Pascale Jeannin

Involvement of LOX-1 in Dendritic Cell-Mediated Antigen Cross-Presentation

Some exogenous antigens, such as heat shock proteins or apoptotic bodies, gain access to the MHC class I processing pathway and initiate CTL responses, a process called cross-priming. To be efficient in vivo, this process requires endocytosis of the antigen by dendritic cells via receptors which remain unidentified. Here, we report that scavenger receptors are the main HSP binding structures on human dendritic cells and identify LOX-1 as one of these molecules. A neutralizing anti-LOX-1 mAb inhibits Hsp70 binding to dendritic cells and Hsp70-induced antigen cross-presentation. In vivo, to target LOX-1 with a tumor antigen using an anti-LOX-1 mAb induces antitumor immunity. Thus, the scavenger receptor LOX-1 is certainly a promising target for cancer immunotherapy.

The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps

The long pentraxin (PTX) 3 is produced by macrophages and myeloid dendritic cells in response to Toll-like receptor agonists and represents a nonredundant component of humoral innate immunity against selected pathogens. We report that, unexpectedly, PTX3 is stored in specific granules and undergoes release in response to microbial recognition and inflammatory signals. Released PTX3 can partially localize in neutrophil extracellular traps formed by extruded DNA. Eosinophils and basophils do not contain preformed PTX3. PTX3-deficient neutrophils have defective microbial recognition and phagocytosis, and PTX3 is nonredundant for neutrophil-mediated resistance against Aspergillus fumigatus. Thus, neutrophils serve as a reservoir, ready for rapid release, of the long PTX3, a key component of humoral innate immunity with opsonic activity.

Direct Stimulation of Human T Cells via TLR5 and TLR7/8: Flagellin and R-848 Up-Regulate Proliferation and IFN-γ Production by Memory CD4+ T Cells

TLRs are involved in innate cell activation by conserved structures expressed by microorganisms. Human T cells express the mRNA encoding most of TLRs. Therefore, we tested whether some TLR ligands may modulate the function of highly purified human CD4+ T lymphocytes. We report that, in the absence of APCs, flagellin (a TLR5 ligand) and R-848 (a TLR7/8 ligand) synergized with suboptimal concentrations of TCR-dependent (anti-CD3 mAb) or -independent stimuli (anti-CD2 mAbs or IL-2) to up-regulate proliferation and IFN-γ, IL-8, and IL-10 but not IL-4 production by human CD4+ T cells. No effect of poly(I:C) and LPS, ligands for TLR3 and TLR4, respectively, was detected. We also observed that CD4+CD45RO+ memory T cell responses to TLR ligands were more potent than those observed with CD4+CD45RA+ naive T cells. Moreover, among the memory T cells, CCR7− effector cells were more sensitive to TLR ligands than CCR7+ central memory cells. These data demonstrate for the first time a direct effect of TLR5 and TLR7/8 ligands on human T cells, and highlight an innate arm in T cell functions. They also suggest that some components from invading microorganisms may directly stimulate effector memory T cells located in tissues by up-regulating cytokine and chemokine production.

Histamine Polarizes Human Dendritic Cells into Th2 Cell-Promoting Effector Dendritic Cells

Allergic disorders are characterized by allergen-specific Th2-biased responses. Signals controlling Th2 cell polarization, especially those acting by polarizing dendritic cells (DC) into Th2-promoting DC (DC2), are not well known. Histamine, a mediator released by allergen-stimulated mast cells from allergic subjects, has been reported to activate human immature DC. We have therefore tested whether histamine affects DC polarization. We report here that histamine inhibits LPS-induced IL-12 production and polarizes uncommitted maturing DC into effector DC2. DC matured in the presence of histamine fail to produce IL-12 upon subsequent stimulation and prime Th2 responses, even in presence of IFN-γ, a potent DC1-driving factor. All these effects are mediated through both H1 and H2 receptors. These data show that histamine is a potent DC2-polarizing factor and provide evidence for a novel mechanism that explains the initiation and maintenance of a predominant Th2 response in allergic disorders.

