Ma thèse a permis de développer une nouvelle approche pour étudier, par imagerie in vivo, le comportement des cellules présentatrices d’antigènes (CPA) de la peau suite à la vaccination par voie intradermique chez le primate non-humain. Le ciblage des CPA a été réalisé par injection in vivo d’un anticorps monoclonal anti-HLA-DR fluorescent. L’effet sur les CPA d’un adjuvant (R-848, ligand du TLR7/8) et l’immuno-ciblage des cellules de Langerhans par une protéine de fusion vaccinale anti-VIH (anti-langérine-VIHGag) ont ainsi été évalués par imagerie in vivo, vidéomicroscopie confocale ex vivo et cytométrie en flux. Ce travail a contribué à améliorer nos connaissances immunologiques sur les effets locaux et précoces des vaccins et /ou adjuvants. / My pHD project allowed the development of in vivo imaging approaches to study the skin antigen presenting cell (APC) behavior post-intradermal vaccination in non-human primates. APC targeting was performed by in vivo injection of fluorescent anti-HLA-DR monoclonal antibody. The effect of an adjuvant (R-848, ligand of TLR7/8) on skin APC and the immunotargeting of Langerhans cells by anti-HIV vaccinal fusion protein (anti-langerin-HIVGag) were assessed by in vivo fluorescent imaging, ex vivo confocal videomicroscopy and flow cytometry. This work contributed to improve immunological knowledge on local and early events post-vaccination with or without adjuvant.

La compréhension des mécanismes immunologiques précoces mis en place suite à l’administration de vaccins est encore de nos jours largement méconnue. Pourtant de plus en plus d’études démontrent l’importance de ces mécanismes très précoces faisant intervenir les acteurs de l’immunité innée dans la génération d’une réponse spécifique efficace après vaccination. La peau est un organe intéressant pour l'administration de vaccins du fait de sa richesse en cellules présentatrices d'antigènes (APC), qui sont des cellules essentielles dans la mise en place de la réponse immunitaire. L'administration par voie intradermique du vaccin ADN de type auxoGTU induit des réponses immunitaires fortes et persistantes, en particulier en association avec une électroporation (EP) locale chez le macaque cynomolgus. Le but de ce travail de thèse fut de caractériser les réponses immunitaires locales précocement mises en place suite à l’administration par voie intradermique du vaccin ADN auxo-GTU en association avec une EP. Dans un premier temps, nous avons décrit les populations de cellules immunitaires présentes dans la peau normale chez le macaque cynomolgus.L'épiderme contient des cellules de Langerhans (LC) qui sont : CD1a+ CD1c- et des lymphocytes T caractérisés par l’expression du CD3. Le derme contient des cellules CD1a+CD1c-, qui présentent des similitudes avec les LC et correspondent donc probablement à des LC en migration à travers le derme. Il contient également des cellules dendritiques dermales (DDC) CD1a+CD1c+, des macrophages résidants CD163highCD11b+ et les lymphocytes T CD3+. Chez certains animaux, nous avons mis en évidence la présence de granulocytes CD66+ dans le derme sain. Les populations de cellules immunitaires identifiées chez le macaque sont similaires à celles identifiées chez l’homme malgré de légères différences phénotypiques. Cette caractérisation nous a ensuite permis d'étudier l’impact de la vaccination sur les populations immunitaires de la peau.Nous avons démontré que la vaccination induit le recrutement de granulocytes et de monocytes/macrophages inflammatoires dans l'épiderme et dans le derme, ainsi qu’un recrutement plus tardif de cellules dendritiques inflammatoires épithéliales (IDEC) dans l'épiderme. Dans l'épiderme, 24h après immunisation, nous avons observé une augmentation transitoire des LC accompagnée d’une surexpression de HLA-DR, de CD86 et de CD83, ce qui démontre leur