Cluster of differentiation 1 (CD1) d ist ein antigenpräsentierendes Glykoprotein, das verschiedene Lipidklassen (z.B. Glycerophospholipide und Sphingolipide) bindet. CD1d zeigt intrazellulär eine Verteilung sowohl im sekretorischen als auch im endolysosomalen Kompartiment und bindet dort endogene (körpereigene) und exogene (körperfremde, z.B. mikrobiellen) Lipide, die an natürliche Killer T-Zellen, eine Gruppe lipidreaktiver T-Zellen, präsentiert werden. Nach Antigenerkennung zeigen NKT-Zellen eine schnelle Zytokinsekretion, was wiederum zu einer breiten Aktivierung anderer angeborener und adaptiver Immunzellpopulationen wie dendritischer Zellen, natürlicher Killerzellen, B-Zellen und konventioneller T-Zellen führt. In meiner Dissertation untersuchte ich die Rolle von CD1d und NKT-Zellen im Kontext der intestinalen Tumorentstehung (Kapitel 1). Darüber hinaus untersuchte ich CD1d-abhängige Effekte auf den hepatischen Lipidmetabolismus, verbunden mit der Frage ob diese Effekte zumindest partiell in NKT-Zell-unabhängiger Weise vermittelt werden (Kapitel 2). CD1d und NKT-Zellen in der intestinalen Tumorentwicklung NKT-Zellen beeinflussen CD1d-abhängig entzündliche Prozesse im Darm sowie die intestinale Tumorentwicklung. Verschiedene Modelle und Strategien, die sich mit der Klärung der Rolle der NKT-Zelluntergruppen in diesen Erkrankungen beschäftigten, zeigten, dass hierbei eine komplexe Regulierung durch spezifische NKT-Zelluntergruppen, nämlich invariante (i)NKT-Zellen und diverse (d)NKT-Zellen, mit teils gegensätzlichen Effekten zu beobachten ist. CD1d zeigt eine ubiquitäre Expression und kann in zellspezifischer Weise in die NKT-Zell-Aktivierung eingreifen. So vermittelt CD1d im Kontext der intestinalen Entzündung regulatorische NKT-Zell-Signale wenn die Antigenpräsentation von intestinalen Epithelzellen (IECs) ausgeht, während CD1d-Signale von professionellen Immunzellen intestinale Entzündung in NKT-Zell-abhängiger Weise fördern. Das Ziel des ersten Teils meiner Arbeit (Kapitel 1) war die Analyse zelltypspezifischer Effekte von CD1d in der Aktivierung von NKT-Zellen im Rahmen der intestinalen Tumorentstehung. Unter Verwendung des Cre-lox-Systems zur Erzeugung von IEC- und myeloidspezifischen CD1d-defizienten Mäusen und der ApcMin/+ und Apcfl/wt-Mausemodelle intestinaler Tumorentwicklung untersuchte ich die Wirkung der zelltypspezifischen CD1d-Deletion auf die NKT-Zell-Immunantwort im Rahmen der intestinalen Tumorentwicklung. Ich konnte dabei zeigen, dass CD1d in NKT-Zell-abhängiger Weise das intestinale Tumorwachstum fördert. Während die intestinal-epitheliale Deletion von CD1d keine Effekte auf die Tumorentwicklung hatte, führte die myeloide Deletion von CD1d zumindest zu einem partiell reduzierten Tumorwachstum. Diese Daten zeigen, dass myeloide Zellen zum CD1d- und NKT-abhängigen Tumorwachstum beitragen. Darüber hinaus ist anzunehmen, dass weitere, bislang uncharakterisierte Zellen zur CD1d-abhängigen Regulation der Tumorentwicklung beitragen. NKT-Zell-unabhängige Effekte von CD1d im hepatischen Lipidmetabolismus. Der zweite Teil meiner Dissertation (Kapitel 2) befasste sich mit der Rolle von CD1d in der Regulierung des hepatischen Fettstoffwechsels unter konstitutiven Bedingungen sowie im Kontext der nichtalkoholischen Fettleberkrankheit (NAFLD). Mausmodelle mit konstitutiver Deletion von CD1d zeigten dabei, dass diese Prozesse in CD1d-abhängiger Weise vermittelt werden. Da die Deletion von CD1d mit einem Verlust von NKT-Zellen verbunden ist, wurde daraus geschlossen, dass NKT-Zellen zur Pathogenese metabolischer und inflammatorischer Veränderungen bei NAFLD beitragen. Ob CD1d auch in NKT-Zell-unabhängiger Weise zur Regulation des hepatischen Metabolismus beitragen kann, wurde bislang nicht untersucht. CD1d wird ubiquitär und abundant von verschiedenen Zelltypen einschließlich Enterozyten, Adipozyten und Hepatozyten exprimiert und interagiert mit verschiedenen Lipidtransferproteinen. Ich untersuchte daher, ob CD1d auch in direkter, NKT-Zell-unabhängiger Weise Einfluss auf den hepatischen Lipidmetabolismus nimmt. Hierzu wurden CD1d-exprimierende und CD1d-defiziente Mäuse auf einem genetischen Hintergrund mit Defizienz des recombination activating gene 1 (Rag1) untersucht, in dem aufgrund der fehlenden VDJ-Rekombination reife T- und B-Zellen einschließlich NKT-Zellen fehlen. Meine Ergebnisse zeigen, dass CD1d den hepatischen Lipidstoffwechsel unter konstitutiven Bedingungen wie auch im Kontext der nicht-alkoholischen Fettleber in einer NKT-Zell-unabhängigen Weise regulieren kann. Die Mechanismen über die diese Regulation vermittelt wird, werden derzeit experimentell untersucht. Zusammenfassend habe ich in dieser Arbeit die Rolle von epithelialem und myeloiden CD1d in der intestinalen Tumorentstehung charakterisiert. Darüber hinaus konnte ich zeigen, dass CD1d in NKT-Zell-unabhängiger Weise den hepatischen Lipidmetabolismus reguliert.:Zusammenfassung Summary General introduction 1 The CD1 family of antigen presenting proteins 1.1 Structure of CD1 proteins 1.2 Trafficking of CD1 proteins 1.3 Lipid transfer proteins 1.4 CD1 associated lipid repertoire 2 CD1d-restricted T cells 2.1 Lipid antigens presented to CD1 restricted T cells 2.2 NKT cell subsets 2.3 NKT cells in homeostasis and disease