A fibrose pulmonar é uma via comum de várias doenças agudas e crônicas do interstício pulmonar que pode resultar na cicatrização anormal do pulmão. Há acúmulo excessivo das proteínas da matriz extracelular levando a desestruturação das paredes alveolares, e consequente perda das trocas gasosas pelos pulmões. As células NKT são grande fonte de citocinas e podem ser cruciais na polarização de macrófagos para o fenótipo M2. O projeto tem a hipótese de que as células NKT podem influenciar o desenvolvimento da fibrose pulmonar via modulação de macrófagos. Para isso, animais selvagens e knockout para células NKT invariante (Ja18-/-) foram submetidos ao protocolo de indução de fibrose pulmonar pela bleomicina. Os resultados indicam que o grupo Ja18-/- assim como os grupos experimentais que receberam agonistas para células NKT apresentaram uma proteção contra a fibrose pulmonar uma vez que houve menor síntese de hidroxiprolina, deposição de colágeno, citocinas pró-fibróticas e a manutenção de macrófagos M1 no tecido pulmonar. / Pulmonary fibrosis is a common pathway of various acute and chronic interstitial lung diseases that may result in abnormal healing of the lung. There is excessive accumulation of extracellular matrix proteins, leading to disruption of the alveolar walls and the consequent loss of gas exchange through the lungs. NKT cells are a big source of cytokines and may be crucial in the polarization of macrophages to the M2 phenotype. This project has hypothesized that NKT cells can influence the development of pulmonary fibrosis through modulation of macrophages. For this, wild and knockout invariant NKT cells (Ja18-/-) mice were subjected to the protocol of bleomycin induced pulmonary fibrosis. The results indicate that the group Ja18-/- as well as the experimental groups receiving agonists for NKT cells showed protection against lung fibrosis since there was less synthesis of hydroxyproline, collagen deposition, pro-fibrotic cytokines and maintenance of macrophages M1 in lung tissue.

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

The immune system is a complex network of tissues, cells and proteins which protects us against infections and invading pathogens we encounter every day. Immunosenescence refers to age-related impairments in immune function which may contribute to increased prevalence and severity of infectious disease in the elderly. How and why ageing affects the immune system is not fully understood. Using a naturally aged mouse model, work in this thesis shows that the abundance of a rare type of lymphocyte, known as NKT cells, increased across multiple immune organs. Additionally, macrophage abundance was also altered in the lymph nodes of aged mice. Invariant NKT (iNKT) cells express an invariant T cell receptor (TCR) which recognises lipids presented on the CD1d molecule. iNKT cells can be activated and respond to invading pathogens either by recognition of antigens through TCR-CD1d interactions or cytokine-dependent means. Less is known about NKT-like cells, which also express NK cell-associated surface markers, such as CD49b, but lack an invariant TCR. Data within this thesis show that both iNKT and NKT-like cell populations are abundant in the spleen and liver of aged mice. iNKT and NKT-like cells can be divided into subpopulations based on their expression of surface markers or transcription factors, and data suggests that not all subpopulations of these cells are affected by age equally. For instance, flow cytometry showed that while spleen-derived iNKT cells are significantly increased in aged mice, within the iNKT cell population the percentage representation of CD4+ cells are significantly reduced with age. Additionally, data indicates that both iNKT and NKT-like cells from aged mice show compromised responses to in vitro stimulation compared to young controls. Using bone marrow chimeras, where either young cells are reconstituted within an aged mouse or old cells are reconstituted within a young mouse, provided the opportunity to determine whether the aged environment contributes to this diminished response. Data demonstrates that the aged environment plays at least a partial role in these age-related changes to response to stimulation, however the young environment seems unable to reverse these changes. Macrophages are phagocytes which are found within all organs of the body. Studies in this thesis show that CD169+ macrophages have diminished numbers in the lymph nodes of aged mice, but this did not seem to affect the capture of the model antigen, dextran. Further studies revealed ageing affects macrophage populations differently in the different tissues within the body. For example, macrophage numbers remain constant in the spleen with ageing, but appear to increase in density in the lungs. To conclude, ageing can cause dramatic changes to the numbers and function of different cells of the immune system across multiple organs. Furthering our understanding of the ageing immune system and the underlying mechanisms which cause age-related decline in immune function is important to design strategies to improve the quality of the lives of the elderly.

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.

