Edith Dériaud

Coactivation of Syk Kinase and MyD88 Adaptor Protein Pathways by Bacteria Promotes Regulatory Properties of Neutrophils

Neutrophils are one of the first lines of defense against microbial pathogens and are rapidly recruited at the infection site upon inflammatory conditions. We show here that after bacterial stimulation, and in contrast to monocytes and macrophages, murine neutrophils contributed poorly to inflammatory responses; however, they secreted high amounts of the anti-inflammatory cytokine IL-10 in a DAP12 adaptor-Syk kinase and MyD88 adaptor-dependent manner. Cotriggering of TLR-MyD88- and C-type lectin receptor (CLR)-Syk-dependent pathways led to a quick and sustained phosphorylation of p38 MAP and Akt kinases in neutrophils. In vivo, both Gram-negative bacteria and mycobacteria induced the recruitment of neutrophils secreting IL-10. In acute mycobacterial infection, neutrophil-derived IL-10 controlled the inflammatory response of dendritic cells, monocytes and macrophages in the lung. During a chronic infection, neutrophil depletion promoted inflammation and decreased the mycobacterial burden. Therefore, neutrophils can have a previously unsuspected regulatory role during acute and chronic microbial infections.

Dendritic Cells Are Host Cells for Mycobacteria In Vivo That Trigger Innate and Acquired Immunity

In the present study, we investigated in vivo the infection and APC functions of dendritic cells (DC) and macrophages (Mφ) after administration of live mycobacteria to mice. Experiments were conducted with Mycobacterium bovis bacillus Calmette-Guerin (BCG) or a rBCG expressing a reporter Ag. Following infection of mice, DC and Mφ were purified and the presence of immunogenic peptide/MHC class II complexes was detected ex vivo on sorted cells, as was the secretion of IL-12 p40. We show in this study that DC is a host cell for mycobacteria, and we provide an in vivo detailed picture of the role of Mφ and DC in the mobilization of immunity during the early stages of a bacterial infection. Strikingly, BCG bacilli survive but remain stable in number in the DC leukocyte subset during the first 2 wk of infection. As Ag presentation by DC is rapidly lost, this suggests that DC may represent a hidden reservoir for mycobacteria.

A fully synthetic therapeutic vaccine candidate targeting carcinoma-associated Tn carbohydrate antigen induces tumor-specific antibodies in nonhuman primates.

We recently developed an efficient strategy based on a fully synthetic dendrimeric carbohydrate display (multiple antigenic glycopeptide; MAG) to induce anticarbohydrate antibody responses for therapeutic vaccination against cancer. Here, we show the superior efficacy of the MAG strategy over the traditional keyhole limpet hemocyanin glycoconjugate to elicit an anticarbohydrate IgG response against the tumor-associated Tn antigen. We highlight the influence of the aglyconic carrier elements of such a tumor antigen for their recognition by the immune system. Finally, we additionally developed the MAG system by introducing promiscuous HLA-restricted T-helper epitopes and performed its immunological evaluation in nonhuman primates. MAG:Tn vaccines induced in all of the animals strong tumor-specific anti-Tn antibodies that can mediate antibody-dependent cell cytotoxicity against human tumor. Therefore, the preclinical evaluation of the MAG:Tn vaccine demonstrates that it represents a safe and highly promising immunotherapeutic molecularly defined tool for targeting breast, colon, and prostate cancers that express the carbohydrate Tn antigen.

Type I interferons protect neonates from acute inflammation through interleukin 10–producing B cells

Newborns and infants are highly susceptible to viral and bacterial infections, but the underlying mechanism remains poorly understood. We show that neonatal B cells effectively control the production of proinflammatory cytokines by both neonatal plasmacytoid and conventional dendritic cells, in an interleukin (IL) 10–dependent manner, after Toll-like receptor (TLR) 9 triggering. This antiinflammatory property of neonatal B cells may extend to other TLR agonists (Pam3CSK4, lipopolysaccharide, and R848) and viruses. In the absence of B cells or of CD5+ B cell subsets, neonatal mice developed stronger inflammatory responses and became lethally susceptible to CpG challenge after galactosamine sensitization, whereas wild-type (WT) mice were resistant. Paradoxically, interferon (IFN)-α/β enhanced the inflammatory response to CpG challenge in adult mice, whereas they helped to control neonatal acute inflammation by stimulating the secretion of IL-10 by neonatal B cells. Finally, WT neonatal B cells rescued IL-10−/− neonates from a lethal CpG challenge, whereas IFN-α/β receptor–deficient B cells did not. Our results show that type I IFNs support a negative regulatory role of neonatal B cells on TLR-mediated inflammation, with important implications for neonatal inflammation and infection.