Chapter I: CD1d in intestinal tumor development Introduction 1 The role of CD1d and NKT cells in intestinal homeostasis 1.1 The intestine: structure and function 1.2 Immune cell populations in the intestine 1.3 Interplay between iNKT cells and intestinal microbiota 1.3.1 The intestinal microbiota shapes mucosal iNKT cells 1.3.2 Effect of the microbiota on systemic iNKT cells 1.3.3 Bacterial lipid antigens influence iNKT cell-dependent mucosal immunity 1.3.4 Effect of CD1d deficiency on commensals 2 CD1d & NKT cells in cancer 2.1 Enhancing anti-tumor immunity 2.2 Suppressing anti-tumor immunity 3 CD1d & NKT cells in colorectal cancer 3.1 Spontaneous tumorigenesis 3.2 Intestinal inflammation and inflammation-induced cancer Aim of the study Materials and Methods Results 1.1. Validation of the conditional CD1d knockout mouse lines 1.2. Analysis of tumorigenesis in the ApcMin/+ and Apcfl/wt models 1.3. The impact of myeloid cell-specific deletion of CD1d on spontaneous tumor development 1.4. The impact of intestinal epithelial cell specific deletion of CD1d on spontaneous tumorigenesis 1.5. Analysis of constitutive deletion of CD1d in spontaneous tumorigenesis model Discussion Chapter II: CD1d and hepatic lipid metabolism in non-alcoholic fatty liver disease (NAFLD) Introduction 1 Metabolic diseases as a multi-organ pathology 2 Lipid metabolism and inflammation in metabolic diseases 3 Non alcoholic fatty liver disease (NAFLD) 3.1 Mouse models of NAFLD 4 NKT cells in metabolic diseases 4.1 NKT cells in obesity 4.2 NKT cells in NAFLD 5 Potential NKT cell-independent roles of CD1d Materials and methods Results 2.1 Absence of CD1d on the Rag1-deficient background under constitutive conditions reduces neutral lipid accumulation in the liver 2.2 Deletion of CD1d on a Rag1-deficient background reduces hepatic neutral lipid accumulation in response to a HFD and protects from liver injury 2.3 Choline-deficient HFD as a model of NASH shows no difference between CD1d-deficient Rag1-deficient mice and CD1d-proficient littermates Discussion References Appendix 132 List of abbreviations 132 List of tables 137 List of figures 138 Acknowledgments Anlage 1 Anlage 2 / Cluster of differentiation 1 (CD1) d is an atypical antigen-presenting glycoprotein which binds diverse lipid classes including glycerophospholipids and sphingolipids. Trafficking through secretory and endolysosomal compartments, CD1d broadly surveys the cell for endogenous (self) and exogenous (e.g. microbial) lipids and presents those lipids to a subset of T cells, named natural killer T (NKT) cells. NKT cells exhibit rapid and abundant cytokine secretion upon antigen recognition, leading to a broad activation of other innate and adaptive immune cell populations such as dendritic cells, natural killer cells, B cells, and conventional T cells. My thesis studied CD1d and NKT cells in the context of intestinal tumorigenesis (chapter I) and investigated a novel NKT cell-independent role of CD1d in the regulation of hepatic lipid metabolism (chapter II). CD1d and NKT cells in intestinal tumor development NKT cells modulate intestinal inflammation and tumor development in a CD1d-dependent manner. Different models and strategies have been used to elucidate the role of NKT cell subsets in these processes, highlighting a complexity of regulation by specific NKT cells subsets, namely invariant (i)NKT cells and diverse (d)NKT cells, and other immune cells and mediators in the tumor microenvironment. In addition, CD1d, which is ubiquitously expressed, can elicit cell-type specific effects on NKT cell subsets as shown in intestinal inflammation, where intestinal epithelial cell (IEC) CD1d provide regulatory cues, while CD1d signal from bone marrow-derived cells promote intestinal inflammation. The first part of my thesis (chapter I) aimed at further dissecting potential cell type-specific effects of CD1d in the activation of NKT cells in the context of intestinal tumorigenesis. Using the Cre-lox system to generate IEC- and myeloid-specific CD1d-deficient mice and the ApcMin/+ and Apcfl/wt mouse models of intestinal tumorigenesis, I investigated the effects of cell type-specific CD1d deficiency on iNKT cell immune responses and tumor development. My findings show that CD1d, presumably through iNKT cells, promotes tumor growth as shown in a model of constitutive CD1d deletion. While epithelial CD1d did not contribute to NKT cell-dependent tumor growth, myeloid deletion of CD1d was associated with a trend towards reduced tumor growth. These results suggest that myeloid CD1d promotes NKT cell-dependent tumor growth and that other, yet uncharacterized cells, have additional contributions to this process. NKT cell-independent roles of CD1d in the regulation of liver metabolism The second part of my thesis (chapter II) tackled the role of CD1d in the regulation of hepatic lipid metabolism under constitutive conditions and in the context of non-alcoholic fatty liver disease (NAFLD), a prevalent metabolic liver disease which is associated, in a subset of individuals, with immune-mediated progression to liver fibrosis and cirrhosis. Inflammation has an important role in the progression of NAFLD and metabolic diseases, and iNKT cells have been linked to these