Natural killer T (NKT) cells are a subset of T cells that modify a variety of immune responses. NKT cells recognize glycolipid antigen presented by a molecule called CD1d, a nonclassical antigen-presenting molecule. The best known subset of CD1d-dependent NKT cells expresses an invariant T cell receptor Vα (TCR-α) chain. These are referred to as type I or invariant NKT (iNKT) cells. When stimulated by a glycolipid, NKT cells rapidly release large amounts of cytokines. Cytokines released by NKT cells can induce either Th1 or Th2 responses. Th1 cytokines are effective in regulating bacterial, parasitic, and viral infections. But Th1 responses are also involved in some autoimmune diseases, such as type 1 diabetes, lupus, rheumatoid arthritis, and allergen-induced asthma. Th2 cytokines can attenuate proinflammatory Th1 responses and therefore prevent some autoimmune diseases. Lysosomal processing and CD1d loading is very important in regulating the stimulatory properties of antigens. Analogs of KRN7000, with small molecules appended on C6” position of the galactose portion, do not significantly change stimulation of NKT cells. The question is if the substitution at this position would influence the lysosomal processing. Two sets of mono- and disaccharides with and without substitution at C6” position were prepared and evaluated the NKT cell stimulatory properties. The substitution at the C6” position of the sugar moiety of glycolipids do not significantly impact the stimulatory properties of glycolipids and their processing in lysosome. Small changes at C6” are well tolerated. A double bond in the acyl chain and modification of the C6” functional group helped the glycolipid loading into CD1d and NKT cells stimulation. PBS57 is 100 times more active than KRN 7000 in stimulation of NKT cells responses in vitro and in vivo. This improvement is probably due to increasing solubility and improving binding ability with the CD1d.

Le développement de nouveaux ligands capables d’orienter sélectivement la réponse des cellules Natural Killer T invariantes (iNKT) vers un profil immunostimulant (Th1) ou immunorégulateur (Th2) est un challenge important qui peut mener à de nouvelles opportunités thérapeutiques dans le traitement de nombreuses maladies auto-Immunes et de cancers. Dans ce contexte, il est indispensable de comprendre le mécanisme de polarisation des iNKT. L’hypothèse principale est que l’intensité et la nature de la réponse biologique dépendent de la stabilité de l’interaction CD1d/TCR (Récepteur des Cellules T) qui serait elle-Même influencée par la forme du CD1d et donc par l’antigène qui y est chargé. C’est pourquoi nous avons étudié l’impact des activateurs des iNKT sur la structure du CD1d dans le but de faciliter la conception de molécules induisant sélectivement un profil de réponse Th1 ou Th2.En s’aidant des relations structure-Activité, du « docking-Scoring » et de dynamiques moléculaires de complexes CD1d/ligand analysées par différents outils, nous avons comparé la structure des CD1d humain et murins en fonction des profils de réponse.A partir de l’analyse des trajectoires de dynamique moléculaire, nous avons identifié des différences notables dans le comportement des CD1d humains et murins, les plus remarquables étant des changements dans les distances inter-Hélice et une mobilité du ligand accrue dans les systèmes humains. Un autre résultat majeur est l’identification d’une conformation spécifique de la tête polaire du ligand qui pourrait être corrélée à une réponse préférentiellement Th2.Ces différentes méthodes et la combinaison des descripteurs protéiques et moléculaires utilisés pour analyser la dynamique des complexes binaires nous donne suffisamment d’indices structuraux pour tenter de prédire le comportement de ligands dans le CD1d et aider à la conception de nouveaux modulateurs des cellules iNKT. / Development of new ligands able to switch invariant Natural Killer T (iNKT) cells toward an immunostimulant Th1 or an immunoregulative Th2 profile is a great challenge that can lead to new therapeutic opportunities in the treatment of various auto-Immune diseases or cancers. In this context, understanding the polarizing effect of iNKT ligands is of a major interest. We hypothesized that the intensity and nature of the biological response could depend on the stability of the CD1d-T Cell Receptor (TCR) interactions under the influence of the antigen which could modulate the shape of CD1d. Thus, our goal was to study the impact of iNKT ligands on the structure of the CD1d molecule and find clues to help design Th1/Th2 selective ligands.Using structure-Activity relationships, docking and molecular dynamic analyzed by a mix of classical and in house tools, we compared the structural behavior of the human and mouse CD1d molecule loaded with different ligands inducing Th1 or Th2 immune response profile. From the analysis of our molecular dynamics trajectories, it appears that there are noticeable differences in the behaviour of human and mouse CD1d molecules, mainly caracterized by changes at the inter-Helix distance and an increase ligand mobility for human systems.One major result is the identification of a specific conformational state of the ligand sugar group which could be correlated, in the present study, to the biological Th2 biased response. The different methods and combinations of ligand and protein descriptors used to analyze the dynamics of the binary complexes provide a structural basis for predicting the very different dynamical behaviors of ligands in CD1d and might aid in the future design of new iNKT modulators.