maturation. Entre 24h et 72h, le nombre de LC diminue, ce qui suggère que les LC matures quittent l’épiderme pour migrer vers les nœuds lymphatiques. Ces événements cellulaires sont majoritairement dus à l’EP, indépendamment de la présence du vaccin à ADN. L’analyse du microenvironnement mis en place dans la peau suite à la vaccination révèle une libération de facteurs solubles pro-inflammatoires, comme MCP-1, IL-18 , IL-15, IL-8 et de facteurs solubles anti- inflammatoires comme IL-1RA et sCD40L dès 24h, dont certains sont considérablement augmentés par la présence de l’ADN vaccinal. Nos résultats suggèrent que l’EP, indépendamment de la présence de l'ADN, est suffisante pour induire la mobilisation des cellules et la maturation des DC au niveau du site de vaccination, ce qui montre un important rôle adjuvant de l’EP. Cependant, il semble que l'ADN soit nécessaire pour générer un microenvironnement favorable à l'activation optimale des APC. Ce travail fournit des éléments importants sur les mécanismes de l'inflammation locale et ouvre de nouvelles possibilités pour les stratégies vaccinales. / Mechanisms involved in early vaccine response are poorly understood. However, more and more studies show the importance of innate immunity in the very early times following vaccine administration in the generation of an optimal specific immune response. Skin is an interesting target for vaccine delivery because of its richness in antigen presenting cells (APC) which are essential cells in immune responses. The intradermal delivery of auxoGTU DNA vaccine was shown to induce strong and persistent immune responses, especially in association with electroporation in cynomolgus macaque. The aim of this work was to characterize the early local immune responses followed intradermal auxoGTU DNA vaccination in association with EP in cynomolgus macaque. In a first step, we have described immune cell populations present in the normal skin in the cynomolgus macaques. The epidermis contains CD1a+CD1c- Langerhans cells (LCs), and CD3+ T cells. The dermis contains CD1a+CD1c- cells, which present similarities with LCs and probably correspond to LC in migration through dermis. It also contains CD1a+CD1c+ dermal dendritic cells (DDCs), CD163highCD11b+ resident macrophages, and CD3+ T cells. We found CD66+ polymorphonuclear cells in healthy dermis in some of the animals. Immune cell populations in the macaque are similar to those in humans despite moderate differences in phenotype. This characterization has allowed us to study the impact of vaccination on immune populations of the skin. We have demonstrated a recruitment of granulocytes and inflammatory monocytes/macrophages in epidermis and dermis, as well as a population of inflammatory dendritic epithelial cell (IDEC) in epidermis after vaccination. In epidermis, 24h after treatment, we have observed an initial increase of LC with an up-regulation of HLA-DR, CD86 and CD83, demonstrating their maturation. Between 24h and 72h, LC number decreased, suggesting that mature LC has leaved epidermis to migrate to skin draining lymph node. All these cellular events were almost due to EP process, independently of DNA vaccine presence. The skin microenvironment reveals a release of pro-inflammatory soluble factors, as MCP-1, IL-18, IL-15, IL-8 and anti-inflammatory mediators as IL-1RA and sCD40L by 24h, all considerably enhanced in the presence of DNA.Our results suggest that EP, independently of the presence of DNA, is sufficient to induce cells mobilization and DC maturation at the vaccinated site, suggesting an important adjuvant effect of EP. However, it seems that DNA is required to generate a favorable microenvironment essential for correct APC activation. This work provides important clues to local inflammation mechanisms and opens up new possibilities for vaccine strategies.