processes. Specifically, constitutive deletion of CD1d, which is associated with loss of NKT cells, has been demonstrated to influence hepatic lipid metabolism and the progression of NAFLD. In this thesis, I investigated whether the effects of CD1d are indeed dependent on NKT cells or whether CD1d has direct, NKT cell-independent effects on liver metabolism. CD1d is expressed ubiquitously and abundantly by various cell types including enterocytes, adipocytes and hepatocytes, and it binds to a plethora of endogenous cellular lipids through the interaction with lipid transfer proteins, which are important regulators of lipid metabolism. To investigate CD1d-mediated effects that are independent from NKT cells, CD1d-proficient and CD1d-deficient mice were analyzed on a recombination activating 1 (Rag1)-deficient background, which lacks mature T and B cells including NKT cells due to the lack of VDJ recombination. My results demonstrate that CD1d can regulate hepatic lipid metabolism in an NKT cell-independent manner under constitutive conditions and in the context of models of NAFDL. The mechanisms by which CD1d can directly regulate hepatic lipid metabolism are currently being addressed. In conclusion, in this thesis I have characterized the cellular contributions to CD1d- and NKT cell-dependent regulation of intestinal tumor development. In addition, I have identified a novel, NKT cell-independent effect of CD1d on hepatic lipid metabolism.:Zusammenfassung Summary General introduction 1 The CD1 family of antigen presenting proteins 1.1 Structure of CD1 proteins 1.2 Trafficking of CD1 proteins 1.3 Lipid transfer proteins 1.4 CD1 associated lipid repertoire 2 CD1d-restricted T cells 2.1 Lipid antigens presented to CD1 restricted T cells 2.2 NKT cell subsets 2.3 NKT cells in homeostasis and disease Chapter I: CD1d in intestinal tumor development Introduction 1 The role of CD1d and NKT cells in intestinal homeostasis 1.1 The intestine: structure and function 1.2 Immune cell populations in the intestine 1.3 Interplay between iNKT cells and intestinal microbiota 1.3.1 The intestinal microbiota shapes mucosal iNKT cells 1.3.2 Effect of the microbiota on systemic iNKT cells 1.3.3 Bacterial lipid antigens influence iNKT cell-dependent mucosal immunity 1.3.4 Effect of CD1d deficiency on commensals 2 CD1d & NKT cells in cancer 2.1 Enhancing anti-tumor immunity 2.2 Suppressing anti-tumor immunity 3 CD1d & NKT cells in colorectal cancer 3.1 Spontaneous tumorigenesis 3.2 Intestinal inflammation and inflammation-induced cancer Aim of the study Materials and Methods Results 1.1. Validation of the conditional CD1d knockout mouse lines 1.2. Analysis of tumorigenesis in the ApcMin/+ and Apcfl/wt models 1.3. The impact of myeloid cell-specific deletion of CD1d on spontaneous tumor development 1.4. The impact of intestinal epithelial cell specific deletion of CD1d on spontaneous tumorigenesis 1.5. Analysis of constitutive deletion of CD1d in spontaneous tumorigenesis model Discussion Chapter II: CD1d and hepatic lipid metabolism in non-alcoholic fatty liver disease (NAFLD) Introduction 1 Metabolic diseases as a multi-organ pathology 2 Lipid metabolism and inflammation in metabolic diseases 3 Non alcoholic fatty liver disease (NAFLD) 3.1 Mouse models of NAFLD 4 NKT cells in metabolic diseases 4.1 NKT cells in obesity 4.2 NKT cells in NAFLD 5 Potential NKT cell-independent roles of CD1d Materials and methods Results 2.1 Absence of CD1d on the Rag1-deficient background under constitutive conditions reduces neutral lipid accumulation in the liver 2.2 Deletion of CD1d on a Rag1-deficient background reduces hepatic neutral lipid accumulation in response to a HFD and protects from liver injury 2.3 Choline-deficient HFD as a model of NASH shows no difference between CD1d-deficient Rag1-deficient mice and CD1d-proficient littermates Discussion References Appendix 132 List of abbreviations 132 List of tables 137 List of figures 138 Acknowledgments Anlage 1 Anlage 2

Lectin-saccharide interactions are involved in many biological processes essential for the survival and proper function of multicellular organisms. C-type lectin-like receptors, predominantly expressed by cells of the innate immune system, recognize saccharide structures on microbes and also aberrant glycosylation pattern of cancer cells. The NKR-P1 receptor family was among the first natural killer (NK) receptor families that were identified, however ligands for some of members remain still elusive. Recently, publications describing N-acetylglucosamine-terminated oligosaccharide structures as possible ligands for NKR-P1 receptor have been subjects for correction/retractions after investigation of the Ethical Committee of the Institute of Microbiology, ASCR, v. v. i. and Charles University in Prague. Re-evaluation of glycodendrimer effect, particularly effect of N-acetyl-D-glucosamine octabranched dendrimer on polyamidoamine scaffold (GN8P), revealed mostly indirect role of NK cells on modulation of immune responses. Properly folded soluble recombinant rat NKR-P1A and mouse NKR-P1C lack binding activity to neoglycoproteins modified with GlcNAc-terminated structures. Moreover, new possible target cell populations (NKT cells and macrophages) for saccharide binding were identified.