In Vivo Induction of a High-Avidity, High-Frequency Cytotoxic T-Lymphocyte Response Is Associated with Antiviral Protective Immunity

ABSTRACT Many approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8+ cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8+ class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8+ T-cell epitopes, bound to 1-μm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503–7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4+T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies.

Anti-Tumor Immunity Provided by a Synthetic Multiple Antigenic Glycopeptide Displaying a Tri-Tn Glycotope

In many cancer cells the alteration of glycosylation processes leads to the expression of cryptic carbohydrate moieties, which make them good targets for immune intervention. Identification of cancer-associated glycotopes as well as progress in chemical synthesis have opened up the way for the development of fully synthetic immunogens that can induce anti-saccharide immune responses. Here, we synthesized a dendrimeric multiple antigenic glycopeptide (MAG) containing the Tn Ag O-linked to a CD4+ T cell epitope. This MAG is based on three consecutive Tn moieties (tri-Tn) corresponding to the glycotope recognized by an mAb (MLS 128) produced against the LS180 colon carcinoma cell line. The Abs induced by this MAG recognized murine and human tumor cell lines expressing the Tn Ag. Prophylactic vaccination using MAG provided protection of mice against tumor challenge. When used in active specific immunotherapy, the MAG carrying the tri-Tn glycotope was much more efficient than the mono-Tn analogue in promoting the survival of tumor-bearing mice. Furthermore, in active specific immunotherapy, a linear glycopeptide carrying two copies of the tri-Tn glycotope was shown to be poorly efficient compared with the dendrimeric MAG. Therefore, both the clustering of carbohydrate Ags and the way they are displayed seem to be important parameters for stimulating efficient anti-saccharide immune responses.

Characterization of T‐cell immune responses of Echinococcus multilocularis‐infected C57BL/6J mice

Specific and non‐specific parasite‐induced changes in lymphocyte responses were analysed in C57BL/6J mice after intrahepatic infection with Echinococcus multilocularis. Spleen cells harvested at selected times after infection were in vitro stimulated with mitogens or a crude soluble parasitic extract (EmAg) at an optimized dose. Cell proliferative responses to Con‐A were not modified by the infection over the first 22 weeks. In contrast, LPS‐induced responses were decreased from the 13th week. A strong CD4 + proliferative T‐cell response to the parasitic extract of infected mouse spleen cells was observed at the early stage of infection. This response then progressively decreased but remained significantly higher than that of control mice until the 19th week of infection. Cytokine production was investigated after in vitro EmAg stimulation of spleen cells. IFN‐γ, IL‐2. IL‐5 were produced within the first weeks after infection whereas the detection of IL‐10 was slightly delayed. Thus, the promotion of the disease does not appear associated with the expansion of one rather than another T‐cell subset in C57BL/6J mice. A general immunosuppression affecting both mitogenic and parasite‐specific T‐cell responses was observed at the end of the infection.

A recombinant virus-like particle system derived from parvovirus as an efficient antigen carrier to elicit a polarized Th1 immune response without adjuvant

Hybrid virus‐like particles (VLP) were prepared by self‐assembly of the modified porcine parvovirus (PPV) VP2 capsid protein carrying a CD8+ or CD4+ T cell epitope. Immunization of mice with a single dose of these hybrid pseudo‐particles, without adjuvant, induced strong cytotoxic T lymphocyte and T helper (Th) responses against the reporter epitope. The Th response was characterized by a Th1 phenotype. We also analyzed in vitro the uptake mechanism of these parvovirus‐like particles and the processing requirements associated with presentation by MHC molecules. Although previously shown to be presented by MHC class I molecules, these particles also enter very efficiently the MHC class II endocytic pathway, and behave as conventional exogenous antigens. Indeed, the processing of chimeric PPV : VLP was performed in endosomal/lysosomal acidic vesicles and the presentation of the foreign epitope carried by these particles was sensitive to brefeldin A and cycloheximide, showing that the foreign peptide was loaded on nascent MHC class II molecules. These results give some indication of how PPV : VLP can be presented by MHC class I and class II molecules, and underscore the wide potency of such VLP system to deliver foreign antigens for vaccine design.