In this thesis, I examined the expression of CD1d on respiratory epithelial cells (REC) in human and explored its potential role in mucosal immunity in the lungs. Hitherto, there have been no published reports of CD1d expression on REC though it has been observed on other epithelial surface (notably intestinal epithelial cells). This observation, and work in my supervisor’s laboratory demonstrating CD1d-restricted natural killer T cells (iNKT) cells as early players in the lungs of influenza A virus (IAV)–infected mice prompted my interest in this area. I hypothesized that CD1d is expressed on REC and that it contributes to activation of iNKT cells in the lungs via presentation of endogenous or pathogenic glycolipids. I asked following questions – i) is CD1d expressed on REC ii) can this expression be regulated and iii) does CD1d expression on REC have a function. This thesis provides the first evidence for CD1d expression on human RECs (in cell lines and primary RECs) and also presence of alternatively spliced variants. CD1d expression was inducible by viral-associated signals in vitro and despite being non-professional antigen presenting cells, RECs can present glycolipid (α–GC) to, and activate iNKT cells in a CD1d-dependent process resulting in production of both Th1 and Th2 cytokines. Using whole genome expression profiling, I then showed that iNKT cells expressed a distinct profile of genes while in direct contact with α–GC-bound CD1d on RECs compared to cells separated by transwell membrane. Here early biological pathways were dominated by cytokine and chemokine related genes (JAK-STAT signaling pathways, cytokine-responsive elements and cytokine/chemokine genes) and apoptosis-related genes. This suggested that glycolipid-bound CD1d on REC was capable of inducing a programme of immune activation in iNKT cells. I concluded my work by examining if CD1d expression on RECs influenced its active role in immunity. Using wild type and CD1d-deficient transgenic mice challenged with IAV, I showed that CD1d expression is induced on REC in vivo after viral challenge, and in the absence of CD1d, mice showed worse outcome. RECs isolated from CD1d-deficient mice had a much stronger gene expression profile for pro-inflammatory genes. This suggested that CD1d expression on REC could have a bi-directional effect – on the RECs that expressed CD1d (preventing excessive immune-related genes activation) and on the iNKT cells that it engaged (activation, with pro-immunity effects). The thesis concludes with discussion of the potential implications of these findings and future work to examine hypotheses generated from this work.

