In ongoing trials with peptide loaded DC (> 60 pat.) routine immunomonitoring at frequent intervals showed a high correlation between different assays (IFN gamma ELISPOT; modified MLPC = combination of limiting dilution, multiple peptide restimulation, tetramer detection). Both assays demonstrated declining responses upon extension of vaccination intervals. We suggest: IFN gamma ELISPOT as basic assay (costs, workload) that can be frequently performed in every patient for routine monitoring Modified MLPC (combination of limiting dilution, multiple peptide resti...

In ongoing clinical trials (multipeptide loaded cytokine matured moDC +/- CD40L activation in >60 pat.) we have obtained valuable information with high impact on future DC-trials: 1) the presence of 2% DMSO in the thawed final vaccine has no negative impact on the quantity and quality of induced immune responses; thus thawed DMSO containing vials do not have to be centrifuged, but simply diluted; 2) class I peptide loaded cytokine matured DC induce de novo or expand preexisting CD8+ T cell IFN gamma responses in the majority of melanoma patients (>70%), surpri...

In 2008, much effort was spent to optimize protocols for RNA transfection in human sDCs. - fusion proteins of the DC specific receptor DC-SIGN fused to GFP - use of rna to express melanoma associated antigens in dendritic cells to prepare better vacines for the treatment of melanoma patients.

To determine ROS production we used the modified version by Choi et al. (2006) of the microscopic Nitroblue tetrazolium (NBT, Sigma-Aldrich) assay. Briefly, DCs were stimulated with spores or yeasts, washed, supplemented with 0.1 mg/ml of NBT, which allows the precipitation of formazan particles in presence of ROS. Blue formazan particles were dissolved using 2M KOH and DMSO and its absorbance was measured using a microplate reader at 620 nm. The absorbance of dissolved NBT increased in proportion to cell number, incubation time, and stimulus concentration.

We performed experiments to set up the optimal conditions for both transient transfection of synthetic siRNAs and lentiviral transduction of shRNAs into DC. To optimize transfection efficiency, while minimizing toxicity, non targeting FITC-conjugated siRNA were transfected in two different experimental settings: a) fresh monocytes and b) immature DCs (iDCs) obtained from monocytes in the presence of GM-CSF/IL-4. In both experimental settings a considerably high transfection efficiency (up to 75%) was achieved in the absence of significant cell death. Moreov...

A single step procedure for the in vitro maturation and antigen loading of human moDC was optimized. DC were electroporated with mRNA encoding tumor antigens and a putative TLR3 ligand. In vitro stimulation of naïve T cells indicated the superiority of this approach compared to in vitro stimulation with DC matured with inflammatory cytokines followed by electroporation with TAA encoding mRNA.

We have worked out SOPs for the cytometric assessment of Th-cell immunity based on antigen-induced CD154 expression, a technology developed by us in the frame of previous DC-THERA activities. Now the entire antigen-specific Th-cells specific for defined antigens can be monitored simultaneously with respect to quantity and quality. Tests can either be performed on standard 4 colour cytometers or on 12 colour high-end cytometric analysers. Tests that can be either performed to analyse fixed or isolate live antigen-specific Th-cells have been adapted also to be used ...

(Basic manufacturing and QC SOPs (Generation of Peptide loaded DCs, Generation of cytokines and media components, Generation of autologous plasma and serum, Staining of cells for FACS Analysis, Cell Counting by Trypanblue Staining) have been provided to DC-THERA participants and associated partners via DC-THERA intranet. )

Argos successfully developed a manual DC manufacturing method with excellent reproducibility in terms of quality, yield, and viability of DCs. The process developed has the following unique advantages: - The manufacturing process has been optimized for producing DC from non-frozen day-old pheresis material which allows centralized manufacturing. The pheresis product is sent in a specially designed shipper to the manufacturing facility. - The final vaccine product is formulated for direct injection (i.e., no clinical site participation required except for bedside thaw and i.d. injection)

We are progressing steadily towards the delivery of a standardized method for the immuno-monitoring of DC vaccination trials using a finite number of defined tumor antigens. Our starting point was the method that we had developed previously to estimate blood frequencies of CD8 T lymphocytes against one defined antigenic peptide. It was based on an in vitro restimulation of blood lymphocytes with the peptide and growth factors over two weeks followed by detection with the appropriate HLA class I tetramer. In order to estimate frequencies of responding cells, these ...

