Malgré la stabilité historiquement associée à la méthylation de l’ADN, cette modification épigénétique subit des changements rapides importants dans les types cellulaires plus plastiques. Par exemple, l’infection de cellules dendritiques humaines est associée à des milliers de changements dans le paysage de méthylation, principalement des pertes de méthylation dans les amplificateurs. Cette déméthylation est corrélée à l’apparition de marques actives d’histones et à l’activation des gènes à proximité. Dans la présente thèse, le rôle plus précis de cette déméthylation active a été investigué. Une étude temporelle de l’infection de cellules dendritiques par Mycobacterium tuberculosis a révélé que les changements d’expression génique et la liaison des facteurs de trancription classiquement activés en réponse à une infection bactérienne, comme les familles de NF-B et AP-1, étaient préalables aux changements de méthylation, suggérant un rôle minimal de la déméthylation dans la réponse à l’infection. D’autres résultats allaient dans le même sens, soit en inhibant TET2, une enzyme participant à la déméthylation active de l’ADN dans les cellules dendritiques via sa conversion en intermédiaires. En effet, l’impact d’une telle inhibition sur la réponse transcriptionnelle à l’infection par Salmonella typhimurium était minime. Cependant, l’inhibition de TET2 entraîne un niveau basal d’expression de cytokines pro-inflammatoires plus élevé, ce qui m’entraîne à proposer que la déméthylation puisse participer au retour à la normale post-infection. Elle pourrait également avoir une fonction dans la mémoire immunitaire innée en permettant une réponse plus rapide à une seconde infection, un phénomène surtout démontré dans les monocytes et les macrophages, mais mis en évidence dans les cellules dendritiques dans la présente thèse. Ainsi, cette étude ouvre de nombreuses perspectives dans la thérapie épigénétique contre les maladies auto-immunes, le développement de vaccins ciblant le système immunitaire inné, et l’utilisation de la méthylation de l’ADN comme biomarqueur d’infections. / DNA methylation has historically been perceived as a highly stable epigenetic modification. However, rapid and important changes in its landscape occur in cells with higher plasticity. For example, human dendritic cells infection associated with thousands of changes of DNA methylation, majoritarily losses in enhancer regions. This demethylation is correlated with gain of active histone marks and nearby gene upregulation. The present dissertation investigated the precise role of this demethylation. Firstly, a temporal study of dendritic cells infected with Mycobacterium tuberculosis revealed that gene expression changes and the classical immune transcription factors binding, such as members of the NF-B or AP-1 families, occurred prior to DNA methylation changes. This suggests a minimal function of demethylation in response to infection. Results from a TET2 inhibition an enzyme participating to active demethylation by converting 5-methylcytosine to intermediates were concordant with this hypothesis. Indeed, this inhibition has a very small impact on transcriptional response to an infection by Salmonella typhimurium. Nevertheless, the pro-inflammatory cytokine production of TET2-inhibited non-infected cells is higher. I therefore propose that demethylation might play a role in the return to basal state after an infection is cleared. It could also participate to innate immune memory by allowing cells to respond faster to a second infection. This phenomenon has especially been demonstrated in monocytes and macrophages but is also highlighted in dendritic cells in the present work. This dissertation opens numerous perspectives in epigenetic therapy of auto-immune diseases, the development of vaccines targeting the innate immune system and the use of DNA methylation as a biomarker.

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)

Multimodal treatment adding immunotherapy and photodynamic treatment (PDT) to standard therapy might improve the devastating therapeutic outcome of glioblastoma multiforme patients. As a first step, we provide investigations to optimize dendritic cell (DC) vaccination by using PDT and ionizing radiation (IR) to achieve maximal synergistic effects. In vitro experiments were conducted on murine glioblastoma GL261 cells, primary DCs differentiated from bone marrow and T cells, isolated from the spleen. Induction of cell death, reactive oxygen species, and inhibition of proliferation by tetrahydroporphyrin-tetratosylat (THPTS)-PDT and IR were confirmed by WST-1, LDH, ROS, and BrdU assay. Tumor cargo (lysate or cells) for DC load was treated with different combinations of THPTS-PDT, freeze/thaw cycles, and IR and immunogenicity analyzed by induction of T-cell activation. Cellular markers (CD11c, 83, 86, 40, 44, 69, 3, 4, 8, PD-L1) were quantified by flow cytometry. Cytotoxic T-cell response was evaluated by calcein AM assay. Immunogenicity of THPTS-PDT-treated GL261 cells lysate was superior to IR-treated lysate, or treated whole cells proven by increased DC phagocytosis, T-cell adhesion, proliferation, cytolytic activity, and cytokine release. These data strongly support the application of PDT together with IR for optimal immunogenic cell death induction in tumor cell lysate used to pulse DC vaccines.

