Salaheddine Mécheri

Mast cell-derived exosomes induce phenotypic and functional maturation of dendritic cells and elicit specific immune responses in vivo

Mast cells (MCs) are considered major players in IgE-mediated allergic responses, but have also recently been recognized as active participants in innate as well as specific immune responses. Recent work provided evidence that MCs are able to activate B and T lymphocytes through the release of vesicles called exosomes. Here we demonstrate that exosomes, which are located in the endocytic pathway, harbor exogenous Ags that associate with other molecules endowed with immunomodulatory functions, including 60- and 70-kDa heat shock proteins. Administration to naive mice of Ag-containing exosomes in the absence of conventional adjuvants elicits specific Ab responses across the MHC II haplotype barrier. We demonstrate that MC-exosomes induce immature dendritic cells (DCs) to up-regulate MHC class II, CD80, CD86, and CD40 molecules and to acquire potent Ag-presenting capacity to T cells. Uptake and processing of Ag-associated exosomes by endogenous DCs were also demonstrated. Finally, exosome-associated heat shock proteins are critical for the acquisition by DCs of the Ag-presenting function. This work demonstrates a heretofore unrecognized collaborative interaction between MCs and DCs leading to the elicitation of specific immune responses.

Mast Cell-Dependent B and T Lymphocyte Activation Is Mediated by the Secretion of Immunologically Active Exosomes

Mitogenic activity of bone marrow-derived mouse mast cells and mast cell lines P815 and MC/9 on B and T lymphocytes is present in their culture supernatants. To identify this activity, mast cells were incubated in serum-free medium and the supernatant was subjected to differential centrifugation, which resulted in two fractions, the hypodense and dense fraction (pellet). When analyzed for their mitogenic activity on spleen cells, all activity was found to be associated with the dense fraction. Electron microscopy studies revealed the presence in this fraction of small vesicles called exosomes with a heterogeneous size from 60 to 100 nm of diameter. When cocultured with spleen cells, purified exosomes induced blast formation, proliferation, as well as IL-2 and IFN-γ production, but no detectable IL-4. Similar data were obtained by injecting exosomes into naive mice. In contrast to mast cell lines, a pretreatment with IL-4 is required for bone marrow-derived mast cells to secrete active exosomes. Structurally, exosomes were found to harbor immunologically relevant molecules such as MHC class II, CD86, LFA-1, and ICAM-1. These findings indicate that mast cells can represent a critical component of the immunoregulatory network through secreted exosomes that display mitogenic activity on B and T lymphocytes both in vitro and in vivo.

Mast Cell-Dependent Down-Regulation of Antigen-Specific Immune Responses by Mosquito Bites

While probing host skin to search for blood vessels, the female Anopheles mosquito delivers Plasmodium parasites in the presence of saliva. Saliva from various blood-feeding vectors which contains several pharmacologically active components is believed to facilitate blood feeding as well as parasite transmission to the host. Recently, we found that mosquito saliva has the capacity to activate dermal mast cells and to induce local inflammatory cell influx. Our main objective in the present work is to investigate whether saliva, through mosquito bites, controls the magnitude of Ag-specific immune responses and whether this control is dependent on the mast cell-mediated inflammatory response. Using a mast cell knockin mouse model, we found that mosquito bites consistently induced MIP-2 in the skin and IL-10 in draining lymph nodes, and down-regulate Ag-specific T cell responses by a mechanism dependent on mast cells and mediated by IL-10. Our results provide evidence for new mechanisms which may operate during Plasmodium parasite transmission by mosquito bites.

Unravelling the mast cell dilemma: culprit or victim of its generosity?

Abstract Recent data have demonstrated that mast cells are qualified to achieve specific immune responses by providing costimulation to CD4 and CD8 T cells, as well as by delivering proliferation and differentiation signals to B cells. Here, Salaheddine Mecheri and Bernard David discuss how manipulating mast cell functions may help to decipher a wide spectrum of physiological and immunopathological processes.

