Laurent Mascarell

Oral dendritic cells mediate antigen-specific tolerance by stimulating TH1 and regulatory CD4+ T cells

BACKGROUND A detailed characterization of oral antigen-presenting cells is critical to improve second-generation sublingual allergy vaccines. OBJECTIVE To characterize oral dendritic cells (DCs) within lingual and buccal tissues from BALB/c mice with respect to their surface phenotype, distribution, and capacity to polarize CD4(+) T-cell responses. METHODS In situ analysis of oral DCs was performed by immunohistology. Purified DCs were tested in vitro for their capacity to capture, process, and present the ovalbumin antigen to naive CD4(+) T cells. In vivo priming of ovalbumin-specific T cells adoptively transferred to BALB/c mice was analyzed by cytofluorometry in cervical lymph nodes after sublingual administration of mucoadhesive ovalbumin. RESULTS Three subsets of oral DCs with a distinct tissue distribution were identified: (1) a minor subset of CD207(+) Langerhans cells located in the mucosa itself, (2) a major subpopulation of CD11b(+)CD11c(-) and CD11b(+)CD11c(+) myeloid DCs at the mucosal/submucosal interface, and (3) B220(+)120G8(+) plasmacytoid DCs found in submucosal tissues. Purified myeloid and plasmacytoid oral DCs capture and process the antigen efficiently and are programmed to elicit IFN-gamma and/or IL-10 production together with a suppressive function in naive CD4(+) T cells. Targeting the ovalbumin antigen to oral DCs in vivo by using mucoadhesive particles establishes tolerance in the absence of cell depletion through the stimulation of IFN-gamma and IL-10-producing CD4(+) regulatory T cells in cervical lymph nodes. CONCLUSION The oral immune system is composed of various subsets of tolerogenic DCs organized in a compartmentalized manner and programmed to induce T(H)1/regulatory T-cell responses.

A regulatory dendritic cell signature correlates with the clinical efficacy of allergen-specific sublingual immunotherapy.

BACKGROUND Given their pivotal role in the polarization of T-cell responses, molecular changes at the level of dendritic cells (DCs) could represent an early signature indicative of the subsequent orientation of adaptive immune responses during immunotherapy. OBJECTIVE We sought to investigate whether markers of effector and regulatory DCs are affected during allergen immunotherapy in relationship with clinical benefit. METHODS Differential gel electrophoresis and label-free mass spectrometry approaches were used to compare whole proteomes from human monocyte-derived DCs differentiated toward either regulatory or effector functions. The expression of those markers was assessed by using quantitative PCR in PBMCs from 79 patients with grass pollen allergy enrolled in a double-blind, placebo-controlled clinical study evaluating the efficacy of sublingual tablets in an allergen exposure chamber over a 4-month period. RESULTS We identified several markers associated with DC1 and/or DC17 effector DCs, including CD71, FSCN1, IRF4, NMES1, MX1, TRAF1. A substantial phenotypic heterogeneity was observed among various types of tolerogenic DCs, with ANXA1, Complement component 1 (C1Q), CATC, GILZ, F13A, FKBP5, Stabilin-1 (STAB1), and TPP1 molecules established as shared or restricted regulatory DC markers. The expression of 2 of those DCs markers, C1Q and STAB1, was increased in PBMCs from clinical responders in contrast to that seen in nonresponders or placebo-treated patients. CONCLUSION C1Q and STAB1 represent candidate biomarkers of early efficacy of allergen immunotherapy as the hallmark of a regulatory innate immune response predictive of clinical tolerance.

IL-10-Inducing Adjuvants Enhance Sublingual Immunotherapy Efficacy in a Murine Asthma Model

Background: IL-10-inducing adjuvants could enhance the efficacy of allergy vaccines in establishing allergen-specific tolerance.The aim of this study wasto identify such adjuvants using in vitro cultures of human and murine cells and to evaluate them in a therapeutic murine model of sublingual immunotherapy (SLIT). Methods: Adjuvants stimulating IL-10 gene expression by human or murine immune cells were tested sublingually in BALB/c mice sensitized to ovalbumin (OVA), assessing the reduction in airway hyperresponsiveness (AHR) by whole-body plethysmography. The induction of regulatory T cells (Treg) was evaluated using phenotypic and functional assays. T-cell proliferation in cervical lymph nodes (LNs) was assessed following intravenous transfer of CFSE-labelled OVA-specific T cells and FACS analysis. Results: A combination of 1,25-dihydroxyvitamin D3 plus dexamethasone (VitD3/Dex) as well as Lactobacillus plantarum were found to induce IL-10 production by human and murine dendritic cells (DCs). The former inhibits LPS-induced DC maturation, whereas L. plantarum induces DC maturation. Following stimulation with VitD3/Dex-pretreated DCs, CD4+ naïve T cells exhibit a Treg profile.In contrast, a Th1/Treg pattern of differentiation is observed in the presence of DCs treated with L. plantarum. Both adjuvants significantly enhance SLIT efficacy in mice, in association with either induction of Foxp3+ Treg cells (for VitD3/Dex) or proliferation of OVA-specific T cells in cervical LNs (for L. plantarum). Conclusions: Both VitD3/Dex and L. plantarum polarize naïve T cells towards IL-10-expressing T cells, through distinct mechanisms. As adjuvants, they both enhance SLIT efficacy in a murine asthma model.