Les cellules iNKT (de l’anglais invariant Natural Killer T) constituent un sous-type particulier de lymphocytes T caractérisé par un profil de type inné. Ces cellules répondent rapidement à des antigènes lipidiques et glycolipidique présentés par le CD1d, une glycoprotéine exprimée par les différentes cellules présentatrices d'antigène. Suite à l’activation, les cellules iNKT sont capables de produire de grandes quantités de cytokines anti-inflammatoires et pro-inflammatoires et elles sont impliquées dans différentes maladies, telles que l'allergie, l'auto-immunité, le cancer et les infections, parmi lesquelles la leishmaniose. Les parasites protozoaires de les espèces Leishmania sont les agents causaux de la leishmaniose, une maladie tropicale négligée dont la manifestation la plus grave affecte les organes viscéraux et qui peut être mortelle si elle n'est pas traitée. Le succès de l'infection dépend de la capacité du parasite à maitriser la réponse immunitaire de l'hôte. Récemment, quelques groupes, y compris le nôtre, ont observé que les parasites Leishmania libèrent des vésicules extracellulaires (VE). Les VE sont formées par une bicouche membranaire lipidique, contenant des lipides, des protéines et du matériel génétique et elles peuvent transmettre des molécules dérivées des pathogènes aux cellules hôte sans contact direct entre les cellules. Les VE produites par les parasites Leishmania et aussi par d’autres protozoaires ont été associés à des effets pro-parasite car elles favorisent un environnement plus permissif à l'établissement de l'infection. Dans cette thèse, nous avons étudié l'effet du matériel extracellulaire (ME), correspondant aux VE et aux molécules non-associées aux VE, libéré par les promastigotes de L. infantum sur les cellules iNKT. Dans le début de ce travail, il a été observé que le ME de L. infantum empêche l'expansion ex-vivo des cellules iNKT humaines à partir de cellules mononucléaires du sang périphérique. Cela a mis en évidence la communication entre les cellules iNKT et le ME de L. infantum, ce qui a été exploré par la suite. Le ME de L. infantum module la capacité très importante des cellules iNKT à produire des cytokines. En effet, le ME de L. infantum empêche la production des différentes cytokines par les cellules iNKT, comme par exemple IL-4 et IFNγ. De plus, nous avons aussi démontré que le ME de L. infantum compète avec l’α-GalCer, un agoniste très puissant des cellules iNKT, pour la liaison à la molécule CD1d, ce qui justifie l’effet inhibiteur dans l'activation des cellules iNKT. Nous avons aussi montré que les lipides qui sont présents dans chaque fraction du ME de L. infantum ont un rôle très important dans l’inhibition de l'activation et l'expansion des cellules iNKT. Ainsi, le ME de L. infantum, par ces lipides, peut participer à l’altération de l’activation des cellules iNKT dépendante du CD1d. Cela ajoute une nouvelle évidence de la contribution du ME de L. infantum dans la subversion de la réponse immunitaire de l’hôte. La communication entre le ME libéré par un pathogène et les cellules iNKT a été étudié pour la première fois, ce qui a suggéré un mécanisme de modulation de ces cellules qui n’avait jamais été décrit. Ce travail ouvre des perspectives pour l'étude de l'interaction de ME libéré par d'autres pathogènes avec des cellules iNKT. De plus, l'analyse des lipides contenus dans le ME de L. infantum pourra aboutir à la découverte de nouvelles molécules spécifiques pour inhiber les cellules iNKT. Cela apporterait des avantages significatifs dans les approches cliniques ciblant la modulation de l'activation des cellules iNKT. / The invariant natural killer T (iNKT) cells constitute a particular subset of T lymphocytes characterized by an innate-like profile. These cells rapidly respond to lipid and glycolipid antigens bound by the glycoprotein CD1d expressed by different antigen presenting cells. Once activated, they release large amounts of anti- and proinflammatory cytokines. Thus, iNKT cells are endowed with a remarkable immunomodulatory potential and they have been implicated in different disorders, such as allergy, autoimmunity, cancer and infection, among which is leishmaniasis. Leishmania spp. are a group of protozoan parasites that includes the causative agents of leishmaniasis. This is a neglected tropical disease in which the most severe form of manifestation affects visceral organs and could be fatal if left untreated. Importantly, the success of Leishmania infection relies on the capacity of the parasite to subvert host immune responses. Recently, a few groups, including ours, observed that Leishmania parasites release extracellular vesicles (EVs). EVs are vesicles formed by a lipid bilayer membrane, containing other lipids, proteins and genetic material on their surface as well as in their lumen. Due to their potential to transmit messages between pathogens and host cells without a direct cell contact, they have been a focus of great interest regarding infection. EVs derived from Leishmania and other protozoan parasites have been associated with pro-parasite effects, by creating a more permissive environment to the establishment of the infection. Herein, we studied the effect of the extracellular material (ExM), which encloses both EVs and vesicle-depleted material, released by L. infantum promastigotes in iNKT cells. In the first steps of this work, it was observed that L. infantum ExM is capable of impairing the expansion of human iNKT cells ex vivo from peripheral blood mononuclear cells. This evidenced the cross-talk between iNKT cells and L. infantum ExM that we explored further. L. infantum ExM also modulates the important capacity of iNKT cells to release cytokines, impairing the production of different cytokines, such as IL-4 and IFNγ by these cells. Furthermore, we also show that L. infantum ExM competes with α-GalCer, a potent iNKT cell agonist, for CD1d binding, which justifies its effect in the impairment of iNKT cell activation. Additionally, we also proved the lipids present in each fraction of L. infantum ExM take an important role in the inhibition of iNKT cell activation and expansion. Thus, L. infantum ExM, through their lipids, is suggested to participate in the impairment of CD1d-mediated activation of iNKT cells, adding a new evidence regarding the contribution of the parasite ExM to subvert host immune responses. To the best of our knowledge, this is the first time that the cross-talk between the ExM released by a microbe and iNKT cells was assessed, shedding light on a mechanism of iNKT cell modulation that remained unexplored so far. This opens new perspectives regarding the study of the interaction of the ExM released by other pathogens with iNKT cells. Moreover, a further analysis of the lipid content of L. infantum ExM might allow the finding of new inhibitory molecules specific to iNKT cells, which can bring significant advantages in clinical approaches targeting the modulation of iNKT cell activation.