Le diabète de type 1 (T1D) est une maladie auto-immune caractérisée par la destruction des cellules β du pancréas par les lymphocytes T auto-réactifs. Durant ma thèse, nous nous sommes intéressés au rôle des cellules de l’immunité innée dans le T1D à l’aide d’un modèle murin de la maladie : la souris NOD. Au contraire des cellules du système adaptatif (lymphocytes T et B), les cellules de l’immunité innée constituent la première ligne de défense de l’organisme lors d’une infection. Cette population est constituée entre autre de neutrophiles, cellules dendritiques plasmacytoïdes (pDC), macrophages, mais aussi de lymphocytes T et B non conventionnels tel que les cellules iNKT et B-1a. Précédemment, notre laboratoire a mis en lumière le rôle des lymphocytes iNKT dans le développement du T1D. Durant la première partie de ma thèse, nous avons démontré que les lymphocytes iNKT17, une sous-population des lymphocytes iNKT, ont un rôle délétère dans le T1D chez la souris NOD. Ces cellules infiltrent le pancréas et y produisent de l’IL-17, une cytokine pro-inflammatoire. Grâce à des expériences de transferts, nous avons mis en évidence que les lymphocytes iNKT17 exacerbent la maladie via la production d’IL-17. Dans la deuxième partie de ma thèse, nous nous sommes intéressés aux mécanismes qui induisent l’activation des lymphocytes T auto-réactifs. Nous avons observé chez la souris NOD, que la mort physiologique des cellules β conduit à l’activation de cellules de l’immunité innée : les neutrophiles, les lymphocytes B-1a et les pDC. La coopération entre ces cellules conduit à l’activation des pDC qui produisent de l’IFNα. Cette cytokine active les lymphocytes T auto-réactifs qui vont détruire les cellules β du pancréas. Nos résultats montrent que l’immunité innée est un acteur important dans la physiopathologie du diabète sucré. / The type 1 diabetes ( T1D ) is an autoimmune disease characterized by the destruction of β cells in the pancreas by autoreactive T lymphocytes. During my thesis, we are interested in the role of cells of innate immunity in T1D using a mouse model of the disease: NOD mice. In contrast to cells of the adaptive system (T and B lymphocytes ) cells of innate immunity is the first line of defense of the body during infection . This population consists of neutrophils , among other , plasmacytoid dendritic cells ( pDC ) , macrophages , T lymphocytes but not conventional B as iNKT cells and B -1a.Previously, our laboratory has highlighted the role of iNKT cells in the development of T1D . During the first part of my thesis , we demonstrated that iNKT17 cells, a subpopulation of iNKT cells, have a deleterious role in T1D in NOD mice . These cells infiltrate the pancreas and there produce IL -17 , a proinflammatory cytokine. Through transfer experiments , we demonstrated that lymphocytes iNKT17 exacerbate disease through the production of IL-17 . In the second part of my thesis , we investigated the mechanisms that induce the activation of autoreactive T lymphocytes. We observed in NOD mice , the physiological death of β cells leads to activation of innate immunity cells : neutrophils, lymphocytes B- 1a and pDCs . The cooperation between these cells leads to activation of pDC that produce IFNa . This cytokine activates autoreactive T cells which will destroy the β cells of the pancreas. Our results show that innate immunity is an important player in the pathogenesis of diabetes mellitus.

La survenue de la maladie du greffon contre l’hôte aiguë (GVHa) après l’allogreffe de cellules souches hématopoïétiques (CSH) reste une source majeure de mortalité et morbidité pour laquelle des avancées thérapeutiques restent indispensables. Les lymphocytes NKT invariants (iNKT) possèdent de multiples propriétés à potentiel immuno-régulateur et anti-tumoral, mais peu de données existent chez l’homme dans le cadre de l’allogreffe de CSH. Nous avons récemment montré qu’une bonne reconstitution en iNKT post greffe ainsi qu’une bonne richesse et capacité d’expansion des iNKT du greffon, notamment du sous type CD4-, étaient corrélés à une moindre survenue de GVHa chez les patients. Ce travail avait pour objectif de déterminer quel sous type de iNKT humain pouvait réguler la réponse allo-immune et ses mécanismes d’action. Dans le modèle murin humanisé de xéno-GVH, chez la souris NSG, nous avons mis en évidence que le sous-type iNKT CD4- humain ,contrairement au sous type CD4+, permettait de diminuer la survenue de GVH et d’améliorer la survie des souris NSG greffées avec des PBMC humains enrichis ou non en lymphocytes iNKT. Nous avons montré de façon concordante, in vivo et in vitro, que les iNKT CD4- humains diminuent le profil Th1 et Th17 des lymphocytes T conventionnels et leur marqueurs d’activation après