OmpA targets dendritic cells, induces their maturation and delivers antigen into the MHC class I presentation pathway.

We analyzed the interaction between a bacterial cell wall protein and dendritic cells (DCs). Outer membrane protein A from Klebsiella pneumoniae (kpOmpA) specifically bound to professional antigen presenting cells and was endocytosed by immature DCs via a receptor-dependent mechanism. kpOmpA signaled through Toll-like receptor 2, induced DCs to produce interleukin 12 and induced maturation of DCs. Whole antigen that was coupled to kpOmpA and injected into mice was taken up by DCs and delivered to the conventional cytosolic MHC class I presentation pathway. kpOmpA also primed antigen-specific CD8+ CTLs in the absence of CD4+ T cell help or adjuvant and elicited therapeutic immunity to antigen-expressing tumors. Thus, OmpA belongs to a class of proteins that are able to elicit CTL responses to exogenous antigen.

Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity

BACKGROUND Current cytotoxic assays, including Cr release and fluorescent assays, do not directly measure the proportion of target cells which are killed by apoptosis. Cell-mediated cytotoxicity induced by CTLs and NK cells is mainly regulated by the perforin-granzyme, the Fas ligand (Fas L), and the Tumor Necrosis Factor (TNF)-alpha pathways. Perforin generates pores in the membrane of target cells, allowing granzyme B to enter and initiate apoptosis. The other effectors, Fas L and TNF-alpha act by an apoptosis mechanism, leading to DNA fragmentation. A three color flow cytometric method to measure cell-mediated cytotoxicity induced by CTLs or NK cells is described. METHODS The fluorochromes used are: PKH-26, a stable membrane dye for the labeling of the effector cells, annexin V-FITC which allows the direct evaluation of early apoptotic cells and propidium iodide which distinguishes membrane permeabilized and late apoptotic cells. RESULTS By eliminating through gating PKH-26 positive effector cells, we obtain a direct estimation of the percentage of target cells in the early stages of apoptosis as well as the percentage of target cells dying after late apoptosis and membrane permeabilization. The cytotoxic activity of IL-2 stimulated PBL against K562, Jurkat and KYM-1 was evaluated. CONCLUSIONS This rapid and novel assay permits the discrimination of the cell death mechanisms occurring during a cytotoxic response and to precisely evaluate the contribution of apoptosis in the early phases of cell-mediated cytotoxicity.

Interferon-γ reverses the immunosuppressive and protumoral properties and prevents the generation of human tumor-associated macrophages

Tumor‐associated macrophages (TAM) are M2d‐polarized cells (IL‐10high, IL‐12low, ILT3high, CD86low) that accumulate in tumor microenvironment. TAM inhibit antitumor T lymphocyte generation and function, contribute to tumor tolerance and are trophic for tumors. In this study, we investigated whether some immunological factors may reverse TAM immunosuppressive properties. Among 32 cytokines, we have identified IFNγ on its ability to switch immunosuppressive TAM into immunostimulatory cells. Upon IFNγ exposure, TAM purified from ovarian cancer ascites recover a M1 phenotype (IL‐10low, IL‐12high), express high levels of CD86 and low levels of ILT3, enhance the proliferation of CD4+ T lymphocytes and potentiate the cytotoxic properties of a MelanA‐specific CD8+ T cell clone. IFNγ‐treated TAM also secreted reduced levels of mediators promoting suppressive T cell accumulation (CCL18) and trophic for tumors (VEGF and MMP9). As TAM derive from the local differentiation of peripheral blood monocytes, we investigated whether IFNγ may also affect TAM generation. In the presence of ovarian ascites, IFNγ skewed monocyte differentiation from TAM‐like cells to M1‐polarized immunostimulatory macrophages. Together, these data show that IFNγ overcomes TAM‐induced immunosuppression by preventing TAM generation and functions. These data highlight that IFNγ used locally at the tumor site could potentiate the efficacy of antitumor immunotherapies based on the generation of effector T cells.

