Beta-glucan receptor, mannan receptor and chitin receptor are blocking by a pre-treatment of DCs with the ligand antagonist laminarin (500 micrograms/ml) mannan (500 micrograms/ml) and chitin (500 micrograms/ml) respectively before exposure to fungi.

Phagocytosis assay Yeasts/spores were biotinylated using 10 mg/ml sulfo-NHS-LC-biotin (Sigma-Aldrich) in 50 mM NaHCO3 pH 8.5 for 2 hours at 4°C. The remaining reactive biotin molecules were inactivated by incubation in 100 mM Tris-HCl pH 8.0 for 40 minutes at 4°C. DCs were then treated with biotinylated spores/yeasts. After 1 hour, cells were permeabilized and labeled with aHLA-DR-FITC. After zymolyase treatment, intracellular yeasts/spores were detected using APC-labeled streptavidin and analyzed by flow cytometry. Receptor mediated-internalization assay I...

We previously described mRNA electroporation as an efficient gene delivery method to introduce tumor-antigens (Ag) into murine immature dendritic cells (DC). We have further optimized the protocol and evaluated the capacity of mRNA-electroporated DC as a vaccine for immunotherapy.

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

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.

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...

GMP SOPs covering the protocol are also available.

IL-12p70 inhibition by cytochalasin D DCs were exposed to cytochalasin D (10 microg/ml, TebuBio) for 30 minutes at 4°C. After washing with cold PBS, DCs were stimulated with S. cerevisiae yeast cells or spores at a DC:stimuli ratio of 4:1 for 24 hours. IL-12p70 production was assessed by ELISA. IL-12p70 blocking assay In order to assess the importance of IL-12p70 in balancing Th1/Th17 response two different experiments were performed. In one case, DCs were stimulated for 8 hours with live spores of S. cerevisiae and C. albicans hyphae at a stimuli:DC ratio of 4:...

We have developed and optimized a robust electroporation procedure to load monocyte derived and cytokine cocktail matured DC with RNA encoding defined tumor antigens (MelanA, Mage3, Survivin).

DC activation is induced by Saccharomyces cerevisiae yeast in different culture conditions, Candida albicans yeast or hyphae at different ratio stimuli:DCs. As control stimuli DCs are stimulated with LPS, Curdlan or Zymosan.

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

FACS analysis Type I IFN- production We established a very sensitive, fibroblast based assay for the detection of interferon (outside collaboration) Add the supernatant from the uptake experiments to these reporter cells. We use the Luciferase Assay System from Promega for 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...

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.

We set up a method to detect, count, and eventually clone CD4 or CD8 T cells against any peptide encoded by a given gene and presented by any HLA molecule. PBMC are stimulated in limiting dilution conditions with a pool of overlapping peptides (15 amino acids) covering the protein. After 2 rounds of in vitro stimulation, the microcultures are left without stimulation for 2 weeks, and all the individual microcultures are then screened for recognition of autologous EBV-B cells transduced with a retrovirus encoding the protein. Non transduced EBV-B cells are used as...

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).

We have adapted the cDNA amplification method of Kurimoto et al. (Nat Protoc 2007, 2:739) to cDNA extracted from small numbers (±100) of cells microdissected from human tumor sections. We are currently checking the accuracy and reproducibility of microarray (Affymetrix) data obtained through this procedure.