These T cells stem from an elutriation process from a metastatic lesion of stage III/IV melanoma patients. Subsequently, these T cells will be expanded by co-culture with tumor lysate loaded DC.

Commercially available siRNA Smart Pool from Dharmacon targeted against the STAT3 transcription factor have been successfully transfected in these human MDDCs.

The cell populations which infiltrate progressive versus regressing P815 mastocytoma were examined in order to identify cells which display immunosuppressive properties. We found changes in regulatory T cells populations as well as in the “myeloid suppressor cells”.

An HSV vector expressing full length tyrosinase, gp100 and MART-1 was used to transduce DC from melanoma patients which were then to be used for vaccination.

We have efficiently transfected DC with RNA encoding a functional protein (E/L-Selectin) which allows entry of DC into LN from HEV. The novel adherence capacity of human E/L-S transfected DC was demonstrated via sticking to sialyl-LewisX coated slides using a parallel plate flow microscope. RNA transfected human DC could be frozen and thawed without loosing their functionality.

We have efficiently transfected DC with RNA encoding a functional protein (E/L-Selectin). The mouse E/L-S transfected DC, when given i.v., homed to the lymph nodes, whereas non transfected DC home only to the spleen.

A vaccination trial with RNA transfected autologous DC was done with overlapping peptides covering the whole protein sequence of the tumor antigens MageA3, MelanA and Survivin.

The expression and function of murine FcgammaR in these CD11c+CD11b-B220+ plasmacytoid DCs was investigated.

These Treg were induced after treatment of primed mice with intact anti-CTLA-4 antibodies. 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.

These monocytes were extracted from a Leukapheresis product from cancer patients using elutriation.

These DC were generated by culturing enriched monocytes for 5 days.

The receptors of these DC were blocked by addition of laminarin, mannan and chitin.

These DC still have functional receptors and are exposed to the receptor antagonist laminarin in order to block them.

The DC were matured with inflammatory cytokines followed by electroporation with TAA encoding mRNA. They were used to compare different maturation methods.

Cells stained in PE-alpha DC80 in PBS + 2 % FCS for FACS analysis for activation markers.

These cells are incubated in Fc-block (1:50 in PBS + 2 % FCS) 30 min on ice, to be stained for FACS.

Dendritic cells were matured with TNF alpha, IFN gamma and PGE2. These DC were used to produce a DC vaccine.

These DC were used in a maturation step for subsequent production of a DC vaccine.

These cells are to be stained and are the input for FACS staining for activation markers.

By selecting for cytokine receptor expression relevant to DC development, we were able to identify highly cycling Lin–c-KitintFlt3+M-CSFR+ cells with a distinct gene-expression profile in mouse bone marrow that, on a clonal level in vitro and as a population both in vitro and in vivo, efficiently generated plasmacytoid and conventional DCs but no other lineages, which increased in number after in vivo injection of the cytokine Flt3 ligand.