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

No description available.

Type 1 diabetes is an autoimmune disorder determined both by genetic and environmental factors. The Non-obese diabetic (NOD) mouse is one of the best animal models of this disease. It spontaneously develops diabetes through a process resembling the human pathogenesis. The strong association of NOD Type 1 diabetes to the MHC region and the existence of other diabetes susceptibility loci are also in parallel with the human disease. The identity of the genetic factors and biological function mediated by these loci remain, however, largely unknown. Like in other autoimmune diseases, defects in tolerance mechanisms are thought to be at the origin of type 1 diabetes. Accordingly, defects in both central and peripheral tolerance mechanisms have been reported in the NOD mouse model. Using a subphenotype approach that aimed to dissect the disease into more simple phenotypes, we have addressed this issue. In paper I, we analyzed resistance to dexamethasone-induced apoptosis in NOD immature thymocytes previously mapped to the Idd6 locus. Using a set of congenic mice carrying B6-derived Idd6 regions on a NOD background and vice-versa we could restrict the Idd6 locus to an 8cM region on the telomeric end of chromosome 6 and the control of apoptosis resistance to a 3cM region within this area. In paper II, further analysis of diabetes incidence in these congenic mice separated the genes controlling these two traits, excluding the region controlling the resistance to apoptosis as directly mediating susceptibility to diabetes. These results also allowed us to further restrict the Idd6 locus to a 3Mb region. Expression analysis of genes in this chromosomal region highlighted the Lrmp/Jaw1 gene as a prime candidate for Idd6. Lrmp encodes an endoplasmatic reticulum resident protein. Papers III and IV relate to peripheral tolerance mechanisms. Several T cell populations with regulatory functions have been implicated in type 1 diabetes. In paper III, we analyzed NOD transgenic mice carrying a diverse CD1d-restricted TCR αVa3.2b9), named 24abNOD mice. The number of nonclassical NKT cells was found to be increased in these mice and almost complete protection from diabetes was observed. These results indicate a role for nonclassical NKT cells in the regulation of autoimmune diabetes. In paper IV, we studied the effects of introducing the diverse CD1d-restricted TCR (Va3.2b9) in immunodeficient NOD Rag-/- mice (24abNODRag-/- mice). This resulted in a surprising phenotype with inflammation of the ears and augmented presence of mast cells as well as spleenomegaly and hepatomegaly associated with extended fibrosis and increased numbers of mast cells and eosinophils in the tissues. These observations supported the notion that NKT cells constitute an “intermediary” cell type, not only able to elicit the innate immune system to mount an inflammatory response, but also able to interact with the adaptive immune system affecting the action of effector T cells in an autoimmune situation. In this context the 24abNODRag-/- mice provide an appropriate animal model for studying the interaction of NKT cells with both innate and adaptive components of the immune systemα.

No description available.

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.

