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...

Untouched naive CD4+ T cells are co-coltured with fungi -pulsed DCs and then the polarization is assessed by flow cytofluorimetry and cytokine production.

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

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.

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.

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)

Protocols were developed to refold in vitro CD1 molecules, which were used for the generation of CD1 tetramers. The ability to generate CD1d tetramers has provided us with the opportunity of comparing a broad panel of CD1d binding compounds for their ability to stimulate iNKT cells.

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

The vaccine is composed by autologous DCs pulsed with apoptotic autologous ovarian carcinoma cells. Apoptosis is induced by UV.

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

A single step procedure for the in vitro maturation and antigen loading of human moDC was optimized. DC were electroporated with mRNA encoding tumor antigens and a putative TLR3 ligand. In vitro stimulation of naïve T cells indicated the superiority of this approach compared to in vitro stimulation with DC matured with inflammatory cytokines followed by electroporation with TAA encoding mRNA.

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.

We have efficiently transfected DC with RNA encoding a functional protein (E/L-Selectin) which allows entry of DC into LN from HEV. RNA transfected human DC could be frozen and thawed without loosing their functionality.

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

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).

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 are progressing steadily towards the delivery of a standardized method for the immuno-monitoring of DC vaccination trials using a finite number of defined tumor antigens. Our starting point was the method that we had developed previously to estimate blood frequencies of CD8 T lymphocytes against one defined antigenic peptide. It was based on an in vitro restimulation of blood lymphocytes with the peptide and growth factors over two weeks followed by detection with the appropriate HLA class I tetramer. In order to estimate frequencies of responding cells, these ...

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.

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.