Bertrand Dubois

Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells

Enteropathogenic bacteria elicit mucosal innate and adaptive immune responses. We investigated whether gut epithelial cells played a role in triggering an adaptive immune response by recruiting dendritic cells (DCs). Immature DCs are selectively attracted by the CCL20 chemokine. The expression of the CCL20 gene in human intestinal epithelial cell lines was up-regulated by pathogenic bacteria, including Salmonella species, but not by indigenous bacteria of the intestinal flora. The Salmonella machinery for epithelial cell invasion was not required for CCL20 gene activation. Flagellin but not the lipopolysaccharide was found to be the Salmonella factor responsible for stimulation of epithelial CCL20 production. CCL20 in turn triggered a specific migration of immature DCs. Our data show that crosstalk between bacterial flagellin and epithelial cells is essential for the recruitment of DCs, a mechanism that could be instrumental to initiate adaptive immune responses in the gut.

RESPECTIVE INVOLVEMENT OF TGF-BETA AND IL-4 IN THE DEVELOPMENT OF LANGERHANS CELLS AND NON-LANGERHANS DENDRITIC CELLS FROM CD34+ PROGENITORS

In vivo, dendritic cells (DC) form a network comprising different populations. In particular, Langerhans cells (LC) appear as a unique population of cells dependent on transforming growth factor β (TGF‐β) for its development. In this study, we show that endogenous TGF‐β is required for the development of both LC and non‐LC DC from CD34+ hematopoietic progenitor cells (HPC) through induction of DC progenitor proliferation and of CD1a+ and CD14+ DC precursor differentiation. We further demonstrate that addition of exogenous TGF‐β polarized the differentiation of CD34+ HPC toward LC through induction of differentiation of CD14+ DC precursors into E‐cadherin+, Lag+CD68−, and Factor XIIIa−LC, displaying typical Birbeck granules. LC generated from CD34+ HPC in the presence of exogenous TGF‐β displayed overlapping functions with CD1a+ precursor‐derived DC. In particular, unlike CD14+‐derived DC obtained in the absence of TGF‐β, they neither secreted interleukin‐10 (IL‐10) on CD40 triggering nor stimulated the differentiation of CD40‐activated naive B cells. Finally, IL‐4, when combined with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), induced TGF‐β‐independent development of non‐LC DC from CD34+ HPC. Similarly, the development of DC from monocytes with GM‐CSF and IL‐4 was TGF‐β independent. Collectively these results show that TGF‐β polarized CD34+ HPC differentiation toward LC, whereas IL‐4 induced non‐LC DC development independently of TGF‐β. J. Leukoc. Biol. 66: 781–791; 1999.

Dendritic cells directly modulate B cell growth and differentiation

A cardinal feature of Langerhans cells or dendritic cells (DC) located within the mucosal epithelium is to capture foreign antigens after tissue injury and subsequently initiate immune responses. While migrating through the draining afferent lymph into the proximal secondary lymphoid organ, DC process the antigens. Within paracortical areas of the secondary lymphoid organs, DC [referred to in this localization as interdigitating dendritic cells (IDC)] select the rare antigen-specific T and B cells [1, 2]. IDC have the unique capacity to stimulate antigen-specific naive T cells to proliferate, secrete cytokines, and express CD40L [3–5]. In murine models, immunohistological studies have demonstrated that primary T cell-dependent B cell responses were initiated within the T cell/IDC-rich areas [6]. Activated T cells stimulate antigen-specific naive B cells to proliferate and to differentiate into germinal center founder cells or into short-lived plasma cells producing essentially immunoglobulin M (IgM) [7]. The germinal center reaction starts with the colonization of primary follicles by germinal center founder cells. Antigen transporting cells have been identified that trap immune complexes in the lymph and move these complexes onto the follicular dendritic cell network within the follicles [8]. The nature of such antigen transporting cells has not yet been formally identified. Within the follicles, follicular dendritic cells, which are probably not of hemopoietic origin, retain immune complexes, thus allowing B cells to endocytose, process, and present the antigen to CD4 T cells. After intense proliferation of centroblasts in the dark zone, irreversible events occur during the germinal center reaction that lead to isotype-switched B cells with high affinity for the antigen that eventually differentiate into either memory B cells or plasma cells. Apart from the critical role of DC in the initiation of cellular immune responses [4], several experiments strongly support their direct involvement in the regulation of humoral responses. Our studies now provide evidence that human DC directly interact with B cells in vitro and regulate mature B cell responses at various stages of their differentiation.