stimulation allo- et xéno-génique. Par ailleurs, les iNKT CD4- humains induisent la mortalité des cellules dendritiques in vitro et in vivo, contrairement au sous-type iNKT CD4+ qui induit leur maturation. Par contre, les iNKT CD4- n’ont pas d’impact sur les lymphocytes T régulateurs, ni sur la prolifération des T conventionnels. Ils n’altèrent pas non plus la qualité de la prise de greffe des cellules humaines dans la souris NSG. Nous concluons, que contrairement aux iNKT murins, le sous-type iNKT CD4- humain, et non pas CD4+, est capable de diminuer la survenue de xéno-GVH dans le modèle murin NSG par une régulation directe de la réponse immune allogénique. Ces résultats obtenus dans ce modèle préclinique ouvrent la possibilité d’une nouvelle thérapie cellulaire utilisant ces cellules chez l’homme pour prévenir la survenue de GVH aiguë. / The occurrence of acute graft versus host disease (aGVHD) after allogeneic stem cell transplantation (allo-SCT) is a major source of mortality and morbidity and new therapeutic advances are urgently needed. Despite the numerous immunomodulatory properties depicted for the iNKT lymphocytes, there are few studies of these lymphocytes in human allo-SCT and GVHD setting. We previously reported that a higher iNKT reconstitution in patients after allo-SCT and higher graft CD4- iNKT expansion rates, were associated with reduced risk of aGVHD in patients. In this study we aimed to assess a direct immune regulatory role of CD4+ or CD4- iNKT subsets and their mechanisms of action during the allo-immune response. We demonstrated that the human iNKT CD4- subset, but not the CD4+ subset, was associated with reduced xeno-GVHD and prolonged survival of humanized NSG mice infused with human PBSC with or without iNKT cells. We also concomitantly showed, both in vitro and in vivo, that human iNKT CD4- could dampen Th1 and Th17 conventional T cell allo and xeno-responses as well as T cell activation markers. In addition, whereas iNKT CD4+ could stimulate DC maturation, iNKT CD4- induced their apoptosis, in vitro and in vivo. We did not observe any impact on regulatory T cells and conventional T cell proliferation. iNKT CD4- did not impact the engraftment of human cells in NSG mice. We conclude that, by contrast with mice, in humans, the CD4- subset and not their CD4+ counterpart can directly down-regulate the allo-immune response. These results obtained in a robust humanized preclinical xeno-GVH mice model could open new strategies of cellular therapy in the prevention of acute GVHD.

In den letzten Jahren ergaben sich vermehrt Hinweise, dass dendritische Zellen (DCs) zu einer Stimulation von Natürlichen „Killer“ (NK)-Zellen in der Lage sind, die als zytotoxische Effektorzellen des angeborenen Immunsystems Tumorzellen eliminieren. Aus diesem Grund bestand ein wesentliches Ziel dieser Arbeit in der Analyse der Wechselwirkungen zwischen nativen DCs und NK-Zellen. Dazu wurden slanDCs verwendet, welche die größte DC-Subpopulation des Blutes repräsentieren. Zunächst wurde evaluiert, ob slanDCs eine effiziente Aktivierung von NK-Zellen bewirken. Als ein Ergebnis zeigte sich, dass Lipopolysaccharid (LPS)-stimulierte slanDCs sowohl zu einer verstärkten Expression des Aktivierungsmarkers CD69 auf der Oberfläche von NK-Zellen als auch zur Induktion der NK-Zell-Proliferation führen. Darüber hinaus wurde erstmals die slanDC-abhängige Erhöhung der Expression von aktivierenden Rezeptoren (NKp46, NKp44, NKp30) und Korezeptoren (2B4, DNAM-1) auf NK-Zellen demonstriert, welche essentiell für die NK-Zell-vermittelte Erkennung und Lyse von Tumorzellen sind. In weiteren Untersuchungen induzierten LPS-aktivierte slanDCs eine erhebliche Produktion von Interferon (IFN)-gamma in NK-Zellen, welches proliferationshemmend auf Tumorzellen und aktivierend auf T-Lymphozyten wirkt. Funktionelle Analysen ergaben, dass aktivierte slanDCs das zytotoxische Potential von NK-Zellen gegenüber der Tumorzelllinie K-562 deutlich verstärken. Untersuchungen der zugrunde liegenden Mechanismen zeigten die herausragende Bedeutung von IL-12, das sowohl die Steigerung der IFN-gamma-Sekretion als auch die Zunahme der zytolytischen Aktivität von NK-Zellen induzierte. Darüber hinaus konnte erstmals gezeigt werden, dass LPS-aktivierte slanDCs eine Zytotoxizität von NK-Zellen gegenüber frisch etablierten Blasten von Patienten mit akuter myeloischer Leukämie induzieren. In weiteren Untersuchungen wurde evaluiert, ob NK-Zellen ihrerseits die immunstimulatorischen Eigenschaften von slanDCs beeinflussen. Die Analysen zeigten erstmals, dass unstimulierte NK-Zellen die Expression von MHC-Klasse II-Molekülen, kostimulatorischen Molekülen und Adhäsionsmolekülen auf slanDCs deutlich erhöhen und somit ihre Fähigkeit zur Aktivierung von CD8+ T-Lymphozyten