From the CD14- PBMCs, untouched naive CD4+ T cells were isolated with a combination of magnetic sorting.

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.

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.

These HIV strains were used to infect Human Hemato-Lymphoid System Rag2gc-/- mice to closely resemble HIV infection in humans.

We found that CCR6–deficient (CCR6-KO) mice were resistant to induction of EAE but became susceptible when transferred with small number of CCR6-sufficient T cells. CCR6 was found to be required on a first wave of TH-17 cells that entered the CNS through the choroid plexus epithelial cells, which constitutively expressed CCL20 in both mice and humans.

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.

Three patients were enrolled in a clinical trial of a DC vaccine administered into irradiated tumours in RCC. However only this one patient received the vaccine due to cancer progression in the other two patients. The DC were labelled with 111In. The following patients will receive DC labelled with Endorem and the vaccine will be imaged with MRI. The patient treated is in complete remission 6 months after treatment.

The Pharmacological compound 1?,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of Vitamin D3, and its analogues, was evaluated for its effect on the DC differentiation/activation pathway induced by type I IFN. The suppressive effect of 1,25(OH)2D3 was associated with a potent impairment of DC migration in response to inflammatory and lymph node homing chemokines, thus unraveling a novel mechanism involved in 1,25(OH)2D3-mediated immunomodulation.

We are collecting blood (and ascitic fluid) of ovarian cancer patients to study the presence of immature myeloid populations as compared to healthy donor blood. We have identified two candidate populations that will be tested for the ability to suppress T cells.

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

Blood was collected from >10 melanoma patients and the myeloid derived suppressor cells (MDSC) phenotype and function were compared with healthy donor derived cells. This blood was used to investigate phenotypic markers that can be used to further characterize MDSC, focusing on molecules that are related to their suppressive function.

The yeast cells were resuspent in 3.6 ml AE Buffer and then transferred in 15 ml tubes wit 200 µl 10% SDS (w/v). 2 ml of preheated acid phenol (pH 4.3) were added and the falcon were mixed by vortex.

The organization of bmDC, B lymphocytes and plasma cells in the BM of the long bones was studied by immunochistochemistry. Using CX3CR1GFP mice, in which bmDC are green fluorescent (GFP), the bmDC localization was analyzed and it was found that the cells were organized in unique clusters, mainly located in the endosteum of the BM. These DC co-localize with B cells and these clusters are occupying architecturally definable niches and are reminiscent to the niches that were found in the cranial BM parenchyma. By using Fibronectin antibody the stroma of the BM co...

We found that the cooperation between IFNalpha,beta and Flt3L (FL) plays an important role in the defense against Herpes simplex virus type 1 (HSV-1) in neonates. Treatment of neonatal mice with recombinant IFNalpha has a short-term, FL-independent and a long-term, FL-dependent protective effect against HSV-1. In mice lacking FL, neonatal resistance against HSV-1 is very low and DC numbers in the spleen are reduced. In C57BL/6 mice, treatment with rIFNalpha at birth induced both FL and plasmacytoid DC (pDC) that resulted in enhanced resistance against HSV-...

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.

PBMC were isolated from this buffy coat. Subsequently, monocytes were isolated from the PBMC and dendritic cells generated from the monocytes.

These mice were immunized with OVA and used in a DC staining experiment. LN cells were purified 2 days later, and stained with either 2C44, 2F74, 2E60 or 2X8 mAb. None of these mAb stained DC purified from non immunized mice. However, among the 4 mAb, only 2C44 could stain DC in LACK-immunized mice, but not in OVA-immunized mice.

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. BDCA-3+ cells seem not have any capability to respond to TLR agonists.

These mice were studied with regard to responses to viral infections and compared to TLR K/O mice. We used the mouse pox virus Ectromelia and MVA-BN.