CD4 T Cells with Effector Memory Phenotype and Function Develop in the Sterile Environment of the Fetus

Umbilical cord blood from healthy neonates with no known infectious exposure contains T cells with an effector memory–like phenotype. Neonatal T Cells Wise Beyond Their Years The uterine environment was traditionally thought to be sterile, and thus, healthy neonates were not predicted to have any memory T cells, because they have not been exposed to infection. However, Zhang et al. found that, contrary to all expectations, umbilical cord blood from healthy neonates does contain T cells that have all the hallmarks of memory cells. Why those cells are there and what role they serve in the fetal environment is still unknown, but these findings suggest that the fetal immune system may be more complex than had been previously suspected, which may affect future vaccine design and treatments for fetal and neonatal infections. The T cell compartment is considered to be naïve and dedicated to the development of tolerance during fetal development. We have identified and characterized a population of fetally developed CD4 T cells with an effector memory phenotype (TEM), which are present in cord blood. This population is polyclonal and has phenotypic features similar to those of conventional adult memory T cells, such as CD45RO expression. These cells express low levels of CD25 but are distinct from regulatory T cells because they lack Foxp3 expression. After T cell receptor activation, neonatal TEM cells readily produced tumor necrosis factor–α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF). We also detected interferon-γ (IFN-γ)–producing T helper 1 (TH1) cells and interleukin-4 (IL-4)/IL-13–producing TH2-like cells, but not IL-17–producing cells. We used chemokine receptor expression patterns to divide this TEM population into different subsets and identified distinct transcriptional programs using whole-genome microarray analysis. IFN-γ was found in CXCR3+ TEM cells, whereas IL-4 was found in both CXCR3+ TEM cells and CCR4+ TEM cells. CCR6+ TEM cells displayed a genetic signature that corresponded to TH17 cells but failed to produce IL-17A. However, the TH17 function of TEM cells was observed in the presence of IL-1β and IL-23. In summary, in the absence of reported pathology or any major infectious history, T cells with a memory-like phenotype develop in an environment thought to be sterile during fetal development and display a large variety of inflammatory effector functions associated with CD4 TH cells at birth.

DNA‐Mediated Immunization to the Hepatitis B Surface Antigen

The use of plasmid vectors expressing the HBsAg, along with improved protocols for transfection of muscle fibers (Refs. 3-6 and Davis et al., this volume), have provided the reagents and methods with which to investigate the characteristics of the strong immune response given by this antigen after DNA-mediated immunization. Analysis of the fine specificity of the humoral response provides support for the idea that the HBsAg-bearing particles are formed such that the B and T epitopes are presented to the immune system in a way resembling that of the natural viral or subviral particles. As shown here and elsewhere, DNA-mediated immunization with the HBsAg-expressing plasmid vectors induces strong CTL responses as well as a dominant Th1 phenotype among the splenic lymphocytes of immunized mice. The Th1 cytokine profile can be obtained in two different strains of mice and with two types of proteins, HBsAg and beta-galactosidase. One important line of investigation in the future will be to determine the mechanism of this generic Th1 response to DNA-based immunization. Circumstantial evidence, discussed by Pisetsky et al. (this volume), suggests that the chemical nature of DNA may play a role as an adjuvant (see also Ref. 31), and this hypothesis to explain the cytokine profiles observed after DNA-mediated immunization must now be taken seriously. All the questions raised by this novel method of immunization are of interest for the design of future vaccines, even if DNA itself is ultimately not the vaccinating moiety. The question of antigen presentation is particularly intriguing, since the small amounts of protein produced by DNA-mediated immunization (on the order of nanograms) are capable of inducing strong immune responses at the level of B and T cells. Although initially it seemed obvious that endogenous protein synthesis in cells transfected with plasmid DNA would account for the observed induction of CTL activity, this idea must be examined in light of two well established sets of experimental results. First, the primary events in activation of CD8+ (as well as CD4+) T lymphocytes normally require professional APC capable of furnishing co-stimulatory signals to supplement the consequences of interaction of the T-cell receptor with MHC surface molecules. Second, endogenous synthesis and processing is not the only mechanism of class I epitope presentation, and numerous examples are now known whereby particulate exogenous proteins, such as HBsAg, can be taken up and processed in such a way as to allow class I presentation of peptides. Consideration of these two points suggests that a major contribution to the observed CTL induction afforded by DNA-mediated immunization could come from the sustained presence of the antigenic protein in interstitial spaces or in the circulation, coupled with the ability of the exogenous protein to be processed for class I presentation. This could be true for many other proteins in addition to the HBsAg. This hypothesis eliminates the inconvenient notion that muscle fibers (or other nonleukocyte cells) present antigen in a way compatible with primary activation of T cells. However, muscle tissue can be an important reservoir of the antigen because of the potential for prolonged synthesis of the protein; this could therefore explain the immune entrainment observed after DNA-mediated immunization. Muscle fibers or other cells could also serve to present class I epitopes for the purpose of restimulating and thus expanding the pool of activated CD8+ T lymphocytes. These explanations, though certainly plausible, will require experimental investigation. The small numbers of the transfected cells in vivo, as well as the potential mobility of transfected cells other than muscle fibers, may well render such experimentation difficult. DNA-mediated immunization clearly offers opportunities for obtaining novel insights into immunological mechanisms and immunization processes. It is also likely to promote vacc