sowie CD4+ T-Helferzellen fördern. NK-Zellen führen ebenfalls zu einer deutlichen Verstärkung der Produktion von IL-12 durch LPS-stimulierte slanDCs. Darüber hinaus zeigte sich, dass NK-Zellen die Sekretion des immunsuppressiven Zytokins IL-10 durch LPS-stimulierte slanDCs reduzieren. In weiteren Analysen wurde demonstriert, dass die Interaktionen mit NK Zellen die Fähigkeit von LPS-aktivierten slanDCs zur Programmierung naiver CD4+ T-Lymphozyten in IFN-gamma-produzierende T-Helfer-1-Zellen deutlich verstärken. Diese Ergebnisse zeigten deutlich, dass stimulierte slanDCs und NK-Zellen in der Lage sind, sich wechselseitig zu aktivieren. NKT-Zellen repräsentieren eine weitere bedeutende Effektorzellpopulation der frühen antitumoralen Immunabwehr, die durch Sekretion von Zytokinen und ein ausgeprägtes zytolytisches Potential zur Elimination von Tumorzellen beiträgt. Deshalb wurden im Rahmen dieser Arbeit erstmals die Wechselwirkungen zwischen slanDCs und NKT-Zellen analysiert. Dabei verstärkten LPS-stimulierte slanDCs die Expression des Aktivierungsmarkers CD69 auf NKT-Zellen. Darüber hinaus induzierten LPS-aktivierte slanDCs eine deutliche IFN-gamma-Produktion in NKT-Zellen, wobei erneut die zentrale Rolle von IL-12 gezeigt wurde. Diese Ergebnisse demonstrierten, dass stimulierte slanDCs zu einer effektiven Aktivierung von NKT Zellen in der Lage sind. In abschließenden Untersuchungen wurde die Wirkung von NKT-Zellen auf slanDCs evaluiert. Dabei verstärkten NKT-Zellen die Maturierung von slanDCs erheblich und führten zu einer signifikanten Steigerung der IL-12-Produktion sowie zu einer Reduktion der IL-10-Freisetzung in Abhängigkeit von IFN-gamma. Die gewonnenen Daten demonstrierten, dass NKT-Zellen und slanDCs zu einer gegenseitigen Aktivierung befähigt sind. Die im Rahmen dieser Dissertation gewonnenen Erkenntnisse zu den Interaktionen von slanDCs und NK- bzw. NKT-Zellen können einen wesentlichen Beitrag zum Verständnis der Immunabwehr von Tumoren leisten und die Konzeption neuer antitumoraler Therapiestrategien unterstützen.

The use of genome wide ENU mutagenesis screening has uncovered vast numbers of novel genes involved in the control of the immune system. This thesis describes the characterization of a novel mutant, Captain Morgan (CPM), originally identified in an immunization screen designed to evaluate both the initial antibody response to antigen and the ability to sustain antibody production. Mapping of this mutant lead to the identification of a single base pair mutation in a novel guanine nucleotide exchange factor, dedicator of cytokinesis 8 (DOCK8). The mutation was found to result in altered gene splicing of the DOCK8 protein leading to the truncation of the protein and loss of catalytic function. The importance of understanding the role of DOCK8 in host immunity has been recently underlined by the discovery that cohorts of patients suffering from autosomal recessive forms of hyper-IgE syndrome have loss-of-function or deletions in this novel guanine nucleotide exchange factor. Disease in these patients is characterised by recurrent viral and bacterial infections mainly of the skin and lungs, with reduced levels of peripheral CD4⁺ and CD8⁺ T cells in the blood of patients. Patients also have high levels of IgE and eosinophilia in the blood and are highly atopic with increased prevalence of allergic diseases including asthma. Loss of DOCK8 function results in a number of phenotypes in CPM mice, which may help understand the immunodeficiency syndrome experienced by DOCK8 deficient patients. CPM mice, like DOCK8 deficient patients, are lymphopenic with losses of both CD4⁺ and CD8⁺ T cells in the blood and secondary lymphoid organs. Challenge of CPM mice with modified vaccina virus (MVA) and influenza strain X31 demonstrated normal primary anti-viral responses. However, similar to the loss of germinal centre B cells previously described in these mice, memory T cell responses were diminished, which may explain the susceptibility of DOCK8 deficient patients to recurrent infections. In addition to the loss of peripheral T cells, rare populations of lymphocytes such as invariant natural killer T cells (iNKT) were also reduced in the liver and thymus. Due to their roles in bacterial and viral responses and cancer immunosurveillance it is expected that loss of these cells will contribute to disease severity. Together these findings illustrate the importance of the ENU mutagenesis model for generating new mutants, which can enhance our understanding of mammalian genes and create disease models of human disease. Further characterization of DOCK8 deficiency and the molecular mechanisms of DOCK8 function will have important implications for disease diagnosis and ongoing treatment for patients.