Background: A mouse model of combined UVA/riboflavin irradiation to eliminate stromal cells and other antigen-presenting cells in the cornea provides the basis for a probably low risk of corneal transplantation. Methods: After abrasion of the epithelium, the central corneas of mouse eyes were treated with UVA/riboflavin in vitro. Histological studies of hematoxylin-eosin and immunohistochemical staining with caspase 3 were performed. Dissected mouse corneas were analyzed by Western blot. Results: Apoptotic cells were shown on the central corneal stroma; a cell-free zone was displayed in the cornea. Numbers of dead cells increased according to cultivation time. However, the endothelium survived due to the adjustment of the irradiation dose. Conclusions: A cell-free zone in the stroma of the mouse cornea was produced by UVA/riboflavin irradiation in vitro. The technique makes possible to prevent or reduce immunological reactions and the risk of graft rejection by pretreatment of the donor cornea, ultimately prolonging graft survival. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Clinical and experimental studies have shown that estradiol (E2) can enhance protection against sexually transmitted infections such as HSV-2 and HIV-1. Antigen presenting cells (APCs) such as Dendritic cells (DCs) are critical for generating immune responses against these infections, and it is unclear whether unique factors present in the genital mucosa can influence immune responses by directly modulating the phenotype and function of local APCs. To address this, I hypothesized that sex hormones, such as E2 and innate factors in the local microenvironment can regulate the phenotype and function of vaginal APCs. The work summarized in this thesis addressed this central hypothesis. In the first section of the thesis, I examined whether vaginal APCs were distinct in their phenotype and function compared to those in other mucosal tissues or spleen. The results show that the vagina was enriched in CD11c+ CD11b+ MHCII− DCs. Functionally, vaginal tissue cells (TC) and CD11c+ DCs were more potent inducers of Th17 responses in co-cultures with CD4+ T cells, compared to lung, small intestine or spleen APCs. E2 was critical for the conditioning of vaginal DCs to prime these Th17 responses through an IL-1-dependent pathway, indicating that sex hormones such as E2 can directly influence the function of vaginal APCs. In the next section, I determined whether other co-factors in the genital microenvironment such as microflora and innate lymphocytes could also influence vaginal APC functions. We found that while microflora was not essential, IL-17 produced by innate lymphocytes was critical for the induction of IL-1 from DCs, and consequently for potentiating Th17 responses. Finally, I attempted to develop an in vivo mouse model where the effect of E2 on vaginal APCs could be examined in the context of genital HSV-2 infection. I tested a 7-day injectable E2 and a 21-day E2 pellet delivery model, and found that both regimes had limitations for examining E2-effects on anti-viral responses. Yet, subsequent to the work done in this thesis, we were able to confirm our observations of E2-conditioned Th17 responses in vivo in an intranasal immunization model utilizing E2 pellet delivery, and thereby addressed the mechanism underlying enhanced anti-viral protection following E2-treatment. In conclusion, this is the first study to show the effect of E2 on genital tract APCs and their ability to prime Th17 responses. It provides future avenues to examine whether modulation of this microenvironment can help optimize vaccine-induced immune responses against STIs. On a more fundamental level, it highlights the need to consider the inherent distinctions in APC populations among different mucosal tissues. / Dissertation / Doctor of Philosophy (PhD)

<h4> </h4> / <p>Asthma is an inflammatory disorder of the airways, and there has been growing insight into the cellular and molecular mechanisms underlying the inflammatory basis of this disease. Research into the inflammatory mechanisms of asthma has progressively shifted focus from downstream effectors, such as mast cells and eosinophils, up to Th2 lymphocytes and their proallergic cytokines. Even more upstream in the allergic cascade are dendritic cells (DCs), potent APCs that orchestrate immune responses. Evidence supporting a role of DCs in regulating airway allergic inflammation is derived mainly from animal studies. In animal models of asthma, myeloid DCs (mDCs) induce and maintain airway inflammation, while plasmacytoid DCs (pDCs) mediate tolerance and lung homeostasis. It remains uncertain, however, whether this concept of pro-allergic mDCs and anti-allergic pDCs translates from animal to human models. The overall objective of this thesis was to investigate the biology of DC subsets in allergen-induced asthma in asthmatic subjects. Initially, we demonstrate that both mDCs and pDCs increase in the airways of subjects with mild asthma after allergen inhalation. Next, we describe a distinct subpopulation of mDCs, called mDC2s, and demonstrate their association with allergy and asthma severity. Expanding on these findings, we show that mDC2s increase in the airways of mild asthmatics after allergen challenge. Lastly, we explore the potential of pharmacological therapies, anti-OX40L MAb and anti-TSLP MAb, to affect DCs in subjects with mild asthma, and demonstrate no effect of either drug on circulating DC subsets. The studies presented here provide evidence for multiple DC subtypes being involved in the regulation of allergen-induced inflammatory responses, and support continued investigations into the biology of different DC subsets in allergen-induced asthma.</p> / Doctor of Philosophy (Medical Science)

Background Previously, we identified a major myeloid-derived proinflammatory subpopulation of human blood dendritic cells which we termed slanDCs (e.g. Schäkel et al. (2006) Immunity 24, 767–777). The slan epitope is an O-linked sugar modification (6-sulfo LacNAc, slan) of P-selectin glycoprotein ligand-1 (PSGL-1). As slanDCs can induce neoantigen-specific CD4+ T cells and tumor-reactive CD8+ cytotoxic T cells, they appear as promising targets for an in vivo delivery of antigens for vaccination. However, tools for delivery of antigens to slanDCs were not available until now. Moreover, it is unknown whether or not antigens delivered via the slan epitope can be taken up, properly processed and presented by slanDCs to T cells. Methodology/Principal Findings Single chain fragment variables were prepared from presently available decavalent monoclonal anti-slan IgM antibodies but failed to bind to slanDCs. Therefore, a novel multivalent anti-slanDC scaffold was developed which consists of two components: (i) a single chain bispecific recombinant diabody (scBsDb) that is directed on the one hand to the slan epitope and on the other hand to a novel peptide epitope tag, and (ii) modular (antigen-containing) linker peptides that are flanked at both their termini with at least one peptide epitope tag. Delivery of a Tetanus Toxin-derived antigen to slanDCs via such a scBsDb/antigen scaffold allowed us to recall autologous Tetanus-specific memory T cells. Conclusions/Significance In summary our data show that (i) the slan epitope can be used for delivery of antigens to this class of human-specific DCs, and (ii) antigens bound to the slan epitope can be taken up by slanDCs, processed and presented to T cells. Consequently, our novel modular scaffold system may be useful for the development of human vaccines.