Capacity of mouse mast cells to prime T cells and to induce specific antibody responses in vivo

Mouse, human and rat mast cells have been shown to express major histocompatibility complex II molecules and present antigens to specific T‐cell hybridomas in vitro. The purpose of our investigation was to determine whether mouse mast cells are able to initiate specific immune responses in vivo. Induction of anti‐dinitrophenyl (DNP) immunoglobulin G1 (IgG1) and IgG2a antibodies was performed by transferring ovalbumin (OVA)–DNP‐pulsed bone marrow‐derived mast cells (BMMC), B cells, or macrophages into naive mice which were boosted later with soluble antigen. Cultured spleen cells from immunized mice were tested for their cytokine content. Our data show that mast cells were by far better inducers of anti‐DNP IgG1 antibodies than were B cells and macrophages. In contrast, anti‐DNP IgG2a response induced by macrophages was much stronger than that obtained with mast cells whereas B cells were completely unable to elicit this response. In addition to a high index of cell proliferation, spleen cells from mast cell‐injected mice produced more interferon‐γ than those mice who received macrophages or B cells by two‐ to fivefold, and almost 10‐fold, respectively. Mast cell‐deficient Wf/Wf mice were compared with their normal +/+ littermates and with mast cell‐reconstituted Wf/Wf mice to develop delayed‐type hypersensitivity (DTH) reactions as well as humoral immune responses. Mast cell sufficient mice as well as mast cell‐reconstituted Wf/Wf mice developed significantly increased DTH reactions (P = 0·02, and 0·03, repectively) and higher anti‐OVA‐specific antibody responses as compared with Wf/Wf mice. Our data suggest that mast cells have the potential to up‐regulate both humoral and cellular immune responses in vivo.

Expression of functional major histocompatility complex class II molecules on HMC‐1 human mast cells

Mast cells hold a key position in the defensive mechanisms against exogenous intruders. In this study, we investigated whether human mast cells express functional major histocompatibility complex (MHC) class II molecules that can transduce endogenous signals and present staphylococcal enterotoxin A (SEA) to T cells. Similar to HMC‐1 human mast cell line, umbilical cord blood‐derived mast cells express HLA‐DR, ‐DP and ‐DQ molecules on their surface. MHC class II molecules expressed on HMC‐1 cells bind significantly the SEA (a natural MHC class II ligand), and their ligation with specific mAbs or with SEA, leads ultrastructural chages, suggesting their degranulation. Recognition of SEA‐bound MHC class II molecules on HMC‐1 mast cells by the T cell receptor of K25 cells, an SEA‐specific murine T cell hybridoma, triggers significant IL‐2 secretion by these T cell hybridomas. Hence, our data point out the expression of functional MHC class II molecules on human mast cells, reinforcing the implication of these cells in the defense mechanisms of acquired immunity. J. Leukoc. Biol. 64: 791–799; 1998.

Nonspecific B and T cell-stimulatory activity mediated by mast cells is associated with exosomes.

Bone marrow-derived mouse mast cells (BMMC) and mast cell lines P815 and MC9 have recently been shown to induce antigen-independent B and T lymphocyte activation. It has been demonstrated that a physical contact between mast cells and B and T lymphocytes is not necessary since mast cell supernatants contain full activity. Electron microscopy studies revealed the presence in mast cell supernatants of small vesicles called exosomes with a heterogeneous size from 60 to 100 nm of diameter. When cocultured with spleen cells, purified exosomes induce B and T cell blast formation, proliferation as well as IL-2 and IFN-γ production. In contrast to P815 and MC9 mast cell lines, a pretreatment with IL-4 is required for BMMC to produce active exosomes. Structurally, these exosomes were found to harbor immunologically relevant molecules such as MHC class II, CD86, LFA-1 and ICAM-1. Here we provide for the first time the evidence that mast cells use exosomes as sophisticated messengers to communicate with cells of the immune system.

IL-4 mRNA transcription is induced in mouse bone marrow-derived mast cells through an MHC class II-dependent signaling pathway

