Exosomes are nanometer particles (50-100 nm) secreted by most living cells. The first description of exosomes was made in 1987 by Rose Johnstone, who described a vesicle formation during the maturation process of reticulocytes. At this time it has been suggested that exosome release could represent a major route for the externalization of obsolete membrane proteins. A renewed vision of exosome function was raised when Graça Raposo demonstrated in 1996 that exosomes derived from B cells could have immunogenic capacities. Since then, exosomes have been described i...

Dendritic cells (DCs) are pivotal regulators of immune reactivity and immune tolerance. The observation that DCs can recruit naive T cells has invigorated cancer immunology and led to the proposal of DCs as the basis for vaccines designed for the treatment of cancer. Designing effective strategies to load DCs with antigens is a challenging field of research. The successful realization of gene transfer to DCs will be highly dependent on the employed vector system. Here, we review various viral and non-viral gene transfer systems, and discuss their distinct charact...

CD4(+)CD25(+) regulatory T cells (Treg) have been described as an important hurdle for immunotherapy. Engagement of glucocorticoid-induced TNF receptor-related protein (GITR) has emerged recently as an important mechanism to control the suppression of CD4(+)CD25(+) Treg. Furthermore, it has been documented extensively that GITR ligation is costimulatory for naive and activated T cells in the murine setting. However, little is known about the role of the human GITR ligand (huGITRL). We wanted to explore whether huGITRL could enhance antigen-specific T cell priming...

Distinct dendritic cell (DC) subsets have been suggested to be preprogrammed to direct either T helper cell (Th) type 1 or Th2 development, although more recently different pathogen products or stimuli have been shown to render these DCs more flexible. It is still unclear how distinct mouse DC subsets cultured from bone marrow precursors, blood, or their lymphoid tissue counterparts direct Th differentiation. We show that mouse myeloid and plasmacytoid precursor DCs (pDCs) cultured from bone marrow precursors and ex vivo splenic DC subsets can induce the developm...

Flt3-ligand is a nonredundant cytokine in type I interferon-producing cell (IPC) and dendritic cell (DC) development. We demonstrated that IPC and DC differentiation potential is confined to Flt3(+)-hematopoietic progenitor cells, that Flt3-ligand drives development along both lymphoid and myeloid developmental pathways from Flt3(+)-progenitors to Flt3(+)-IPCs and -DCs, and that in vivo pharmacologic inhibition of Flt3-signaling leads to disruption of IPC and DC development in spite of consecutive Flt3-ligand upregulation in treated animals. We here summarize our ...

Over the years, the unique capacity of dendritic cells (DC) for efficient activation of naive T cells has led to their extensive use in cancer immunotherapy protocols. In order to be able to fulfil their role as antigen-presenting cells, the antigen of interest needs to be efficiently introduced and subsequently correctly processed and presented by the DC. For this purpose, a variety of both viral and non-viral antigen-delivery systems have been evaluated. Amongst those, HIV-1-derived lentiviral vectors have been used successfully to transduce DC.This review consi...

Human blood contains two populations of dendritic cells (DCs) termed plasmacytoid and myeloid. Myeloid DCs (mDCs) are subdivided into three subsets using the surface markers CD16, CD1c, and BDCA-3. Their differential role as pathogen sentinels and adjuvant targets was tested by extensive phenotypic and functional analysis. We show that mDC subsets are immature and express mRNA for most TLRs, except for TLR3 in CD16-mDCs. The most represented subsets, CD16 and CD1c-mDCs, are similarly responsive to all TLR agonists. Among 31 cytokines tested, both subsets produce C...

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutic strategies. One of the approaches that has gained prominence in recent years is therapeutic vaccination. We decided to assess the capacity of mature dendritic cells, derived from blood monocytes of HIV-1 infected patients, to generate functional T-cell responses. For this purpose, we constructed a chimeric mRNA encoding the proteins Tat, Rev and Nef. The TaReNef encoding information was linked to the HLA class II-targeting sequence of DC-L...

Tumor regressions have been observed in a small proportion of melanoma patients vaccinated with a MAGE-A3 peptide presented by HLA-A1, administered as peptide, ALVAC canarypox virus containing a MAGE-A3 minigene, or peptide-pulsed dendritic cells (DC). There was a correlation between tumor regression and the detection of anti-MAGE-3.A1 CTL responses. These responses were monoclonal and often of a very low magnitude after vaccination with peptide or ALVAC, and usually polyclonal and of a higher magnitude after DC vaccination. These results suggested that, at least...

