The CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) play an important role in the control of peripheral tolerance by directly inhibiting conventional T cell proliferative and effector functions. However, the mechanisms by which Treg regulate the homeostasis of lymph nodes remain unclear. In this study, we show in a mouse model that Treg control two major checkpoints dictated by the interaction between self-reactive CD4(+) T cells and resident dendritic cell (DC) in secondary lymphoid organs. First, Treg inhibit the production of CCR5 ligands, limiting the CCR5-de...

The origins of dendritic cells (DCs) are poorly understood. In inflammation, DCs can arise from blood monocytes (MOs), but their steady-state origin may differ, as shown for Langerhans cells. Two main subsets of MOs, defined by expression of different chemokine receptors, CCR2 and CX3CR1, have been described in mice and humans. Recent studies have identified the inflammatory function of CCR2highCX3CR1low MOs but have not defined unambiguously the origin and fate of CCR2lowCX3CR1high cells. In this study, we show that rat MOs can also be divided into CCR2highCX3CR1l...

The role of dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) in DC-T cell communication was assessed by analyzing the effect of DC-SIGN-blocking mAb in MLR. The results show that the degree of inhibition by DC-SIGN and LFA-1 mAb depends on the magnitude of the MLR and the maturation status of the DC. Addition of DC-SIGN mAb at several time-points during MLR showed that DC-SIGN is involved early on in DC-T cell contacts. This initial role is masked by strong adhesive and costimulatory mechanisms, indicating a short-lived effect of DC-SIGN in DC-T cell ...

Myeloid dendritic cells (DC) and macrophages evolve from a common precursor. However, factors controlling monocyte differentiation toward DC or macrophages are poorly defined. We report that the surface density of the GM-CSF receptor (GM-CSFR) alpha subunit in human peripheral blood monocytes varies among donors. Although no correlation was found between the extent of GM-CSFR and monocyte differentiation into DC driven by GM-CSF and IL-4, GM-CSFR expression strongly influenced the generation of CD1a(+) dendritic-like cells in the absence of IL-4. CD1a(+) cells gen...

Replicon plasmids encoding an alphavirus RNA replicase constitute an alternative to conventional DNA plasmids with promise for DNA vaccination in humans. Replicase activity amplifies the levels of transgene mRNA through a copying process involving double-stranded (ds) RNA intermediates, which contribute to vaccine immunogenicity by activating innate antiviral responses. Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs). Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of r...

The recognition of microbial stimuli by Toll-like receptors (TLRs) expressed on dendritic cells (DCs) is essential for the regulation of immune responses. DC activation via TLRs leads to the production of proinflammatory cytokines, chemokines and surface molecules that play a key role in the regulation and control of inflammatory reactions and adaptive immunity. Minor imbalances in the feedback control of TLR-activated innate immune cells have been associated with autoimmunity in genetically prone individuals. We review here recent studies indicating how TLR-medi...

Tumor-Associated Macrophages (TAM) represent the major inflammatory component of the stroma of many tumors, able to affect different aspects of the neoplastic tissue. Many observations indicate that TAM express several M2-associated protumoral functions, including promotion of angiogenesis, matrix remodelling and suppression of adaptive immunity. The protumoral role of TAM in cancer is further supported by clinical studies that found a correlation between the high macrophage content of tumors and poor patient prognosis and by evidence showing that long-term use o...

Mice lacking complement components show delayed development of prion disease following peripheral inoculation. The delay could relate to reduced scrapie prion protein (PrP(Sc)) accumulation on follicular dendritic cells (DCs). However conventional DCs (cDCs) play a crucial role in the early pathogenesis of prion diseases and complement deficiency could result in decreased PrP(Sc) uptake by cDCs in the periphery. To explore this possibility, we cultured murine splenic or gut-associated lymph node cDCs with scrapie-infected whole brain homogenate in the presence or...

Cancer-germline genes (CGGs) code for immunogenic antigens that are present in various human tumors and can be targeted by immunotherapy. Their expression has been studied in a wide range of human tumors in adults. We measured the expression of 12 CGGs in pediatric brain tumors, to identify targets for therapeutic cancer vaccines. Real Time PCR was used to quantify the expression of genes MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MAGE-C2, NY-ESO-1 and GAGE-1,2,8 in 50 pediatric brain tumors of different histological subtypes. Protein expres...

The scurfy mutant mouse strain suffers from a fatal lymphoproliferative disease leading to early death within 3-4 wk of age. A frame-shift mutation of the forkhead box transcription factor Foxp3 has been identified as the molecular cause of this multiorgan autoimmune disease. Foxp3 is a central control element in the development and function of regulatory T cells (T reg cells), which are necessary for the maintenance of self-tolerance. However, it is unclear whether dysfunction or a lack of T reg cells is etiologically involved in scurfy pathogenesis and its human...