We have previously shown that mouse bone marrow‐derived mast cells (BMMC) can process and present immunogenic peptides to CD4 T cells. Here, we report on a T cell‐dependent MHC class II‐mediated mast cell activation resulting in IL‐4 transcription and protein release. Presentation of optimal doses of ovalbumin peptide 323 – 339 resulted in IL‐2 production by a specific T cell hybridoma and increase in IL‐4 mRNA transcription in mast cells. IL‐4 mRNA transcription increased by 200‐fold in mast cells treated in IL‐3/IL‐4/granulocyte‐macrophage colony‐stimulating factor (high presenters) whereas only a tenfold increase or no increase were obtained with IL‐3/IL‐4/IFN‐γ‐ or IL‐3‐treated mast cells (low presenters), respectively. Induction of IL‐4 mRNA transcription in purified mast cells by direct ligation of MHC class II molecules, using anti‐I‐A and anti‐I‐E‐coated beads, indicates that MHC class II molecules are critical in this signaling pathway. However, when compared to T cells, anti‐MHC class II‐coated beads were less efficient, indicating a potential role of accessory molecules in this mast cell activation process. IgE‐independent IL‐4 production by mast cells as a result of cognate interaction with CD4 T cells could be critical for the development of type 2 responses. This novel mechanism may contribute to the induction and/or amplification of specific IgE‐mediated allergic responses.

FcεRI-mediated antigen endocytosis turns interferon-γ-treated mouse mast cells from inefficient into potent antigen-presenting cells

Previous studies in our laboratory have shown that bone‐marrow‐derived mast cells (BMMC) could present immunogenic peptides, from soluble antigens endocytosed through fluid phase, only if they were subjected to a 48‐hr treatment with interleukin‐4 (IL‐4) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF). In contrast to GM‐CSF, interferon‐γ (IFN‐γ) which highly upregulates major histocompatibility complex (MHC) class II expression, completely inhibits the generation of immunogenic peptides. We have used this model to study the role of FcεRI‐mediated antigen internalization in the regulation of the antigen‐presenting function of IFN‐γ‐treated mast cells. Here, we report that FcεRI can reverse the IFN‐γ‐treated mast cells from inefficient to highly efficient antigen‐presenting cells. Inhibition of the antigen presenting capacity by piceatannol, a protein tyrosine kinase (PTK) syk inhibitor, indicates that this is an active process resulting from immunoglobulin E (IgE)–antigen–FcεRI engagement which involves tyrosines found in the immunoreceptor tyrosine‐based activation motif (ITAM) embedded in the cytoplasmic tail of the FcεRI β and γ chains. Antigen‐presenting function was also shown to require the activation of phosphatidyl inositol 3 (PI3) kinase, downstream of PTK syk phosphorylation, since this activity was completely blocked by wortmannin, a PI3 kinase inhibitor. These data suggest that signalling generated by FcεRI provides mast cells with IgE‐mediated enhanced antigen presentation to T cells and emphasize a so far unknown immunoregulatory mast‐cell function that might take place in inflammatory sites.

Strain-Transcendent Immune Response to Recombinant Var2CSA DBL5-e Domain Block P. falciparum Adhesion to Placenta-Derived BeWo Cells under Flow Conditions

Background Pregnancy-associated malaria (PAM) is a serious consequence of the adhesion to the placental receptor chondroitin sulfate A (CSA) of Plasmodium falciparum-infected erythrocytes (PE) expressing the large cysteine-rich multi-domain protein var2CSA. Women become resistant to PAM, and develop strain-transcending immunity against CSA-binding parasites. The identification of var2CSA regions that could elicit broadly neutralizing and adhesion-blocking antibodies is a key step for the design of prophylactic vaccine strategies. Methodology Escherichia coli expressed var2CSA DBL domains were refolded and purified prior to immunization of mice and a goat. Protein-G-purified antibodies were tested for their ability to block FCR3CSA-infected erythrocytes binding to placental (BeWo) and monkey brain endothelial (ScC2) cell lines using a flow cytoadhesion inhibition assay mimicking closely the physiological conditions present in the placenta at shear stress of 0.05 Pa. DBL5-ε, DBL6-ε and DBL5-6-ε induced cross-reactive antibodies using Alum and Freund as adjuvants, which blocked cytoadhesion at values ranging between 40 to 96% at 0.5 mg IgG per ml. Importantly, antibodies raised against recombinant DBL5-ε from 3 distinct parasites genotypes (HB3, Dd2 and 7G8) showed strain-transcending inhibition ranging from 38 to 64% for the heterologuous FCR3CSA. Conclusions Using single and double DBL domains from var2CSA and Alum as adjuvant, we identified recombinant subunits inducing an immune response in experimental animals which is able to block efficiently parasite adhesion in a flow cytoadhesion assay that mimics closely the erythrocyte flow in the placenta. These subunits show promising features for inclusion into a vaccine aiming to protect against PAM.