R848 (along with LPS, Curdlan, yeast RNA and yeast cells in different conditions of culture) was used to induce DC activation.

2M KOH were used to dissolve blue formazan particles.

We have chemically coupled the R-epimer of the Pam3CSK4 to long peptides containing a CTL epitope and used this structure to investigate the behaviour of the diastereomer.

In a process of total RNA extraction from S. cerevisia, 2 ml of preheated acid phenol (pH 4.3) were added to yeast cells that had been collected and the pellet resuspend in 3.6 ml AE Buffer (AE=50mM NaAcetate pH 5.2, 10mM EDTA).

We are characterising (in collaboration with Sebastian Amigorena) rat lymph exosomes and will determine if they transport intestinally-delivered scrapie ME7.

Electroporation of immature DCs with poly(I:C(12)U), a dsRNA analogue, resulted in phenotypic as well as functional changes, indicative of DC maturation.

The poly(I:C) was used for stimulating DCs. It was shown in mice that both IL-10 as well as IL-12 production is dependent on the signalling adaptor molecules either MyD88 or TRIF, respectively in response to CpG, LPS or PolyIC. Manufacturer: Invivogen Life Technologies

This PSA was used as a control in the transfection of immature DC. Transfection of immature DCs was done with different constructs encoding for the oncogene Her-2/neu or an adeno virus Her-2 full length construct.

The protein vaccines were injected combined with a combination of iNKT cell-agonist alpha-GalCer and MPL, a detoxified version of LPS that signals through TLR4. This combination treatment stimulates antigen-specific T and B cell responses that are greater than those elicited with alpha-GalCer or MPL alone.

These DC were generated from monocytes after elutriation of leukapheresis product (patients with stage III or stage IV melanoma) and pulsed with irradiated tumor cells (apoptotic bodies) prepared from the removed lesion and matured for 48 hours in presence of TNF-alpha, and subsequently frozen.

For a transcriptional profiling of dendritic cells, DC were stimulated with PolyI:C, pretreated or not with PP2, and microarray analysis was performed.

Differentiation of monocytes into dendritic cells is promoted, along with granulocyte-macrophage colony-stimulating factor, by addition of recombinant IL-4 (1000U/ml, R&D Systems).

A portion of the DCs is pulsed with irradiated tumor cells (apoptotic bodies) prepared from the removed lesion and matured for 48 hours in presence of TNF-alpha.

The kinetic of the transcriptional profiling of purified mouse DC treated with IL10 was analysed.

These primed T cells have been obtained from untouched naive CD4+ T cells co-cultured with fungi -pulsed DCs.

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

These mice were treated with intact anti-CTLA-4 antibodies. This induced the development of regulatory T cells expressing high levels of ICOS and producing IL-10. These regulatory T cells inhibit Th1 responses, in vitro and in vivo, and repress experimental intestinal inflammation, by a mechanism involving IL-10 and IDO.

RCC product that was tested in an RCC phase 2 trial. The product does indeed lead to restoration of both IL-2 and IFN responses and leads to a gradual decline of the immune effector T cells in the peripheral circulation (i.e., weaker signal after the fifth dose). Using the PME-CD40L DC product we have observed tumor regressions in a number of patients in the current study. At present, 6 of 12 patients that have been restaged using RECIST criteria have less tumor burden that when they enrolled in the study (15 weeks earlier).

RCC subjects were deficient in T cell IFN-gamma and IL-2 production pre-vaccination. Their response to DC-based immunotherapy was evaluated.

We showed that, in the absence of additional stimuli, inflammatory signals such as pro-inflammatory stroma-derived cytokines on DC are ineffectual in promoting DC activation and cannot substitute for engagement of innate receptors directly on DC.