IL-10 is a major regulator in inflammatory responses. Although various transcription factors were defined to enhance IL-10, the molecular mechanism for the initiation of Il-10 transcription, remains unknown. mRNA profiling of six distinct primary CD4+ T cell populations showed differential expression of the transcription factor GATA-3 correlated with levels of IL-10 expression. We showed that ectopic expression of GATA-3 in naive primary CD4+ T cells enhanced expression of IL-10 by these cells and uncovered a possible mechanism for this effect. We found that GATA...

Background The generation of Ag-loaded DC under good manufacturing practice (GMP) conditions is logistically challenging and further compounded when the starting precursors need to be purified from B-CLL patients who have overwhelming numbers of circulating B-CLL cells and decreased numbers of monocytes. Methods We have previously demonstrated that DC with endocytosed B-CLL apoptotic bodies are powerful stimulators of anti-leukemic T cells. In this study we compared counterflow elutriation and immunomagnetic separation for enriching monocyte precursors, and eva...

There is a growing interest in using dendritic cells (DC) for vaccine approaches in the treatment of cancer and infectious diseases. This requires a reproducible method for the generation of large numbers of DC in a closed culture system suitable for clinical use and conforming to the current guidelines of good manufacturing practices. We designed a system in which the DC were generated in a closed system from adherent monocytes using Cell Factories (DC-CF). Monocytes were enriched from apheresis products by adherence and then cultured in the presence of AB serum...

We describe here a protocol for the representative amplification of global mRNAs from typical single mammalian cells to provide a template for high-density oligonucleotide microarray analysis. A single cell is lysed in a tube without purification and first-strand cDNAs are synthesized using a poly(dT)-tailed primer. Unreacted primer is specifically eliminated by exonuclease treatment and second strands are generated with a second poly(dT)-tailed primer after poly(dA) tailing of the first-strand cDNAs. The cDNAs are split into four tubes, which are independently d...

The glycosphingolipids globotrihexosylceramide (Gb3, CD77) and isoglobotrihexosylceramide (iGb3) are isomers differing only in one glycosidic bond and have been implicated in several processes of the innate and adaptive immune system. AIMS: 1) To verify the function of Gb3 in the pathogenesis of hemolytic-uremic syndrome as the cellular receptor responsible for cytotoxicity caused by verotoxin (VT) elaborated by Shigella and certain strains of E.coli. 2) To investigate in vivo the previously implicated function of iGb3 as the endogenous lipid ligand responsible f...

Dendritic cells (DCs) play a key role in the orchestration of the immune system by virtue of their capacity to control both immunity and tolerance induction. The functions of DCs depend on the subset as well as their location and activation state. A number of in vitro protocols, developed to recapitulate DC generation from hematopoietic precursors, have suggested the importance of microenvironment and cytokine milieu in driving the generation of DCs endowed with peculiar phenotypic and functional features. Recently, some important concepts on the development of D...

To optimize vaccination strategies, it is important to use protocols that can 'jump-start' immune responses by harnessing cells of the innate immune system to assist the expansion of antigen-specific B and T cells. In this Review, we discuss the evidence indicating that invariant natural killer T (iNKT) cells can positively modulate dendritic cells and B cells, and that their pharmacological activation in the presence of antigenic proteins can enhance antigen-specific B- and T-cell responses. In addition, we describe structural and kinetic analyses that assist in...

Melanoma metastases are characterized by pronounced neo-angiogenesis and spontaneous bleeding frequently occurring within central nervous system metastases. Clinically apparent spontaneous hemorrhage within subcutaneous melanoma metastases, however, is a rare event that coincides with progression of such metastases. We report, to our knowledge the first observation, on regression of subcutaneous metastases with hemorrhage of the overlying skin in three patients with stage IV melanoma who participated in clinical trials on therapeutic vaccination. In two patients,...

To study DNA vaccination directed against human HER-2 in the HHD mouse Tg strain, we created a novel HER-2-expressing syngeneic tumor transplantation model. We found that a DNA vaccine encoding the full length HER-2 DNA protected HHD mice from HER-2(+) tumor challenge by a CTL independent mechanism. A more efficient approach to induce HLA-A2 restricted CTLs, through immunization with a multi-epitope DNA vaccine expressing the HLA-A2 restricted HER-2 369-377, 435-443 and 689-697 epitopes, resulted in high numbers of peptide specific T cells but failed to induce tu...

Mature dendritic cells (DCs) are the most potent antigen-presenting cells within the human immune system. However, Herpes simplex virus type 1 (HSV-1) is able to interfere with DC biology and to establish latency in infected individuals. In this study, we provide new insights into the mechanism by which HSV-1 disarms DCs by the manipulation of CD83, a functionally important molecule for DC activation. Fluorescence-activated cell sorter (FACS) analyses revealed a rapid downmodulation of CD83 surface expression within 6 to 8 h after HSV-1 infection, in a manner stri...