For immuno-monitoring we established methods and protocols for detection of antigen-specific T cell populations in human and mice. Particularly we have identified CD40L as a unifying marker for all antigen-reactive T cells. This will significantly improve the possibilities to characterize specific immune responses, e.g. following vaccinations with dendritic cells.

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.

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...

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 established protocols for the efficient in vitro transfection of primary dendritic cells based on viral and non-viral (nucleofection) methods (A. Mantei). These have been applied to modify DC surface molecules (Notch ligands) in the murine system (S. Vaddakadathou) or deliver antigens in both murine and human DC into either the MHC I or MHC II presentation pathways (A. Sattler, M. Dziubainau). For the latter we used a MHC II presentation pathway targeting vector originally published by Wang et al. 1999. Proof of principle studies for antigen delivery have been ...

This methodology incorporates a unique sample preparation procedure based on 96 well plate complex sample trypsinization, a nano-LC-based peptide separation coupled to MSMS analysis sequencing. This procedure led us to the identification of 600 phagosomal proteins from murine DC phagosomes.

Leukapheresis products from cancer patients was used as a starting material for monocyte enrichment by elutriation technology with ELUTRA® (Gambro). The monocytes were cultured for five days to immature DC (imDC´s) and subsequently transfected with different constructs encoding for the oncogene Her-2/neu and as control PSA using the technology of Amaxa biosystems or an adeno virus Her-2 full length construct. Levels of Her-2 expression was highest following transfection with 3 different truncated Transmembrane-extracellular constructs, in particular a Her-2 ra...

Argos has developed a novel DC maturation method that dramatically improves DC immunopotency as assessed by in vitro assays compared to DCs matured using the common ‘cytokine cocktail’ method. This new maturation protocol involves transfecting CD40L-encoding RNA along with the tumor RNA payload after maturation with TNF-alpha, IFN-gamma, and PGE2. DC prepared by this method secrete large amounts of IL-12 and no IL-10. This DC platform is now known as ArcelisTM.

Sample pre-processing and biotin labeling were performed using the Affymetrix GeneChipH cDNA Synthesis Kit and IVT Labeling Kit (Affymetrix) according to the manufacturer’s protocols. Microarrays were then hybridized on Affymetrix GeneChipH HG-U133A 2.0 microarrays, and scanned according to the manufacturer’s instructions on a GeneChipH Scanner 3000 (Affymetrix). Extraction, hybridization and scanning were performed by the Genopolis consortium (University of Milano-Bicocca, Italy).

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...

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 ...

Lentiviral infection of target cells: Day 1: seed cells Day 2: remove the medium from the cells. Mix the medium containing the virus gently by pipetting and add to the cells (+ 6 ?g/ml of polybrene). Incubate the cells at 37°C overnight. Day 3: change medium and start selection for stably transduced cells

Production of lentiviruses: Producing lentivirus in 293 FT Cells: Day 1: 4 x 106 293 FT in 10 ml DMEM (+10 % FCS (Tet free), +P/S) in a 10cm plate Day 2: transfection Day 3: change of medium Day 4: harvesting

a) Incubate cells in Fc-block (1:50 in PBS + 2 % FCS) 30 min on ice b) Staining of the cells in PEalphaDC80 in PBS + 2 % FCS + c) Fc-block for 30 min on ice d) FACS analysis

PBMC were isolated from buffy coats by density gradient centrifugation using Biocoll (BIOCHROM). Monocytes were isolated from PBMC using MACS anti-CD14 microbeads and a Midi-MACS® magnetic cell sorting device (both from Miltenyi Biotec, Bergisch-Gladbach,Germany). Cells were cultured in RPMI 1640 medium (GIBCO BRL) supplemented with 2 mM L-glutamine (Sigma), 1% (vol/vol) non-essential amino acids, 100 mM sodium pyruvate, 50 U/ml of penicillin and 50 mg/ml of streptomycin (Gibco BRL) containing 10% (vol/vol) FCS (Hyclone). Differentiation of monocytes into dendriti...

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

Monocyte source: leukapheresis product (rarely: buffy coats of healthy blood donors, research use only) => gives high yield of monocytes (15-25 x 10e8) Monocyte isolation: affinity purification (CliniMacs) or counter-flow centrifugation (Elutra) => good recovery, high purity, high viability Culture: in culture bags (Teflon Bags, Cell Genix) in serum free medium (CellGro from Cell Genix) => no contact-mediated activation, no interference by serum proteins For research use (sometimes) generation of adherent DC by plating monocytes on plastic. Cytokines: clinical gra...