INKT cells are non-conventional T lymphocytes that are restricted to glycolipid presenting CD1d molecule. iNKT cells express an invariant TCR a chain (Va14-Ja18 in mice and Va28-Ja18 in humans). Their particularity is to rapidly produce copious amounts of cytokines (IFN-? and IL-4) after activation and to activate other cells of the immune system such as dendritic cells, NK cells and T lymphocytes. iNKT cells, therefore, form a bridge between innate and adaptive immune responses. Type 1 diabetes is an autoimmune disease characterized by the destruction of pancreatic ß cells whose role is to produce insulin. While diabetes development can clearly be associated with genetic polymorphisms, environmental factors were also implicated in the etiology of the disease. Numerous studies suggest that viral infections, particularly infections with Coxsackievirus B4 (CVB4), could be implicated in the development of type 1 diabetes. Our study was performed with NOD mice that develop type 1 diabetes around 15 weeks of age and with proinsulin 2 knockout NOD mice (Pro-ins2-/-) which become diabetic around 8 weeks of age. Our results show that CVB4 infection induces accelerated diabetes in around half of NOD and Pro-ins2-/- mice compared to uninfected mice. However, the activation of iNKT cells with their agonist, aGalactosylceramide (aGalCer), at the time of infection greatly decreases diabetes incidence. CVB4 infection induces a strong recruitment of macrophages into the pancreas. Interestingly, iNKT cell activation modifies the function of these macrophages. Indeed, pancreatic macrophages of CVB4 infected mice strongly express IL-1, IL-6 and TNF-a, indicating their pro-inflammatory character. On the contrary, macrophages of mice infected with CVB4 and treated with aGalCer express low levels of these cytokines, but strong levels of suppressive enzymes iNOS (inducible NO synthase), IDO (Indoleamine 2,3-dioxygenase) and arginase I. The use of inhibitors of these enzymes showed that diabetes prevention is induced by IDO. We have also observed that autoreactive T cells strongly infiltrate the pancreatic islets after CVB4 infection. It is interesting to note that the high diabetes incidence of CVB4 infected mice is associated with an increased frequency of IFN-? producing autoreactive T cells in pancreatic islets. On the contrary, the frequency of these cells is very low in infected mice treated with aGalCer. The inhibition of IFN-? production is dependent on IDO enzyme, since the use of its inhibitor strongly increases IFN-? production by anti-islet T cells and diabetes incidence. To summarize, our results show that iNKT cell activation during the infection with CVB4 induces immunosuppressive macrophages in the pancreas. These cells inhibit the function of autoreactive T cells and prevent diabetes development.

Les cellules NKT invariantes (iNKT) sont des lymphocytes T non conventionnels restreints par la molécule CD1d qui présente des glycolipides. Les cellules iNKT expriment un TCR avec une chaîne a invariante, Va14-Ja18 chez la souris et Va28-Ja18 chez l’homme. Elles ont la particularité de produire de grande quantité de cytokines (IFN-? et IL-4) rapidement après leur activation et peuvent à leur tour stimuler d’autres cellules du système immunitaire comme les cellules dendritiques, les cellules NK et les lymphocytes T. Elles représentent ainsi un pont entre les réponses immunitaires innées et adaptatives. Le diabète de type 1 est une maladie autoimmune caractérisée par la destruction des cellules ß pancréatiques productrices d’insuline. Bien que l’apparition de diabète de type 1 soit associée à des polymorphismes génétiques, les facteurs environnementaux ont également été impliqués dans l’étiologie de cette maladie. De nombreuses études suggèrent que les infections virales, en particulier les infections par le virus de coxsackie B4 (CVB4), pourraient être impliquées dans le développement de cette maladie. Notre étude a été réalisée avec des souris NOD qui développent un diabète de type 1 vers 15 semaines d’âge et des souris NOD déficientes pour la proinsulin 2 (Pro-ins2-/-) développant un diabète vers 8 semaines d’âge. Nos résultats montrent qu’après infection par CVB4, la moitié des souris NOD et Pro-ins2-/- développent un diabète accéléré par rapport à des souris non infectées. Toutefois, une injection de l’agoniste des cellules iNKT, la molécule aGalactosylceramide (aGalCer), au moment de l’infection des souris, diminue fortement l’incidence de diabète. L’infection par CVB4 induit un fort recrutement de macrophages dans le pancréas et l’activation des cellules iNKT modifie la fonction de ces macrophages. En effet, les macrophages pancréatiques des souris infectées par CVB4 expriment fortement les cytokines IL-1ß, IL-6 et TNF-a, révélant leur caractère pro-inflammatoire alors que les macrophages des souris infectées et traitées par aGalCer expriment faiblement ces cytokines inflammatoires et fortement des enzymes immunosuppressives iNOS (inducible NO synthase), IDO (Indoleamine 2,3-dioxygenase) et arginase I. L’utilisation d’inhibiteurs de ces enzymes montre que la protection contre le diabète est induite par IDO. Nous avons également observé une forte infiltration de lymphocytes T autoréactifs dans les îlots pancréatiques des souris infectées. De façon intéressante, l’incidence accrue de diabète du groupe CVB4 est associée à une fréquence élevée de cellules T autoréactives produisant de l’IFN-? dans le pancréas, alors que la production d’IFN-? par les cellules T autoréactives est très faible dans les souris du groupe CVB4+aGalCer. Cette inhibition de la production d’IFN-? est dépendante de l’enzyme IDO, car l’utilisation d’un inhibiteur d’IDO augmente fortement la production d’IFN-? par les lymphocytes T anti-îlots et l’incidence de diabète. Dans l’ensemble nos résultats montrent, que l’activation des cellules iNKT lors de l’infection par CVB4 induit des macrophages immunosuppresseurs dans le pancréas, ces cellules inhibant la fonction des lymphocytes T autoréactifs et ainsi le développement du diabète. / INKT cells are non-conventional T lymphocytes that are restricted to glycolipid presenting CD1d molecule. iNKT cells express an invariant TCR a chain (Va14-Ja18 in mice and Va28-Ja18 in humans). Their particularity is to rapidly produce copious amounts of cytokines (IFN-? and IL-4) after activation and to activate other cells of the immune system such as dendritic cells, NK cells and T lymphocytes. iNKT cells, therefore, form a bridge between innate and adaptive immune responses. Type 1 diabetes is an autoimmune disease characterized by the destruction of pancreatic ß cells whose role is to produce insulin. While diabetes development can clearly be associated with genetic polymorphisms, environmental factors were also implicated in the etiology of the disease. Numerous studies suggest that viral infections, particularly infections with Coxsackievirus B4 (CVB4), could be implicated in the development of type 1 diabetes. Our study was performed with NOD mice that develop type 1 diabetes around 15 weeks of age and with proinsulin 2 knockout NOD mice (Pro-ins2-/-) which become diabetic around 8 weeks of age. Our results show that CVB4 infection induces accelerated diabetes in around half of NOD and Pro-ins2-/- mice compared to uninfected mice. However, the activation of iNKT cells with their agonist, aGalactosylceramide (aGalCer), at the time of infection greatly decreases diabetes incidence. CVB4 infection induces a strong recruitment of macrophages into the pancreas. Interestingly, iNKT cell activation modifies the function of these macrophages. Indeed, pancreatic macrophages of CVB4 infected mice strongly express IL-1, IL-6 and TNF-a, indicating their pro-inflammatory character. On the contrary, macrophages of mice infected with CVB4 and treated with aGalCer express low levels of these cytokines, but strong levels of suppressive enzymes iNOS (inducible NO synthase), IDO (Indoleamine 2,3-dioxygenase) and arginase I. The use of inhibitors of these enzymes showed that diabetes prevention is induced by IDO. We have also observed that autoreactive T cells strongly infiltrate the pancreatic islets after CVB4 infection. It is interesting to note that the high diabetes incidence of CVB4 infected mice is associated with an increased frequency of IFN-? producing autoreactive T cells in pancreatic islets. On the contrary, the frequency of these cells is very low in infected mice treated with aGalCer. The inhibition of IFN-? production is dependent on IDO enzyme, since the use of its inhibitor strongly increases IFN-? production by anti-islet T cells and diabetes incidence. To summarize, our results show that iNKT cell activation during the infection with CVB4 induces immunosuppressive macrophages in the pancreas. These cells inhibit the function of autoreactive T cells and prevent diabetes development.

