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.

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 is 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.

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.

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.

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...

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”.

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, these CD107a functional markers were used and intracellular cytokine staining for the further analysis of individual T-cell populations and T-cell clones.

We have demonstrated that the target of FcgammaRs with the antigen induces the internalization of the immune complex, leading to the maturation of CD11c+CD11b+ conventional DCs. These DCs become thus efficient to present antigenic peptides to helper CD4 T cells and cytotoxic CD8 T cells via cross-presentation pathway.

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

Testing the improved RCC product PME-CD40L DC it is noteworthy that presence of CD28+ T cells in tumor lymphocytic infiltrates correlates with favorable clinical outcome.

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 CD127 surface markers (and additionally, CD8, CD4, CD3, CD45RA, CCR7, CD25, CD27, CD137) 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 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 CD137 surface markers (and additionally, CD8, CD4, CD3, CD45RA, CCR7, CD25, CD27, 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 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 CD27 surface markers (and additionally, CD8, CD4, CD3, CD45RA, CCR7, CD25, 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.

CD1 molecules are refolded in vitro in order to generate CD1 tetramers.

CD1 molecules that are subsequently to be refolded in vitro in order to generate CD1 tetramers.

The CD1d tetramers were generated from in vitro refolded CD1 molecules. 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 properties of this DC population and its production of cytokines in response to different Toll-like receptor (TLR) agonists were evaluated. The TLR agonists used were: PAM3CSK4, Poly I:C, LPS, Flagellin, Imiquimod, Resiquimod, CpG 2216, CpG 2006. We found that CD16 and CD1c produce a number of cytokines in response to these stimuli, with the exception of CpGs. In addition, we found that CD16+ DCs are the major producers of TNF-alpha and IL-6, while CD1c+ DCs produce primarily IL-8.

The properties of this DC population and its production of cytokines in response to different Toll-like receptor (TLR) agonists were evaluated. The TLR agonists used were: PAM3CSK4, Poly I:C, LPS, Flagellin, Imiquimod, Resiquimod, CpG 2216, CpG 2006. We found that CD16 and CD1c produce a number of cytokines in response to these stimuli, with the exception of CpGs. In addition, we found that CD16+ DCs are the major producers of TNF-alpha and IL-6, while CD1c+ DCs produce primarily IL-8.