Langerhans cells protect from allergic contact dermatitis in mice by tolerizing CD8+ T cells and activating Foxp3+ regulatory T cells

Allergic contact dermatitis is the most frequent occupational disease in industrialized countries. It is caused by CD8(+) T cell-mediated contact hypersensitivity (CHS) reactions triggered at the site of contact by a variety of chemicals, also known as weak haptens, present in fragrances, dyes, metals, preservatives, and drugs. Despite the myriad of potentially allergenic substances that can penetrate the skin, sensitization is relatively rare and immune tolerance to the substance is often induced by as yet poorly understood mechanisms. Here we show, using the innocuous chemical 2,4-dinitrothiocyanobenzene (DNTB), that cutaneous immune tolerance in mice critically depends on epidermal Langerhans cells (LCs), which capture DNTB and migrate to lymph nodes for direct presentation to CD8(+) T cells. Depletion and adoptive transfer experiments revealed that LCs conferred protection from development of CHS by a mechanism involving both anergy and deletion of allergen-specific CD8(+) T cells and activation of a population of T cells identified as ICOS(+)CD4(+)Foxp3(+) Tregs. Our findings highlight the critical role of LCs in tolerance induction in mice to the prototype innocuous hapten DNTB and suggest that strategies targeting LCs might be valuable for prevention of cutaneous allergy.

Tumor Promotion by Intratumoral Plasmacytoid Dendritic Cells Is Reversed by TLR7 Ligand Treatment

Plasmacytoid dendritic cells (pDC) are key regulators of antiviral immunity. In previous studies, we reported that pDC-infiltrating human primary breast tumors represent an independent prognostic factor associated with poor outcome. To understand this negative impact of tumor-associated pDC (TApDC), we developed an orthotopic murine mammary tumor model that closely mimics the human pathology, including pDC and regulatory T cell (Treg) infiltration. We showed that TApDC are mostly immature and maintain their ability to internalize antigens in vivo and to activate CD4(+) T cells. Most importantly, TApDC were specifically altered for cytokine production in response to Toll-like receptor (TLR)-9 ligands in vitro while preserving unaltered response to TLR7 ligands (TLR7L). In vivo pDC depletion delayed tumor growth, showing that TApDC provide an immune-subversive environment, most likely through Treg activation, thus favoring tumor progression. However, in vivo intratumoral administration of TLR7L led to TApDC activation and displayed a potent curative effect. Depletion of pDC and type I IFN neutralization prevented TLR7L antitumoral effect. Our results establish a direct contribution of TApDC to primary breast tumor progression and rationalize the application of TLR7 ligands to restore TApDC activation in breast cancer. Cancer Res; 73(15); 4629-40. ©2013 AACR.

Comparative histology and immunohistochemistry of porcine versus human skin

BACKGROUND Porcine skin is increasingly being employed as a model of human skin in various research fields, including pharmacology, toxicology and immunology, with particular interest in percutaneous permeation and organ transplantation. Porcine skin shows several anatomical and physiological similarities, but also some differences, with human skin, but few in depth comparative studies are so far available. OBJECTIVES To study the immunohistochemical properties of normal porcine skin in comparison with human skin. MATERIALS AND METHODS We performed a histological and immunohistochemical study on frozen and formalin-fixed, paraffin-embedded skin biopsies from domestic swine and normal human skin, using a panel of 93 monoclonal or polyclonal antibodies recognizing various human and porcine skin cell types or structures. RESULTS We found that several antibodies used to detect normal human skin cells showed equivalent immunoreactivity on normal porcine skin. However, some antibodies commonly used to detect human skin antigens remained unreactive on porcine skin. CONCLUSIONS Our findings highlight the main immunohistochemical properties of porcine skin in comparison with those of human skin and provide a morphological and immunohistochemical basis useful to researchers using porcine skin.