CD8+ T cells are critical to the adaptive immune response and are a target for vaccine development. However, the complex dynamics of cell proliferation can vary response success, providing uncertainty when designing vaccines. Computer models can provide clarity by simulating these dynamics, tracking millions of cell-cell interactions, a feat that is impractical experimentally. Our group created the STORE.1 model, a probabilistic simulation of the CD8+ T cell response to vaccination. While able to accurately simulate in vivo mouse T cell clonal expansion, intracellular dynamics are absent. Furthermore, there is no mechanism by which cell division ceases. This work builds upon the STORE.1 model by systematically explaining the division dynamics of CD8+ T cells and providing measures of the extracellular environment. The new STORE.2 model has demonstrated an ability to accurately simulate differences in CD8+ T cell expansion in WT mice and mice lacking type I conventional dendritic cells up to 170 hours after vaccination. It is the first model to simulate individual cell cycle regulator protein counts for millions of cells, and the resulting impact on pH for the extracellular microenvironment. Finally, it provides a partial mechanism behind division cessation, an important element for future models seeking to further simulate the end of the T-cell response. / Thesis / Master of Applied Science (MASc) / T-cells are an important component of the human immune system, but currently, there are no vaccines in clinical use that are designed to target them. This is because there are many different dynamics that underpin how T-cells activate, and to what degree they can replicate into a substantial pool of pathogen-clearing cells. Learning which candidate vaccines can properly elicit a strong T-cell response is time and resource consuming. Mathematical models can therefore speed development of candidate vaccines by virtually testing their T-cell responsiveness. This thesis works to improve on an existing mathematical model by introducing immunological mechanisms that determine how T-cells undergo cell division, change the acidity of their immediate surroundings, and respond to their own growing population. By doing so, this new model can be more representative of the immunological reality and begin to probe new dynamics of the T-cell response.

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Dendritische Polymere werden im zunehmenden Maße als nicht-virale Vektoren für virus- oder tumor-assoziierte Antigen-Peptide zur Entwicklung neuer immuntherapeutischer Strategien eingesetzt. Diese beruhen auf der Verwendung von dendritischen Zellen (DCs), welche Schlüsselzellen bei der Induktion und Aufrechterhaltung einer T-Zell-basierten Immunantwort darstellen. Im Rahmen dieser Arbeit wurden Nitrilotriessigsäure-funktionalisierte dendritische Glycopolymere (NTA-DG) für den Transport von Antigen-Peptiden in DCs etabliert. Die Ni(II)-NTA-DGs waren durch definierte Komplexierungs- und Freisetzungseigenschaften charakterisiert. So wurde das Antigen-Peptid bei einem pH-Wert unter 6 vom polymeren Träger freigesetzt. Die gebildeten Polyplexe, zwischen Ni(II)-NTA-DG und dem Antigen-Peptid, bewirkten eine Erhöhung der Antigen-Peptid-Aufnahme in immaturen DCs (iDCs). Dieses war nach der Endozytose im frühen endosomalen und lysosomalen Kompartiment von iDCs lokalisiert. Somit kann das Antigen-Peptid am MHC Klasse II-Molekül im lysosomalen Kompartiment ohne sterische Hinderungen durch die Polymeroberfläche binden. Die Polyplexe bewirkten eine Aktivierung der iDCs durch Aufregulation der kostimulatorischen Moleküle CD86 und CD80 sowie der pro-inflammatorischen Zytokine IL-6 und IL-8. Weiterhin wurde die Migrationsfähigkeit und das pro-inflammatorische Potential der Antigen-Peptid enthaltenen maturen DCs (mDCs) aufrechterhalten. Somit stellen Ni(II)-NTA-DGs ein vielversprechendes polymeres Trägersystem für Antigen-Peptide dar.

