Surface markers were used to establish 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 CD45RA surface markers (and additionally, CD8, CD4, CD3, 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.

Surface markers were used to establish 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 CD8 surface markers (and additionally, 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.

We studied the correlation between CD8 multiple cell surface markers and functional profiles studied at single cell level. For that purpose, we subdivided peripheral CD8 T cells into eleven different cell subtypes based on the association of multiple cell surface markers. In each subtype, we isolated single-cells. In each single cell, we quantified the expression of multiple genes. Moreover, we isolated and studied cells from different normal donors.

The crude population of DC was divided into the three known splenic subsets (CD8+, CD4+, DN DC) using Fluorescence activated cell sorting (BD FACS Aria). This procedure allowed to obtain highly pure fractions of each subset. However, because DC are a very rare population, this method is very material- and cost-intensive. Furthermore, it is not surprising that the obtained cell numbers of each subset after isolation are below 107. However, these cell numbers should be sufficient for proteomic investigations. The proteomes of all three subsets using mass spectrometry is in progress.

These T cells were associated with multiple markers into 14 different cell types to identify subsets. The findings thus describe new CD8+ cell subsets, allow the identification of relatively homogeneous CD8+ subpopulations, provide a predictable and precise correlation between particular cell surface markers and CD8+ T-cell functional properties, and identify effector cells present in both CCR7-CD45RA+ and CCR7-CD45R0+ compartments. The results also indicate that activated cells might modulate the expression of CD45RA/R0 asynchronously rather than CCR7-CD45...

We observed that ICAM-1 expression by mature DCs is critical for long-lasting contacts with CD8+ T cells, but dispensable for short-lived antigen-specific interactions. Serial brief T cell-dendritic cell contacts induced early CD8+ T cell activation, proliferation and effector CTL differentiation in the first few days after immunization.

Down-regulation of CD83 expression on human DC through RNA interference (RNAi) results in a less potent induction of allogeneic T cell proliferation, reduced IFN-gamma secretion by established T cells and decreased capacity in the priming of functional tumor antigen-specific CD8+ T lymphocytes. In addition, CD83 mRNA-electroporated DC are stronger T cell stimulators. However, CD83 overexpression on Melan-A/MART-1-specific tumor-infiltrating lymphocytes (TIL) circumvents the need for CD83 expression on DC. Co-culture of immature DC with TIL or K562 cells overex...

CEA specific CD4+ T cells were found both in normal donors and in patients with high-grade cervical lesions, pancreas adenocarcinoma and advanced melanoma but CD4+ T cells from the patients compared to normal donors showed impaired IFN-gamma production.

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”.

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

Manufacturing of cGMP RNA to be used as an API (active pharmaceutical ingredient) in the planned Tri-Mix DC RNA Trial will be performed after obtaining the manufacturing license for RNA loaded DCs.

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

4 ml of chloroform (Fischer BP1145-1) were added to buffered and incubated S. cerevisia cells in a process of total RNA extractions.

A comprehensive protein inventory of clinical grade immature and cytokine cocktail matured (Il-6, IL-1 beta, TNF alpha, PGE2; 48 hours) monocyte derived human dendritic cells (DC) from a healthy donor has been established by using high accuracy, high sensitivity protein identification technology. We have identified 2794 proteins in DCs by liquid chromatography tandem mass spectrometry. Prior to MS analysis, DC were lysed and divided into a soluble fraction containing cytosolic proteins and into an insoluble fraction enriched for membrane containing proteins. Hi...

CLL-DCV01 is used for multiple injections in patients with previously treated B-cell Chronic Lymphocytic Leukemia.

Our current efforts in a study of functional homogeinity involve a transition to 8 colours surface staining, and the evaluation of antigen-specific cell sets identified with MHC tetramers. For the later process, we initiated data collection of EBV, CMV and HIV specific T cell sets.

Co-cultures of mDCs and pDCs in response to CpGs, LPS and bacterial particles were studied to identify a possible cooperation between myeloid and plasmacytoid DCs in response to different microbial stimuli, and to characterize the mechanisms of this cooperation.

These DC were co-electroporated with constitutively active TLRs and TAA encoding mRNA. They were used to compare different maturation methods.

In the process of total RNA extraction from yeast cells, the supernatants were precipitated in RNase-free centrifuge tube by adding 1/10 volume 3 M NaAcetate (pH 5.3) and 2.5 volumes of cold ETOH 100%.

Long peptides which were covalently conjugated to either the TLR2 ligand or TLR9 ligand, Pam3CysSK4 or CpG have been used to study the uptake, antigen presentation, and induction of specific T-cells.