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).

(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. )

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...

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

We submitted descriptions of protocols (SOPs exist) used for expanding patient derived T cells into tumor reactive CTLs, based on our special skills in monocyte isolation by elutriation or adherence methods and in DC-T cell co-cultures as well as in immunomonitoring (all applied in our clinical studies described in the knowledge portal). We also included information on transfection of mature DC by electorporation or using adenoviral constructs.

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.

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.

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...

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...

The vaccine is composed by autologous DCs pulsed with apoptotic allogenic prostate carcinoma cell line LNCap.

(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. )

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 vaccine is composed by autologous DCs electroporated with mRNA encoding CD40L, CD70, caTLR4 and one tumorantigen (gp100, Tyrosinase, Mage-C2 or Mage-A3).

We have a clinical protocol for immunotherapy of melanoma patients in preparation, however many details are not yet finalized. Everything is carried out under GMP conditions according to SOPs. In brief, patients with stage III or stage IV melanoma will undergo surgical resection of a melanotic lesion and leukapheresis will be performed on the following day. DCs will be generated from monocytes after elutriation of the leukapheresis product. On day 5 of culture immature DC will be pulsed with irradiated tumor cells (apoptotic bodies) prepared from the removed le...

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

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 have a current clinical protocol for active immunotherapy in advanced melanoma patients (GMP SOPs covering the protocols are also available). The vaccine is composed by autologous DCs pulsed with allogenic melanoma peptides (SK-Mel 24, HLA-A1 and HLA-A2 positive) plus KLH as immunological tracer and it is produced under GMP condition at MolMed S.p.A. (DIBIT, San Raffaele, Milan, Italy). HLA-A1 and/or HLA-A2 positive patients with stage III/IV melanoma undergo leukapheresis. DCs are generated from adherent peripheral blood monocytes, at day 6 DC are pulsed with ...