Dendritische Defekte vermitteln Störungen der Erregbarkeit, Modulation und Plastizität von Neuronen, die zur Entwicklung neurodegenerativer Krankheiten führen können. Eine Mutation des Transkriptionsregulators Zeb2 führt zur Entwicklung des Mowat-Wilson-Syndroms, einer Erkrankung, die mit kognitiven Störungen und einem erkennbaren Gesichtsphänotyp einhergeht. Obwohl kognitive Defekte häufig mit Defekten bei der Bildung des dendritischen Baums in Verbindung gebracht werden, wurde die Rolle von Zeb2 bei der dendritischen Entwicklung bisher nicht untersucht. Hier zeige ich, dass Zeb2-defiziente Neuronen in den oberen neokortikalen Schichten einen abnormalen dendritische Baum aufweisen. Außerdem führt der Verlust von Zeb2 zu einer Fehlorientierung des apikalen Dendriten in einer nicht senkrechten Ausrichtung zur Pia. Darüber hinaus habe ich die Signalwege analysiert, die an der Regulierung der Morphologie des Dendritenbaum stromabwärts von Zeb2 beteiligt sind, und zwar durch Deep Sequencing des Transkriptoms von Zeb2-defizienten und Wildtyp-Neokortices sowie durch Massenspektrometrie-Screens auf Veränderungen in der Expression von Zelloberflächenproteinen nach dem Verlust von Zeb2. Für die vielversprechenden Kandidaten habe ich ein in situ-Hybridisierungs-Screening bei E15,5 durchgeführt. Eine Reihe von Genen, die an der neuronalen Morphologie und an Membranproteinen beteiligt sind, darunter Neuropilin1 und Cadherin6, werden in Gehirnen mit Zeb2-Mangel überexprimiert. Insbesondere habe ich die Rolle der neuen nachgeschalteten Zielgene Nrp1, Cdh6 und Wnt5a analysiert. Ich verwendete shRNA von Nrp1, Cdh6 und Wnt5a in neuronale Zellkultur, um zu zeigen, dass Nrp1 und Wnt5a eine erhöhte dendritische Komplexität in den Zeb2-defizienten neuronalen Zellen fördern. Die Überexpression von Nrp1 in Neuronen der oberen Schicht in vivo mittels in utero Elektroporation ist ausreichend, um die dendritische Komplexität zu fördern. Darüber hinaus zeige ich durch in utero Elektroporation einer shRNA gegen Nrp1 in Zeb2-defiziente Tiere, dass die Unterdrückung von Nrp1 durch Zeb2 für die Unterdrückung exzessiver Verzweigungen während der Entwicklung erforderlich ist. Für die Ausrichtung des Dendritenbaum ist sie jedoch nicht erforderlich. Zusammengenommen zeigen diese Daten die wichtige Rolle des Zeb2-Gens bei der Entwicklung des korrekten Dendritenbaum von Neuronen und der Ausrichtung des apikalen Dendriten. / Dendritic defects mediate disturbances in the excitability, modulation and plasticity of neurons, which can lie at the cause of neurodegenerative diseases. Mutation of the transcriptional regulator, Zeb2, causes the development of Mowat-Wilson syndrome, a condition associated with cognitive defects and a recognizable facial phenotype. Although cognitive defects are often associated with defects in the formation of the dendritic tree, the role of Zeb2 in dendritic development had not been studied previously. Here, I show that Zeb2- deficient neurons in the upper neocortical layers have abnormal dendritic trees. Also, loss of Zeb2 results in the mis-orientation of the apical dendrite to a non-perpendicular orientation to the pia. Furthermore, I have analysed the signalling pathways involved in regulation of dendritic tree morphology downstream of Zeb2 by deep sequencing of the transcriptome of Zeb2-deficient and wildtype neocortices and mass spectrometry screens for changes in the expression of cell surface proteins upon loss of Zeb2. For the promising candidates, I have performed in situ hybridization screening at E15.5. A number of genes involved in neuronal morphology and membrane proteins, including Neuropilin1 and Cadherin6, become overexpressed in Zeb2- deficient brains. In particular, I analysed the role of the novel downstream target genes Nrp1, Cdh6 and Wnt5a. I used shRNA of Nrp1, Cdh6 and Wnt5a in cortical neuron cultures to demonstrate that Nrp1 and Wnt5a promote increased dendritic complexity in Zeb2-deficient neuronal cells. Overexpression of Nrp1 in upper layer neurons in vivo, using in utero electroporation, is sufficient to promote dendritic complexity. In addition, I show using in utero electroporation of an shRNA against Nrp1 into Zeb2-deficient animals, that repression of Nrp1 by Zeb2 is required for suppressing excessive branching during development. It is not needed however for the orientation of the dendritic tree. Taken together, these data show the important role of the Zeb2 gene in the development of the correct dendritic tree of neurons and the orientation of the apical dendrite.