Lactic acid bacteria as adjuvants for sublingual allergy vaccines.

We compared immunomodulatory properties of 11 strains of lactic acid bacteria as well as their capacity to enhance sublingual immunotherapy efficacy in a murine asthma model. Two types of bacterial strains were identified, including: (i) potent inducers of IL-12p70 and IL-10 in dendritic cells, supporting IFN-gamma and IL-10 production in CD4+ T cells such as Lactobacillus helveticus; (ii) pure Th1 inducers such as L. casei. Sublingual administration in ovalbumin-sensitized mice of L. helveticus, but not L. casei, reduced airways hyperresponsiveness, bronchial inflammation and proliferation of specific T cells in cervical lymph nodes. Thus, probiotics acting as a Th1/possibly Treg, but not Th1 adjuvant, potentiate tolerance induction via the sublingual route.

Induction of tolerance via the sublingual route: mechanisms and applications.

The clinical efficacy of sublingual immunotherapy (SLIT) with natural allergen extracts has been established in IgE-dependent respiratory allergies to grass or tree pollens, as well as house dust mites. Sublingual vaccines have an excellent safety record, documented with approximately 2 billion doses administered, as of today, in humans. The oral immune system comprises various antigen-presenting cells, including Langerhans cells, as well as myeloid and plasmacytoid dendritic cells (DCs) with a distinct localisation in the mucosa, along the lamina propria and in subepithelial tissues, respectively. In the absence of danger signals, all these DC subsets are tolerogenic in that they support the differentiation of Th1- and IL10-producing regulatory CD4+ T cells. Oral tissues contain limited numbers of mast cells and eosinophils, mostly located in submucosal areas, thereby explaining the good safety profile of SLIT. Resident oral Th1, Th2, and Th17 CD4+ T cells are located along the lamina propria, likely representing a defence mechanism against infectious pathogens. Second-generation sublingual vaccines are being developed, based upon recombinant allergens expressed in a native conformation, possibly formulated with Th1/T reg adjuvants and/or mucoadhesive particulate vector systems specifically designed to target oral dendritic cells.

In Vivo and in Absence of a Thymus, the Enforced Expression of the Notch Ligands Delta-1 or Delta-4 Promotes T Cell Development with Specific Unique Effects

The role of Notch signaling in T cell commitment during lymphoid development is well established. However, the identity of the ligand that triggers this critical signal in vivo is still unclear. By overexpressing Delta-1 and Delta-4 ligands in the hemopoietic cells of athymic nu/nu host mice, we demonstrate that, in vivo and in the absence of a thymus, Delta-1 or Delta-4 expression is sufficient to promote T cell development from the most immature progenitor stages to complete maturation of both CD8+ and CD4+ αβ T cells. The mature T cells developing in a Delta-1- or Delta-4-enriched environment express a diverse TCR repertoire, are able to proliferate upon in vitro TCR stimulation, but show different profiles of cytokine production after in vitro anti-CD3 stimulation.

Induction of Neutralizing Antibodies and Th1-Polarized and CD4-Independent CD8+ T-Cell Responses following Delivery of Human Immunodeficiency Virus Type 1 Tat Protein by Recombinant Adenylate Cyclase of Bordetella pertussis