Dendritic Cells Recruitment and In Vivo Priming of CD8+ CTL Induced by a Single Topical or Transepithelial Immunization Via the Buccal Mucosa with Measles Virus Nucleoprotein

The buccal mucosa, a prototype of pluristratified mucosal epithelia, contains a network of directly accessible class II+ epithelial dendritic cells (DC), similar to skin Langerhans cells. We showed that a single buccal immunization with measles virus nucleoprotein (NP), by either topical application onto or intradermal injection in the buccal mucosa, induced in vivo priming of protective class I-restricted specific CD8+ CTL. Both routes of immunization with NP induced a rapid recruitment of DC into the mucosa, which peaked at 2 h and decreased by 24 h. Treatment of mice with Flt3 ligand resulted in an increased number of DC in the buccal mucosa and enhanced the frequency of IFN-γ-producing NP-specific effectors and the NP-specific CTL response generated after buccal immunization with NP. Finally, NP-pulsed bone marrow-derived DC induced NP-specific IFN-γ-producing cells upon adoptive transfer to naive mice. These data demonstrate that a viral protein delivered to DC of the buccal mucosa induces in vivo priming of protective anti-viral CD8+ CTL.

Functional CD40 Antigen on B Cells, Dendritic Cells and Fibroblasts

During antigen specific immune responses, antigen specific naive B cells undergo a cascade of events including activation, expansion, mutations, isotype switch, selections and differentiation into either antibody secreting plasma cells or memory B cells. These antigendependent events occur in different areas of secondary lymphoid organs, as well as other non-lymphoid organs. It requires the interaction of B cells with antigens and numerous cell types including T cells, dendritic cells (DC) and follicular dendritic cells (FDC). These cells interact with B cells through different cell surface molecules and through the release of polypeptidic mediators called cytokines.

Characterization of a CD44/CD122int Memory CD8 T Cell Subset Generated under Sterile Inflammatory Conditions

Most memory CD8 T cell subsets that have been hitherto defined are generated in response to infectious pathogens. In this study, we have characterized the CD8 T cells that survive priming conditions, devoid of pathogen-derived danger signals. In both a TCR-transgenic model and a model of contact hypersensitivity, we show that the priming of naive CD8 T cells under sterile inflammatory conditions generates memory. The corresponding memory CD8 T cells can be identified by their intermediate expression levels of CD44 and CD122. We also show that CD44/122int memory CD8 T cells spontaneously develop in wild type mice and that they display intermediate levels of several other memory traits including functional (IFN-γ secretion capacity, CCL5 messenger stores), phenotypic, and molecular (T-bet and eomesodermin expression levels) features. We finally show that they correspond to an early differentiation stage and can further differentiate in CD44/122high memory T cells. Altogether, our results identify a new memory CD8 T cell subset that is generated under sterile inflammatory conditions and involved in the recall contact hypersensitivity reactions that are responsible for allergic contact dermatitis.

Human Dendritic Cells Enhance Growth and Differentiation of CD40 Activated B Cells

During an immune response, dendritic cells (DC) capture the antigen at site of injury, and migrate through the afferent lymph stream to the lymph-nodes where they efficiently activate naive T cells. This T cell activation is followed by B cell recruitment, which occurs in the extrafollicular area, where DC home1. Accordingly, we wondered herein whether DC might directly interact with B cells, using DC generated in vitro from CD34+ progenitors, called Dendritic-Langerhans cells (D-Lc). As both DCs2 and B cells3 express functionnal CD40, we used CD40-ligand transfected L cells3 as activated T cells surrogate, to study the effect of DCs on B cell activation. We show that D-Lc enhanced both CD40-L dependent B cell proliferation (3–6 fold) and Ig productions (10–200 fold). In presence of exogenous cytokine such as IL-10, D-Lc further increased Ig production and allowed low numbers of B cells to produce detectable amounts of Igs.