The immune system is essential for keeping the integrity of multicellular organisms. We were able to make a step forward in studying the complex immune reactions in mammals in vivo and/ or in situ using the major histocompatibility complex (MHC) class II/ enhanced green fluorescent protein (EGFP) knock-in mouse model. Due to the EGFP visualization of MHC II expressing cells we were able to observe antigen presenting cells, which are essential for the onset of immune responses, in their natural environment. Thus, we report some original features of the immune system. We have identified MHC II+ cell clusters with unknown, probably unique function, in the intestine. We have also described MHC II+ cell migration to the lactating mammary gland and tested few hypotheses about the role of this phenomenon for the development of the mammary gland, milk secretion or infant immune system establishment. Lastly, we observed residential macrophages in the cornea. The presence of APCs in the cornea is a very contradictory issue due to the fact that cornea is an immunologically privileged tissue and therefore harbors special immune features. key words: antigen presenting cells (APC), major histocompatibility complex class II (MHC II), enhanced green fluorescent protein (EGFP), immune system, knock-in mouse model

L'utilisation des nanobiotechnologies dans le domaine de la santé est en plein essor. Les nanoparticules de poly(acide lactique) (PLA) représentent un nanosystème biocompatible capable d'accroître la spécificité et l'efficacité de traitements thérapeutiques et vaccinaux administrables par voie muqueuse et intraveineuse. Toutefois, l'optimisation de ces nanosystèmes se heurte à une caractérisation incomplète de leur biodistribution in vivo, en particulier à l'échelle cellulaire.L'objectif de ce travail de thèse est d'enrichir les connaissances sur la biodistribution des nanoparticules de PLA in vivo après administration muqueuse ou intraveineuse, dans le but d'élargir les perspectives d'optimisation et d'utilisation. Animal complexe et adapté pour les études sur organisme-entier, le modèle du poisson-zèbre (Danio rerio) a été utilisé. Pour mener à bien ce projet, une méthodologie rigoureuse d'analyse de la biodistribution des nanoparticules de PLA a été développée. Ce qui permit, après administration par balnéation, d'en révéler le fort tropisme inné envers les cellules dendritiques muqueuses. Ces données ont servi à élaborer une stratégie de ciblage, utilisant la lectine agglutinine de cacahuète, capable d'augmenter la prise en charge des nanoparticules de PLA par les branchies et la peau. Enfin, l'étude du devenir de ces nanoparticules après injection intraveineuse, a révélé de nombreuses interactions avec le système circulatoire. Ce travail a permis d'approfondir la connaissance des interactions des nanoparticules de PLA avec le vivant, soulignant le potentiel prometteur de ces nanoparticules pour la vaccination muqueuse / Medecine shows a growing interest regarding nanobiotechnologies. Among them are poly(lactic acid) (PLA) nanoparticles, which represent a biocompatible and competent nanosystem to heighten the specificity and efficacy of diverse therapeutic and vaccine treatments, following mucosal and intravenous administration. However, the further optimization of such nanosystem is poised by the lack of informations regarding their in vivo biodistribution, especially at the cellular level.The main objective of this PhD is to increment the knowledge about PLA nanoparticles biodistribution in vivo, after muquous and intravenous administration, to further expand their optimisation and use perspectives. The zebrafish model has been utilized to perform this research because of his conserved complexity as well as his suitability for whole-organism studies.To fulfill this project, a precise methodology has been developed to analyze the PLA nanoparticles biodistribution. Which allowed, after bathing administriation, to unveil their robust innate tropism toward mucous dendritic cells. From these data has been established a targeting strategy, utilizing the peanut agglutinin lectin, which has been proved to enhance nanoparticle uptakes by both gills and skin mucosae. Finally, the study of PLA nanoparticles behavior and destiny after intravenous injection, revealed numerous elaborated interactions with the circulatory system.Overall, this work has been able to strengthen our understandings of PLA nanoparticles among living organisms, furthermore highlighting their promizing potential as nanovehicles for mucosal vaccines