ABSTRACT HIV-Tat, a conserved protein playing a key role in the early life cycle of the human immunodeficiency virus (HIV) has been proposed as a potential AIDS vaccine. An HIV-Tat-based vaccine should elicit a broad, long-lasting, and neutralizing immune response. We have previously demonstrated that the adenylate cyclase (CyaA) from Bordetella pertussis targets dendritic cells and delivers CD8+ and CD4+ T-cell epitopes into the major histocompatibility complex class I and class II presentation pathways. We have also showed that CyaA induced specific and protective cytotoxic T cell responses in vivo. Here, we designed a prototype vaccine based on the HIV type 1 Tat delivered by CyaA (CyaA-E5-Tat) and tested its capacity to induce HIV-Tat-specific cellular as well as antibody responses. We showed that immunization of mice by CyaA-E5-Tat in the absence of adjuvant elicited strong and long-lasting neutralizing anti-Tat antibody responses more efficient than those obtained after immunization with Tat toxoid in aluminum hydroxide adjuvant. Analyses of the anti-Tat immunoglobulin G isotypes and the cytokine pattern showed that CyaA-E5-Tat induced a Th1-polarized immune response in contrast to the Th2-polarized immune responses obtained with the Tat toxoid. In addition, our data demonstrated that HIV-Tat-specific gamma interferon-producing CD8+ T cells were generated after vaccination with CyaA-E5-Tat in a CD4+ T-cell-independent manner. Based on these findings, CyaA-E5-Tat represents an attractive vaccine candidate for both preventive and therapeutic vaccination involving CyaA as an efficient nonreplicative vector for protein delivery.

Adjuvants and vector systems for allergy vaccines.

Allergen-specific immunotherapy represents a curative treatment of type I allergies. Subcutaneous immunotherapy is conducted with allergens adsorbed on aluminum hydroxide or calcium phosphate particles, whereas sublingual immunotherapy relies on high doses of soluble allergen without any immunopotentiator. There is a potential benefit of adjuvants enhancing regulatory and Th1 CD4+T cell responses during specific immunotherapy. Molecules affecting dendritic cells favor the induction of T regulatory cell and Th1 responses and represent valid candidate adjuvants for allergy vaccines. Furthermore, the interest in viruslike particles and mucoadhesive particulate vector systems, which may better address the allergen(s) to tolerogenic antigen-presenting cells, is documented.

New Aspects of Cyclosporin A Mode of Action: from Gene Silencing to Gene Up-Regulation

Cyclosporin A (CSA) has transformed clinical transplantation, both in term of success and of quality-of-life of the patient. Studies aimed to unfold the site of CSA action have shown that this molecule binds to cytosolic proteins of the cyclophilin family. CSA:cyclophilin complexes have a high affinity for calcineurin, a key enzyme in T-cell activation. By blocking the calcineurin activity, CSA prevents the induction of genes encoding for cytokines and their receptors. Thus, humoral and cellular immune responses are abolished, this resulting in the successful graft acceptance. Disappointingly, CSA and the other molecules as FK506, sharing the capacity to inhibit calcineurin, should be administered for all patient life, as tolerance to alloantigens is not achieved by these molecules. The long term utilization of this class of immunosuppressors increases the incidence of different tumors. The finding that CSA does not interfere with various biochemical pathways has prompted different groups to analyze a possible effect of CSA on molecules that might be involved in different functions of the immune response and/or in tumorogenesis. A new picture of CSA mode of action is emerging in which the immunosuppressor prevents the transcription of a group of genes, concomitantly inducing the transcription of another set. Here, we review the data and discuss the consequences of these new findings in term of T-cell activation mechanisms.

Identification of the cysteine protease Amb a 11 as a novel major allergen from short ragweed

BACKGROUND Allergy to pollen from short ragweed (Ambrosia artemisiifolia) is a serious and expanding health problem in the United States and in Europe. OBJECTIVE We sought to investigate the presence of undescribed allergens in ragweed pollen. METHODS Ragweed pollen proteins were submitted to high-resolution gel electrophoresis and tested for IgE reactivity by using sera from 92 American or European donors with ragweed allergy. Pollen transcriptome sequencing, mass spectrometry (MS), and recombinant DNA technologies were applied to characterize new IgE-binding proteins. RESULTS High-resolution IgE immunoblotting experiments revealed that 50 (54%) of 92 patients with ragweed allergy were sensitized to a 37-kDa allergen distinct from Amb a 1. The full-length cDNA sequence for this molecule was obtained by means of PCR cloning after MS sequencing of the protein combined with ragweed pollen RNA sequencing. The purified allergen, termed Amb a 11, was fully characterized by MS and confirmed to react with IgEs from 66% of patients. This molecule is a 262-amino-acid thiol protease of the papain family expressed as a combination of isoforms and glycoforms after proteolytic removal of N- and C-terminal propeptides from a proform. Three-dimensional modeling revealed a high structural homology with known cysteine proteases, including the mite Der p 1 allergen. The protease activity of Amb a 11, as well as its capacity to activate basophils from patients with ragweed allergy, were confirmed. The production of a nonglycosylated recombinant form of Amb a 11 in Escherichia coli established that glycosylation is not required for IgE binding. CONCLUSION We identified the cysteine protease Amb a 11 as a new major allergen from ragweed pollen. Given the similar physicochemical properties shared by the 2 major allergens, we hypothesize that part of the allergenic activity previously ascribed to Amb a 1 is rather borne by Amb a 11.