was well tolerated and robust p53-specific T cell responses were induced. In the future we intend to combine the p53 vaccine with other cancer therapies such as chemotherapy.

In 2008 we have started a trial in patients with VIN3 disease (similar to the Phase I/II clinical trial on the use of long peptides plus adjuvant for end-stage cervical cancer or VIN patients), in which we are comparing the efficacy of this vaccine with or without local application at the vaccination site of the TLR ligand imiquimod (Aldara). Results and data will become available.

The study protocol that will be used for this trial has been reviewed by the local ethical committee and is now ready to be submitted to Läkemedelsverket, the Swedish regulatory authority for clinical trials. Upon approval by Swedish authorities we plan to include, treat and evaluate 10 patients during a period of one to two years. It will probably start in 2009 and data will become available.

Data has been obtained from our work aiming at defining the role of CCR6 in directing migration of TH17 cells at sites of inflammation or infection. We found that CCR6–deficient (CCR6-KO) mice were resistant to induction of EAE but became susceptible when transferred with small number of CCR6-sufficient T cells. CCR6 was found to be required on a first wave of TH-17 cells that entered the CNS through the choroid plexus epithelial cells, which constitutively expressed CCL20 in both mice and humans. CCR6+ T cells triggered entry of a second wave of T cells that mi...

We have used agonists of the dectin-1/Syk pathway as adjuvants in vivo to induce CD8+ T cell responses and have data available on this. In summary, we have shown that this can result in CTL capable of destroying tumours.

We will explore the immunogenicity of necrotic cells as a tool to dissect the molecular determinants involved in “danger” recognition and will obtain data on this.

We have obtained data based on the characterization of a novel C-type lectin expressed primarily in mouse CD8?+ DC and their putative human equivalents. This lectin, named DNGR-1, can serve as a receptor for antigen targetting to DC. Notably, tumour antigens coupled to anti-DNGR-1 antibodies and given together with a suitable adjuvant elicit potent CTL responses that can promote tumour regression. We will continue to explore the use of DNGR-1 as a tool for targeting antigens to DC in vivo. In particular, we will examine if such targeting in the absence of adjuva...

We will start a collaboration with G. Schuler (P06) to examine the modulation of dendritic cell function by Denileukin Difitox (ONTAK) and will provide pathway analysis data.

A large reference ‘golden experiment’ will be carried out in which the transcriptome of DC vaccines of 50-100 patients will be compared and data transcriptomic data will become available.

The initiation of a multi partner trial is planned and data will be generated. The DC-THERA trial will compare the use of ‘TRI-MIX’ RNA in clinical trials that were already underway at each of the three centres (Nijmegen Medical School, Nijmegen; Friedrich-Alexander University, Erlangen; Vrije Universiteit Brussel, Brussels). It was decided that Friedrich-Alexander University, Erlangen, would apply for a licence to prepare GMP-grade ‘TRI-MIX’ RNA and subsequently supply this for all three centres. The multi-centre trial is a well-standardised, two-arm, m...

We have data available from an analysis of the proteome of the phagocytic/endocytic compartments that are formed upon cell entry of antigens via specific pathways. The ultimate goal is to get a better insight into the protein complexes that are involved in antigen processing, and learn whether the different entry routes for antigens into DCs might have distinct functional characteristics.

Data will be obtained from a study that we plan on DC receptor characteristics and signalling, focussing on antigen receptors.

Imaging data is available from MRI tracking of dendritic cells in melanoma patients for cellular therapy. In vivo magnetic resonance tracking of magnetically labeled cells is feasible in humans for detecting very low numbers of dendritic cells in conjunction with detailed anatomical information. MRI cell tracking using iron oxides appears clinically safe and well suited to monitor cellular therapy in humans.

We obtained data on the control of innate immunity by the mammalian target of Rapamycin (m-TOR) by enhancing TLR driven production of proinflammatory cytokines and type 1 interferons and by repressing posttranslational processing, via the inflammasome, of IL-1beta. We will further analyse the impact of the m-TOR inhibitor Rapamycin on innate immunity.

We obtained data on MHC II transport during human monocyte-derived DC maturation. We have published that the E3-ubiquitin ligase MARCH I is responsible for peptide-loaded MHC II ubiquitination and their subsequent internalization in immature DC.s The ligase MARCH I has been shown to be down-regulated upon activation. (De gassart et al, PNAS 2008). We will also investigate further the role of the ubiquitin ligases of the MARCH family in antigen processing, in particular we will focus on cross-presentation and CD1a molecule transport.