These bone marrow derived murine dendritic cells (BM-DC) were generated with GM-CSF according to Lutz et al. (J Immunol Methods 1999, 223: 77-92) and were used for the investigation of chemokine dependent CCR7 signalling.

To quantify presentation of the epitopes by DC, we used bulk T cells electroporated with TCR-encoding RNA in stimulation assays.

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

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 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.

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.

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.

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.

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.

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...

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.

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...

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.

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

We found that LPS-stimulated mDCs are able to induce up-regulation of co-stimulatory molecules on co-cultured pDCs. Likewise, CpG-stimulated pDCs activate co-cultured mDCs. The cross talk between these two populations of DC is very efficient since it can be observed at mDC/pDC ratio ranging from 1/10 to 10/1.

Monocytes were cultured in RPMI 1640 medium (GIBCO BRL) supplemented with 2 mM L-glutamine (Sigma), 1% (vol/vol) non-essential amino acids, 100 mM sodium pyruvate, 50 U/ml of penicillin and 50 mg/ml of streptomycin (Gibco BRL) containing 10% (vol/vol) FCS (Hyclone).

The cDNA’s encoding these early expressed HIV antigens have been human codon optimized and modified with lysosomal targeting sequences.