Die Funktion und Spezifität von Natürlichen-Killer-T-Zellen (NKT) in angeborener und erworbener Immunität ist nicht vollständig geklärt. Die Mehrheit der NKT-Zellen erkennt alpha-galactosylceramid (alphaGalCer), ein Lipid eines marinen Schwamms mit ungeklärter Relevanz. Verschiedene mykobakterielle Lipide wurden isoliert und auf ihre CD1d-Bindung und NKT-Zell-Aktivierung untersucht. Phospatidylinositol-mannosid (PIM) von Mycobacterium bovis BCG konnte als erstes bakterielles NKT-Zell-Antigen identifiziert werden. PIM aktiviert CD1d-abhängig murine und humane NKT-Zellen zur IFN-gamma aber nicht zur IL-4 Produktion. Mehrere andere Lipid-Fraktionen aktivierten ebenfalls NKT-Zellen. Diese Stimulation war entweder eine direkte, T-Zell-Rezeptor (TZR)-vermittelte und/oder indirekte, Toll-like-receptor 2 (TLR2) vermittelte Aktivierung. Iso-globotrihexosylceramide (iGb3) wurde als das endogene NKT-Zell-Antigen beschrieben. IGb3 ist ubiquitär in Lysosomen vorhanden. Dies wirft die Frage nach der Regulation der Antigen-Verfügkarkeit und der Kontrolle der NKT-Zell-Aktivierung auf. Es konnte gezeigt werden dass die Regulation der Antigen-Verfügbarkeit essentiell für die Regulation der NKT-Zell-Aktivität ist. Unkontrolliertes Auftreten und erhöhte Konzentration von iGb3 führte zu einer substantiellen Reduktion der NKT-Zell-Zahl, vermutlich durch Aktivierungs-induziertem-Zelltod. Mit Hilfe von DNS-Microarray Analysen wurden die Gen-Expressionsprofile von naïven NKT-Zellen und klassischen CD4 T-Zellen, regulatorischen T-Zellen, NK-Zellen und aktivierten NKT-Zellen verglichen. Es konnte sowohl ein NKT-Zell-spezifisches Expressionsmuster etabliert als auch eine gemeinsame Expression von Genen in allen verglichenen Zelltypen identifiziert werden. Naive und aktivierte NKT-Zellen zeigen eine erhöhte Expression von Apoptose-regulierenden Genen welches auf eine starke Selbst-Kontrolle zur präzisen Regulation der eigenen Aktivität hinweist. / The function and specificity of Natural Killer T (NKT) cells in innate and acquired immunity still remains elusive. The vast majority of CD1d restricted NKT cells recognise alpha-galactosylceramid (alphaGalCer), derived from a marine sponge, a lipid of unclear physiological significance. Different mycobycterial glycolipids were isolated and examined for binding to CD1d as well as for their capacity of NKT cell stimulation. Phospatidylinositol-mannoside (PIM) derived from Mycobacterium bovis BCG was identified as the first bacterial lipid antigen presented by CD1d. PIM activated both murine and human NKT cells to secrete IFN-gamma but not IL-4 in a CD1d dependent manner. Additionally, several other lipid fractions with NKT cell activation capacities were identified. This activation was either a direct, T-cell-receptor (TCR) mediated and/or an indirect, toll-like-receptor 2 (TLR2) mediated activation. Iso-globotrihexosylceramide (iGb3) was described as the endogenous NKT cell antigen. iGb3 is a ubiquitously present lysosomal glycolipid which raises the question of regulation of antigen availability and NKT cell activation control. It could be shown that regulation of antigen availability plays a crucial role in regulation of NKT cell activation. Moreover, uncontrolled appearance and increased concentrations of the endogenous antigen iGb3 led to substantial decrease in NKT cell number, presumably by activation induced cell death. Using DNA Microarray analysis, the gene expression profiles of naïve NKT cells and classical CD4 T cells, regulatory T cells, NK cells as well as to activated NKT cells were compared. The profiles revealed a NKT cell specific gene expression pattern as well as expression of genes which NKT cells share with NK cells, conventional CD4+ T cells and Treg cells. Both, naïve and activated NKT cells display elevated expression of apoptosis regulating genes providing NKT cells with high degree of self-control to precisely regulate their own activity.

Le foie est un organe connu empiriquement pour sa capacité à induire une tolérance immunologique des lymphocytes T (LT). Cette propriété pourrait s'exercer en particulier visà- vis d'antigènes alimentaires drainés de l'intestin dans le foie via la veine porte. L'objectif de ce travail de thèse était d'étudier l'implication du foie dans la tolérance périphérique et en particulier la tolérance induite par voie orale, en utilisant un modèle murin d'hypersensibilité retardée de contact (HSRC) spécifique d'haptène (DNFB), induit par des LT CD8+ spécifiques. Nous avons identifié deux types cellulaires enrichis dans le foie et doués de propriétés régulatrices/tolérogènes : i) Les cellules dendritiques plasmacytoïdes, capables d'induire une tolérance des LT CD8+ in vivo et jouant un rôle clef dans l'induction de la tolérance orale, et ii) les cellules NKT capables de réguler la réponse T CD8+ aussi bien au cours de la phase afférente que de la phase efférente de la réponse immunitaire.