CD1 Moleküle spielen eine wichtige Rolle in der Lipidpräsentation und T-Zell-Aktivierung. CD1d fungiert als Restriktionselement für NKT-Zellen, eine T-Zell-Untergruppe, die nach Erkennung von Glykosphingolipide (GSL), IFN-gamma und IL-4 produziert. NKT Zellen steuern folglich anschliessende Immunantworten. Den meisten infektiösen Mikroorganismen mangelt es jedoch an GSL-Antigenen zur Stimulation von NKT-Zellen. Der Wirtsorganismus hat daher einen Mechanismus entwickelt, der die Aktivierung der NKT-Zellen dennoch gewährleistet. NKT-Zellen erkennen auch endogene GSL, die in dendritischen Zellen (DZ) infolge von Toll-like-Rezeptor (TLR)-Stimulation durch Pathogene produziert werden. Bislang war unklar, wie genau TLR-Aktivierung zur Produktion von Selbst-GSL-Antigenen führt. Ziel dieser Arbeit war es die Verknüpfung der beiden Prozesse aufzudecken. Diese Dissertation zeigt, dass alpha-Galaktosidase A (a-Gal A) als lysosomales Schlüsselenzym für den konstitutiven Abbau von Selbst-GSL-Antigenen in DZ fungiert. NKT-Zellen antworteten auf CD1d-restringierte Antigene, die von DZ, denen a-Gal A-Aktivität fehlte, präsentiert wurden. Ferner expandierten NKT-Zellen nach adoptiven Transfer in a-Gal A-defiziente Mäuse in Abhängigkeit von CD1d-Expression im Wirtsorganismus. Diese Arbeit zeigte auch, wie GSL-Antigene dem Abbau durch a-Gal A entkommen und für die NKT-Zell-Aktivierung bei Infektionen verfügbar werden. Unter normalen Bedingungen wurden die GSL durch a-Gal A abgebaut. TLR-vermittelte Signale führten jedoch zu Inhibierung der a-Gal A-Aktivität in DZ und resultierten somit in einer GSL-Akkumulation in den Lysosomen. Wir identifizierten einen neuen Regulationsmechanismus der NKT-Zell-Aktivierung bei Infektionen, der auf der Induktion von lysosomalen GSL-Antigenen durch TLR-vermittelte Hemmung der a-Gal A-Aktivität beruht. Diese Dissertation beantwortet fundamentale Fragen der NKT-Zell-Biologie und ebnet den Weg dieses System für therapeutische Ansätze zu nutzen. / CD1 molecules are pivotal for lipid presentation to T lymphocytes. Notably, CD1d functions as a restriction element for NKT cells, a T-cell lineage that produces IFN-gamma and IL-4 following recognition of glycosphingolipids (GSL). Consequently, NKT cells exert decisive regulatory functions on downstream immune responses. Most microbes potentially causing infection of the host lack GSL antigens to stimulate NKT cells. However, facing this challenge, the host developed a mechanism to ensure NKT-cell activation. This pathway exploits the property of NKT cells to react with self GSLs produced in dendritic cells (DCs) stimulated by pathogens through Toll-like receptors (TLR). How TLR engagement leads to production of self GSL antigens remains elusive. The aim of this study was to provide a mechanistic link between these two processes. Here, we identified alpha-galactosidase A (a-Gal A) as a key lysosomal enzyme required for constitutive degradation of self GSL antigens in DCs. Accordingly, NKT cells exposed to DCs lacking a-Gal A activity were activated in the context of CD1d-presented antigens. In addition, NKT cells underwent robust expansion upon transfer to a-Gal A-deficient mice that required CD1d expression by the host. This study further addressed the critical question as to how GSL antigens escape degradation by a-Gal A, and thus become available for presentation to NKT cells in infection. Accordingly, we found that TLR signaling targeted a-Gal A activity for negative regulation in DCs. Consequently, GSLs degraded by a-Gal A in steady-state conditions were induced in lysosomes. Based on these findings, we propose a new pathway that warrants the activation of NKT cells in infection by self GSL antigens induced through TLR-mediated inhibition of a-Gal A activity. Overall, this dissertation answers fundamental questions in the NKT field, and paves the way toward exploring this antigen presentation axis for therapeutic use.