Surface markers were used to establishe routine monitoring of tumor-specific T-cell subsets by multicolor flow cytometry. For this purpose multimers were used kindly provided by Pierre Coulie (Brussels) together with CD25 surface markers (and additionally, CD8, CD4, CD3, CD45RA, CCR7, CD27, CD137, CD127) in different 8-color panels on the BD FACSCanto II. Additionally, functional markers such as CD107a were used and intracellular cytokine staining for the further analysis of individual T-cell populations and T-cell clones.

These surface markers were used to establishe routine monitoring of tumor-specific T-cell subsets by multicolor flow cytometry. For this purpose multimers were used kindly provided by Pierre Coulie (Brussels) together with surface markers (such as CD8, CD4, CD3, CD45RA, CCR7, CD25, CD27, CD137, CD127) in different 8-color panels on the BD FACSCanto II. Additionally, functional markers such as CD107a were used and intracellular cytokine staining for the further analysis of individual T-cell populations and T-cell clones.

IFN alpha was used in a DC maturation process with the final aim to produce a DC vaccine.

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

Laminarin (500 micrograms/ml) was added to DCs in order to block their beta-glucan receptors.

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

This DC vaccine was produced from matured DC (matured using TNF alpha, IFN alpha and PGE2) which were subsequently transfected with CD40L-encoding RNA and tumor RNA.

This RNA was used to transfect matured DC with the final aim to produce a DC vaccine.

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

This PGE2 was used in a DC maturation process with the final aim to produce a DC vaccine.

The effect of environmental factors on development of high affinity CTL was investigated using a system whereby OT-1 cells were primed in vitro by engineered APC. After a 20 h priming phase, CTL were transferred to recipient mice that were either naïve, or had been injected with activated dendritic cells one day earlier, thus creating a reactive lymph node.

TNF alpha was used for DC maturation with the final aim to produce a DC vaccine.

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

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

PE-alpha DC80 is used for staining of incubated cells 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.

PBS (cells in FC-block 1:50) + 2 % FCS was used in incubation of cells for FACS staining for activation markers.

The FC-block was used for incubation of cells (1:50 in PBS + 2 % FCS) for FACS staining of activation markers.

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

A recombinant Leishmania major parasite expressing a fluorescent tracer was used for the infection of BALB/c (H2-d) mice. This was done to investigate whether infected DC process pathogen-derived antigen and stimulate antigen-specific CD4+ T cells in vivo.