Natural killer (NK) cells are lymphocytes of the innate immune system that exert a potent function against infected and tumor cells. Although NK cells were originally defined by their capacity to lyse target cells and produce interferon (IFN)-gamma without prior activation, more recent studies found that NK cells display also a potent regulatory function. Following engagement of surface receptors by other cells or signalling by soluble molecules, NK cells release cytokines able to influence the outcome of an immune response. Since their discovery in the 1970s, the...

Success of immunotherapy with dendritic cells (DC) to treat cancer is highly dependent on their interaction with and activation of antigen specific T cells. To maximize DC-T cell contact accurate delivery of the therapeutic cells into the lymph node, or efficient trafficking of DC to the lymph nodes of the patient is essential. Since responses are seen in some patients but not in others, monitoring of the injected cells may be of major importance. Tracking of cells with magnetic resonance (MR) imaging is a non-invasive method that provides detailed anatomical info...

Single-stranded versus multimeric phosphorothioate-modified CpG oligodeoxynucleotides (ODNs) undergo differential endosomal trafficking upon uptake into plasmacytoid dendritic cells (pDCs), correlating with Toll-like receptor-9-dependent pDC maturation/activation (single-stranded B-type CpG ODN) or interferon-alpha (IFN-alpha) induction (multimeric A-type CpG ODN), respectively. As was recently shown, IFN-alpha production, other than by CpG ODNs, can also be induced in a sequence-independent manner by phosphodiester (PD) ODNs multimerized by 3' poly-guanosine (po...

Sera from colon carcinoma patients were used to identify tumor-associated antigens (TAAs) by screening tumor proteome resolved by 2D electrophoresis. A panel of six TAAs eliciting a serological immune response in colorectal cancer was identified, showing a modification in antigen recognition by B cells in patients as a function of colon cancer progression. The expression of these proteins was either confined or increased in tumor as compared to normal mucosa.

The use of tumor antigen-loaded dendritic cells (DC) is one of the most promising approaches to inducing a tumor-specific immune response. We compared electroporation of mRNA to lentiviral transduction for the delivery of tumor antigens to human monocyte-derived and murine bone marrow-derived DC. Both lentiviral transduction and mRNA electroporation induced eGFP expression in on average 81% of human DC. For murine DC, eGFP mRNA electroporation (62%) proved to be more efficient than lentiviral transduction (47%). When we used tNGFR as a transgene we observed lenti...

Stable isotope labeling by amino acids in cell culture (SILAC) has become a versatile tool for quantitative, mass spectrometry (MS)-based proteomics. Here, we completely label mice with a diet containing either the natural or the (13)C(6)-substituted version of lysine. Mice were labeled over four generations with the heavy diet, and development, growth, and behavior were not affected. MS analysis of incorporation levels allowed for the determination of incorporation rates of proteins from blood cells and organs. The F2 generation was completely labeled in all org...

We have tested the safety and feasibility of a synthetic long peptide-based HPV16-specific skin test to detect cellular immune responses to HPV16 E2, E6 and E7 in vivo. Women with cervical neoplasia (n = 11) and healthy individuals (n = 19) were intradermally challenged with 8 different pools of HPV16 E2, E6 and E7 peptides. The skin test was safe as the injections were perceived as mildly painful and no adverse events were observed. The majority of skin reactions appeared significantly earlier in HPV16+ patients (<8 days) than in healthy subjects (8-25 days). Th...

The in vivo mechanisms of action of most vaccine adjuvants are poorly understood. In this study, we present data in mice that reveal a series of critical interactions between the cholera toxin (CT) adjuvant and the dendritic cells (DC) of the splenic marginal zone (MZ) that lead to effective priming of an immune response. For the first time, we have followed adjuvant targeting of MZ DC in vivo. We used CT-conjugated OVA and found that the Ag selectively accumulated in MZ DC following i.v. injections. The uptake of Ag into DC was GM1 ganglioside receptor dependent...

If the large collection of microarray-specific statistical tools was applicable to the analysis of quantitative shotgun proteomics datasets, it would certainly foster an important advancement of proteomics research. Here we analyze two large multidimensional protein identification technology datasets, one containing eight replicates of the soluble fraction of a yeast whole-cell lysate and one containing nine replicates of a human immunoprecipitate, to test whether normalized spectral abundance factor (NSAF) values share substantially similar statistical propertie...