These procedures have led to an improvement in processing of small RNA samples. Studies of 3’ IVT microarrays expression profiling are obtainable starting from 2 ng of total RNA.

The optimization for the mass spectrometry analysis methods to be used in the generation of a initial proteomic map of mouse DCs (WP2) and stimuli-related phosphorylation cascades (WP5) was carried out by Lyris M. F. de Godoy (Post-doc) and Jesper V. Olsen (PhD student). Recently, our lab has been developing new techniques in quantitative proteomics and has also acquired new instrumentation to assess these types of questions in depth. This, of course, involves a lot of optimization and, consequently, large amounts of sample. However, to obtain high numbers of dend...

We have developed a technology to obtain from melanoma metastases melanin-free total tumor RNA, suitable for in vitro amplification and DC transfection.

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.

Protocols were developed to measure human immune responses to melanoma antigens to be measured using ELISpot and tetramers. The antigens in question were also inserted into adenovirus, lentivirus and vaccinia vectors for re-stimulation studies.

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...

Untouched naive CD4+ T cells are isolated from CD14- PBMC with a combination of magnetic sorting.

Purified CD8 T cells using MACS cell separation were co-cultured with electroporated DC. After several stimulations, the CD8 T cells become evaluated on their cytokine secretion, cytotoxicity and % of antigen specific T cells.

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

IFNgamma ELISPOT of peptide pulsed, leukapheresis derived PBMC Tumor antigen specific activation of CD8 and CD4 DTH infiltrating T cells (CD137, CD107a, CD154, cytokines TNFalpha and IFNgamma)

HLA-A1 and/or HLA-A2 positive patients are immunized with autologous DC loaded with KLH, as immunological tracer, and an allogeneic peptidome (i.e. natural tumour peptides, NTPs), obtained from melanoma cell line SK-Mel24.

MAGE-3 positive patients are immunized with autologous MAGE-3 and TK transduced activated T blasts.

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.

RNA preparation, labeling, hybridization on a HT12 array (Illumina), and scanning were performed according to Illumina instructions by Genomics Lab, Wellcome Trust Centre for Human Genetics (Univeristy of Oxford).

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:...

(Basic manufacturing and QC SOPs on the Generation of Peptide loaded DCs have been provided to DC-THERA participants and associated partners via DC-THERA intranet. )

(Basic manufacturing and QC SOPs on the Generation of cytokines and media components have been provided to DC-THERA participants and associated partners via DC-THERA intranet. )