The DC were matured with TLR ligand R848 and polyI:C. Using these DC, we obtained best results in our evaluation whether antigens could be rerouted from the endosomal into the cytosolic compartment by combining antigen with cell penetrating peptides to further enhance cross-presentation.

DCs were pulsed with allogenic melanoma peptides (SK-Mel 24, HLA-A1 and HLA-A2 positive) plus KLH as immunological tracer and subsequently used to produce a vaccine for melanoma.

The dead cells were stained and used in the uptake of dead cells by DC.

These DC were electroporated, co-cultured with purified CD8 T cells using MACS cell separation to asses the in vitro CD8 T cell stimulatory capacity of DC.

These dendritic cells were obtained from PBMC from which monocytes had been isolated that differentiated into DC after cell culture.

These marginal zone dendritic cells, interacting with the cholera toxin (CT) adjuvant, lead to effective priming of an immune response in vivo. For the first time, we have followed adjuvant targeting of MZ DC in vivo.

After 6 hour of stimulation, DCs were collected, washed 3 times with PBS, treated with zymolyase, washed twice and cells, lysated with a hypotonic solution (KCl 0.05%), were plated on YPD. Survival of yeast cells, spores or hyphae after uptake was reported as percentage of colony forming units after 3 days relative to the total number of cells growing in the absence of DCs exposure. To assess the importance of ROS production in DC killing ability, DPI (10 microM) was added 30 minutes before stimulation and survival of microorganisms was assessed using the same...

These DCs have been generated from monocytes after elutriation and have not been pulsed with irradiated tumor cells (apoptotic bodies).

The tumor antigen derived peptide pulsed monocyte-derived DC were diluted in medium containing 20 ng/mL TNF-?, plus IL-4 and GM-CSF.

DMSO was used to dissolve blue formazan particles.

The crude population of DC is divided into the three known splenic subsets (CD8+, CD4+, DN DC) using Fluorescence activated cell sorting (BD FACS Aria). This procedure allowed to obtain highly pure fractions of each subset. However, because DC are a very rare population, this method is very material- and cost-intensive. Furthermore, it is not surprising that the obtained cell numbers of each subset after isolation are below 107. However, these cell numbers should be sufficient for proteomic investigations. The proteomes of all three subsets using mass spectrometry is in progress.

To assess the importance of ROS production in DC killing ability, 2-(3,4-Dihydroxyphenylimino)-imidazolidine (10 microM) was added to the dendritic cells 30 minutes before stimulation.

Our current efforts in a study of functional homogeinity involve a transition to 8 colours surface staining, and the evaluation of antigen-specific cell sets identified with MHC tetramers. For the later process, we initiated data collection of EBV, CMV and HIV specific T cell sets.

Three groups of end stage cervical cancer patients (in total N=35) were subcutaneously vaccinated with HPV16 E6 combined with or separated from HPV16 E7 overlapping long peptides in Montanide ISA-51 adjuvant, 4 times at three week intervals. Group 1 received 300 microgram per peptide at a single site, group 2 received 100 microgram per peptide of the E6 peptides in one limb, and 300 microgram per peptide of the E7 peptides in a second limb. Group 3 received separate injections of E6 and E7 peptides, each at a dose of 50 micrograms per peptide. The primary endpo...

These T cells are specific for the 0T-1-OVA antigen. They were used to study the expression of twenty different genes either mediating effector functions, or coding for different receptors involved in T cell differentiation and memory generation.

These T cells are specific for the P14-GP33 antigen. They were used to study the expression of twenty different genes either mediating effector functions, or coding for different receptors involved in T cell differentiation and memory generation.

These monocytes were extracted from a Leukapheresis product from cancer patients using elutriation.

In the process of extracting total RNA from yeast cells, the pellets were washed with 70% Ethanol two times.

The model antigen OVA was fused to the C1C2 exosome-binding domain of MFG-E8/lactadherin. This construct was used in a study on the capture of antigen-bearing exosomes by DCs and cross-presentation of tumour antigen in exosomes.

Exosomes were used to treat a chohort of 77 gastrointestinal stromal tumor (GIST) cancer patients.