Histamine Induces CD86 Expression and Chemokine Production by Human Immature Dendritic Cells

Mast cells and immature dendritic cells (DC) are in close contact in peripheral tissues. Upon activation, mast cells release histamine, a mediator involved in the immediate hypersensitivity reaction. We therefore tested whether histamine could affect human DC activation and maturation. Histamine induces CD86 expression on immature DC in a dose-dependent (significant at 10−7 M) and transient manner (maximal after 24-h stimulation). Histamine also transiently up-regulates the expression of the costimulatory and accessory molecules, CD40, CD49d, CD54, CD80, and MHC class II. As a consequence, immature DC exposed for 24 h to histamine stimulate memory T cells more efficiently than untreated DC. In addition, histamine induces a potent production of IL-6, IL-8, monocyte chemoattractant protein 1, and macrophage-inflammatory protein 1α by immature DC and also up-regulates IL-1β, RANTES, and macrophage-inflammatory protein 1β but not TNF-α and IL-12 mRNA expression. Histamine activates immature DC through both the H1 and H2 receptors. However, histamine-treated DC do not have a phenotype of fully mature cells, as they do neither show significant changes in the expression of the chemokine receptors, CCR5, CCR7 and CXC chemokine receptor 4, nor expression of CD83 de novo. These data demonstrate that histamine activates immature DC and induces chemokine production, thereby suggesting that histamine, via stimulation of resident DC, may participate locally in T cell stimulation and in the late inflammatory reaction associated with allergic disorders.

The Trypanosoma cruzi Tc52-released protein induces human dendritic cell maturation, signals via Toll-like receptor 2, and confers protection against lethal infection.

The intracellular protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas disease. We have recently identified a T. cruzi-released protein related to thiol-disulfide oxidoreductase family, called Tc52, which is crucial for parasite survival and virulence. In vitro, Tc52 in combination with IFN-γ activates human macrophages. In vivo, active immunization with Tc52 relieves the immunosuppression associated to acute infection and elicits a specific immune response. As dendritic cells (DC) have a central role in the initiation of immune responses, we investigated whether Tc52 may modulate DC activity. We show that Tc52 induces human DC maturation. Tc52-treated immature DC acquire CD83 and CD86 expression, produce inflammatory chemokines (IL-8, monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1α), and present potent costimulatory properties. Tc52 binds to DC by a mechanism with the characteristics of a saturable receptor system and signals via Toll-like receptor 2. While Tc52-mediated signaling involves its reduced glutathione-binding site, another portion of the molecule is involved in Tc52 binding to DC. Finally, we report that immunization with Tc52 protects mice in vivo against lethal infection with T. cruzi. Together these data evidence complex molecular interactions between the T. cruzi-derived molecule, Tc52, and DC, and suggest that Tc52 and related class of proteins might represent a new type of pathogen-associated molecular patterns. Moreover, the immune protection data suggest that Tc52 is among candidate molecules that may be used to design an optimal multicomponent vaccine to control T. cruzi infection.

Pattern recognition receptors in the immune response against dying cells

Pattern recognition receptors (PRR), immune sensors that discriminate self from non-self, link innate to adaptive immunity. PRR are involved in microbe internalization by phagocytes (soluble PRR and endocytic receptors) and/or cell activation (signaling PRR). PRR also recognize dying cells (i.e. modified self). Apoptotic cell recognition involves soluble bridging molecules (e.g. pentraxins) and endocytic receptors (e.g. scavenger receptors, the CD91-calreticulin complex). Apoptotic cells induce an immunosuppressive signal, avoiding the initiation of an autoimmune response. By contrast, necrotic cells, via the release of stimulatory molecules [heat shock protein (HSP), high-mobility group box 1 protein (HMGB1)], activate immune cells. This review summarizes the PRR involved in the recognition of dying cells and the consequences on the outcome of the immune response directed against dying cell antigens.