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.

Pathway analysis performed with Eu.Gene (Cavalieri et al., 2005), following the algorithm developed by Beltrame et al. (2009).

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).

(Basic manufacturing and QC SOPs on the Generation of autologous plasma and serum 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. )

(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. )

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...

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.

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:...

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 met...

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...

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.

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...

(A new protocol will probably be started in the near future.)

GMP SOPs covering the protocol are also available.

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.

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.

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.

A retrotranscriptional reaction is performed on RNA extracted from T cells or DCs. Transcripts for interested genes are quantified by real-time quantitative PCR on an Perkin-Elmer Applied Biosystems with predesigned TaqMan Gene Expression Assays according to the manufacturer’s instructions.

Cytokine production by DCs and T cells is assessed by ELISA assay according to the manufacturer’s instructions of ELISA kit. After the times indicated, supernatants were collected and cytokine detection was performed. MesoScale Assay 7-spot (Meso Scale Discovery) or Luminex® Assay (Invitrogen) were used for detection of IL-1beta, IL-8, IL-6, TNFalpha, IL-10, IL-12p70, IFNg and IL-17A according to the manufacturer’s instructions. These procedures allowed simultaneously measurement of different cytokines in the same supernatants. The multiplex assay format di...