Made available in DSpace on 2015-07-22T20:49:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-27. Added 1 bitstream(s) on 2015-08-11T03:25:52Z : No. of bitstreams: 1 Publico-10949.pdf: 1710290 bytes, checksum: 4f0e0bd77ebdd477f06366315fe3b8f9 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / As características da resposta imunológica em pacientes infectados pelo Mycobacterium leprae e pelo virus da imunodeficiência humana (HIV) não estão bem elucidadas. O objetivo geral desta tese foi avaliar diferentes parâmetros imunológicos na interação destas doenças. Inicialmente avaliamos a imunidade celular em quatro grupos de pacientes dividos em: indivíduos saudáveis, monoinfectados pelo Mycobacterium leprae, monoinfectados pelo virus da imunodeficiência humana e coinfectados pelo M. leprae e HIV. Observamos que o grupo coinfectado apresentou diminuição da razão de células CD4:CD8, aumento de níveis de ativação em células T CD8+, aumento da razão de células T V1:V2 e diminuição da porcentagem de células dendríticas plasmocitóides, comparadas com o grupo de indivíduos monoinfectados pelo HIV-1. A produção de IL-4 por linfócitos T CD4+ foi correlacionado positivamente com a porcentagem de subpopulações de memória efetora de células T CD4+, sugerindo diferenciação antigênica da população de células T em ambas as infecções de HIV-1 e M. leprae. A coinfecção por M. leprae pode exacerbar a imunopatologia da doença induzida pelo HIV-1. Houve uma tendência na expressão de citocinas Th2 na resposta de células T CD4+ em ambas infecções de M. leprae e HIV-1, mas não obtivemos efeitos aparentes nos pacientes coinfectados. No trabalho subsequente, avaliamos de forma quantitativa e qualitativa as células NKT da imunidade inata nos indivíduos saudáveis e infectados pelo HIV. A frequência de células NKT que secretam IFN- e TNF- estava significantemente diminuída em pacientes HIV-1 quando comparados com os indivíduos saudáveis. A magnitude da resposta de IFN- teve correlação inversa com o número de anos da infecção, sugerindo que a função das células NKT está diminuída progressivamente ao longo do tempo. Não houve alteração na resposta das células NKT dos indivíduos infectados pelo HIV após tratamento com Forbol12-Miristato13-Acetato (PMA) e ionomicina, sugerindo um defeito no sinal do TCR prejudicando a produção de citocina. Foi observado uma diminuição na magnitude da resposta com produção de citocinas Th1 pelas células NKT quando correlacionado com a expressão de CD161, sugerindo um mecanismo inibitório deste receptor na regulação da resposta de células NKT. Por último, nós demonstramos que pacientes coinfectados tem redução da frequência de células NKT no sangue periférico, quando comparados com indivíduos saudáveis e pacientes monoinfectados pelo M. lepare. Por outro lado, as células NKT de pacientes coinfectados secretam mais IFN- quando comparadas com pacientes monoinfectados pelo M. lepare. Estes resultados sugerem que as células NKT têm atividade aumentada em pacientes coinfectados, contudo em frequência diminuída no sangue periférico. / The immune response characteristics in patients infected with Mycobacterium leprae and human immunodeficiency virus (HIV) is not elucidated. The aim of this study was to evaluate different immune parameters in the overlapping of both diseases. In the first paper we observaded The co-infected group exhibited lower CD4:CD8 ratio, higher levels of CD8 T-cell activation, increased V1: V2 T cell ratio and lower percentage of plasmacytoid dendritic cells, compared to HIV-1 infected subjects. Across infected groups, IL-4 production by CD4 T lymphocytes was positively correlated with the percentage of effector memory CD4 T cells, suggesting antigenically-driven differentiation of such T cell population in both HIV-1 and M. leprae infections. Co-infection with M. leprae may exacerbate the immunopathology of HIV-1 induced disease. A T helper 2 (Th2) bias in the CD4 T-cell response was evident in both HIV-1-infection and leprosy, but no additive effect was apparent in co-infected patients. Subsequently, we evaluated quantitatively and qualitatively the NKT cells from innate immune response in HIV-infected subjects and healthy controls. The frequencies of NKT cells secreting IFN- and TNF- were significantly lower in HIV-1-infected subjects and the magnitude of the IFN- production was negatively correlated with the number of years of infection, suggesting that NKT cell function is progressively lost over time. NKT cell responses in HIV-1 infected subjects were essentially normal after treatment with Phorbol12-Myristate13- Acetato (PMA) and ionomycin, suggesting that defective TCR-signaling was the underlying defect in the cytokine production. The lower levels of the NKT Th1 response correlated with higher CD161 expression, suggesting a role for this inhibitory receptor in regulating NKT cell responsiveness. Finally, we have investigated the NKT cells in the context of HIV and M. leprae coinfection. The volunteers were enrolled into four groups: twenty-seven healthy controls, seventeen HIV seropositive patients, seventeen patients with leprosy, and twenty-three co-infected patients with leprosy and HIV-1 infection. Flow cytometric and ELISPOT assays were performed in stored PBMC. We demonstrated that coinfected patients have reduced NKT cells in the peripheral blood when compared to healthy subjects and leprosy monoinfected patients. On the other hand, NKT cells from coinfected patients secrete more IFN- when compared to leprosy monoinfected patients. These results suggest that NKT cells are highly active in coinfected patients, although occurring in lower frequency in the peripheral blood. / TEDE / BV UNIFESP: Teses e dissertações

NKT cells are an important part of human immune system and recognize a specific set of antigens called glycolipids. Only a handful of "natural" NKT cell antigens are known till date. Although NKT cells play a protective role against pathogenic organisms, imbalances in NKT cell functions are implicated in many diseases including asthma. Allergic asthma, a Th2 driven inflammation of airways, is primarily caused by inhalation of environmental allergens. In the last decade, inhaled allergen Aspergillus fumigatus has been under scrutiny for the presence of NKT cell antigens that might trigger asthma. We successfully isolated, characterized and synthesized a "natural" antigenic glycolipid which activates NKT cells in CD1d dependent manner. When this glycolipid is administered intranasally to mice, WT but not CD1d-/- mice developed airway hyperreactivity (AHR), which is a cardinal feature of asthma. Our results indicate that this glycolipid also triggers the production of key cytokines responsible for development of airway hyperreactivity, namely IL-4 and IL-13. Widespread use of antibiotics has convoluted the problem of antimicrobial resistance. Our research group has developed a novel class of antimicrobial peptide mimics called Ceragennins. These cholic acid based antimicrobial compounds have many desirable properties including low MICs, effectiveness against biofilms, and relatively low manufacturing cost. In order to advance the clinical development of Ceragennins, we developed analytical methods for qualitative and quantitative determination of these compounds in complex biological matrices. These methods were also used for carrying out the stability studies of Ceragenins under varying pH and temperatures