Different strains of Saccharomyces cerevisiae were cultured and collected in different conditions: * different growth phase (exponential and stationary phase) * different growth media (standard and promoting pseudohyphal growth) * different cell form (spheroplast, spore and whole cell) Monocyte-derived DCs were added at a final concentration of 5x105 cells/ml into 96-well plates. Serial diluition of yeast cells and preparations were added to the MoDCs. Cytokine accumulation was evaluated in the supernatants at 24h by ELISA, according to a standard protocol and ...

The company has now successfully developed a technique for the large-scale generation of DC in serum-free medium under GMP conditions, and are willing to share their know-how with Network Partners.

For quantitative proteomic studies, our group recently described the SILAC (Stable Isotope Labelling by Amino Acids in Cell Culture) approach. In this method, independent populations of cells are grown in medium containing different non-radioactive isotope forms of essential amino acids (e.g. arginine and lysine). Because the cells cannot synthesize these amino acids, after some generations all proteins are labelled and therefore can be distinguished and quantified by the mass spectrometer.

The cells were collected and the pellet were resuspended in 3.6 ml AE Buffer (AE=50mM NaAcetate pH 5.2, 10mM EDTA) and then transferred in 15 ml tubes wit 200 µl 10% SDS (w/v). 2 ml of preheated acid phenol (pH 4.3) were added and the falcon were mixed by vortex. After 10’ of incubation in 65 °C water bath, the samples were incubated on ice and then centrifugated at 5000 rpm in an tabletop centrifuge. The supernatants were transferred to a new tube and added 2 ml of acid phenol to repeat the extraction. After the centrifugation (10’ at 5000 rpm) the supernata...

We have efficiently transfected DC with RNA encoding a functional protein (E/L-Selectin) which allows entry of DC into LN from HEV. RNA transfected human DC could be frozen and thawed without loosing their functionality.

Staining of D2SC1/Flt3-DC and staining of the cells to be taken up by Mini67-1KT or Mini26-1KT (PKH67/PKH26 green/red fluorescent cell linker mini kit for general cell membrane labelling) from Sigma Staining of GM-DC/Raw with anti-CD11c antibodies Coculture of DCs and cells to be taken up (1(-2)x105 + 1(-2)x105 cells in 12-well) for 4 h to 24 h at 37°C Take supernatant and freeze at -80°C for analysis of DC-stimulation Trypsinize cells have and fix in 4 % PFA (or formalin) FACS analysis

Protocol for the documentation and the analysis of the interaction between RIG-I, a viral sensing protein, and NS1, an inhibitor of interferon production that is encoded by influenza A virus. This protocol has been made available.

1 Saccharomyces cerevisiae cell stimuli Saccharomyces cerevisiae strain SK1 (MATa/alpha HO gal2 cupS can1R BIO, Kane SM and Roth J. 1974 Bacteriol. 118: 8-14) was cultured in complete medium (YPD, 2% yeast extract, 1% peptone, 2% glucose) for 18 hours, then collected, washed twice with sterile water and resuspended at 108 cells/ml. S. cerevisiae strains BY4741 (genotype, Mata his3delta1 leu2delta0 met15delta0 ura3delta0) and BY4741 och1 (Mata his3delta1 leu2delta0 met15delta0 ura3delta0 OCH1::kanMX4) were cultured in complete medium till exponentially phase and t...

Array construction: The yeast oligonucleotide array was constructed using the S. cerevisiae Genome Oligo Set ™ (Operon Technologies, CA, USA) composed of 6240 optimized oligonucleotides (70mers) each representing one yeast gene. Probe preparation and hybridization: We used the indirect labeling method. Briefly, the reactive amine derivative of dUTP, 5-(3- aminoallyl)-2?-deoxyuridine 5?-triphosphate (Sigma) was incorporated into cDNA using the Superscript II reverse transcriptase (Invitrogen) and oligo dT (Invitrogen) and random examers (Roche). After synthesi...