Background Previously, we identified a major myeloid-derived proinflammatory subpopulation of human blood dendritic cells which we termed slanDCs (e.g. Schäkel et al. (2006) Immunity 24, 767–777). The slan epitope is an O-linked sugar modification (6-sulfo LacNAc, slan) of P-selectin glycoprotein ligand-1 (PSGL-1). As slanDCs can induce neoantigen-specific CD4+ T cells and tumor-reactive CD8+ cytotoxic T cells, they appear as promising targets for an in vivo delivery of antigens for vaccination. However, tools for delivery of antigens to slanDCs were not available until now. Moreover, it is unknown whether or not antigens delivered via the slan epitope can be taken up, properly processed and presented by slanDCs to T cells. Methodology/Principal Findings Single chain fragment variables were prepared from presently available decavalent monoclonal anti-slan IgM antibodies but failed to bind to slanDCs. Therefore, a novel multivalent anti-slanDC scaffold was developed which consists of two components: (i) a single chain bispecific recombinant diabody (scBsDb) that is directed on the one hand to the slan epitope and on the other hand to a novel peptide epitope tag, and (ii) modular (antigen-containing) linker peptides that are flanked at both their termini with at least one peptide epitope tag. Delivery of a Tetanus Toxin-derived antigen to slanDCs via such a scBsDb/antigen scaffold allowed us to recall autologous Tetanus-specific memory T cells. Conclusions/Significance In summary our data show that (i) the slan epitope can be used for delivery of antigens to this class of human-specific DCs, and (ii) antigens bound to the slan epitope can be taken up by slanDCs, processed and presented to T cells. Consequently, our novel modular scaffold system may be useful for the development of human vaccines.

L’électroporation (EP) in vivo est utilisée comme stratégie d’amélioration de la réponse immune induite par les vaccins ADN. Cependant son effet sur les acteurs du système immunitaire inné reste méconnu. Dans l’objectif de mettre en évidence le comportement cellulaire sur le site de la vaccination, nous avons développé des approches d’imagerie par fluorescence in vivo chez le macaque. Nos résultats montrent que l’EP locale, augmente non seulement la quantité et la distribution de l’antigène vaccinal, mais induit également la mobilisation et la migration des cellules de Langerhans. De plus, l’EP cause un recrutement de leucocytes dans la peau et le tissu sous-cutané et favorise la production de cytokines pro-inflammatoires dans la peau. Ces évènements précoces, qui résultent de l’utilisation de l’EP en tant que système de délivrance des vaccins ADN, mettent en évidence le potentiel de l’EP en tant qu’adjuvant vaccinal. / In vivo electroporation (EP) is used as a strategy to improve the immune response induced by DNA vaccines. However, its local effect on the innate immune cells has not been fully described. We developed in vivo fluorescence imaging approaches to highlight the cell behavior in the site of vaccination in macaques. Our results show that the local EP not only increases the amount and the distribution of the vaccine antigen, but also induces the mobilization and migration of Langerhans cells. Furthermore, EP causes the recruitment of leukocytes into the skin and subcutaneous tissue and promotes the production of pro-inflammatory cytokines. These early events that result from the use of the EP as a delivery system for DNA vaccines, highlight its potential as a vaccine adjuvant.