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

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Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2007. / Despite the ongoing global tuberculosis (TB) problem and extensive research into protective immunity against this intracellular pathogen, mechanisms of protective immunity against Mycobacterium tuberculosis (Mtb) in humans have not been fully clarified. Numerous reports have addressed the potential immunological defect(s) in infected individuals that have developed active TB in comparison to those who have remained healthy in spite of infection. Markers of treatment response phenotypes are still elusive. The aims of this study were to define lymphocyte subsets in the peripheral blood of TB patients and controls, to determine intracellular interferon-γ (IFN-γ) and interleukin-4 (IL-4) production and to find correlations of these data with microbiologically-defined treatment response. Methods Whole blood tests were done on 30 HIV-negative, smear-positive pulmonary TB patients and 18 healthy skin test positive volunteers resident in the same community. Immunophenotyping was performed by flow cytometry, combined with routine haematology, for the enumeration of peripheral blood immune cell subtypes. Whole blood was also stimulated in vitro with anti-CD3 monoclonal antibody and intracellular IFN-γ and IL-4 determined by flow cytometry. Lymphocyte proliferation in response to heat-killed Mtb was determined by tritiated thymidine incorporation. Routine microbiological monitoring by sputum smears and culture was done throughout the patients’ 26 weeks of treatment. Results Compared to healthy controls, absolute numbers of peripheral blood lymphocytes and lymphocyte subsets were significantly depressed in patients at diagnosis but normalized during treatment with the exception of natural killer (NK) cells and natural killer T (NKT) cells. A novel subset of the latter was found to correlate significantly with treatment response. IFN-γ-producing T cells after a 4-hour T cell receptor stimulation were significantly higher in patients at diagnosis and normalized during treatment. Supplementary kinetic experiments showed that IFN-γ production in patients at diagnosis seemed to be accelerated. Lymphocyte proliferation was lower in patients at diagnosis and normalized during treatment. Neither IFN-γ production nor lymphocyte proliferation correlated with treatment response. Low intracellular IL-4 production was constitutive in patients and controls, was insignificantly lower in patients at diagnosis than in controls and, in the slow responder patient group, it was significantly lower than in the fast responder group. High IL-4 expression was found in low numbers of T cells in patients and controls and supplementary experiments showed co-expression of active caspase-3 in these cells, which signified apoptosis. Conclusions Lymphocyte subset phenotypes associated with TB are largely abnormal only during active infection and only a novel subset of NKT cells showed correlation with treatment response. Intracellular IFN-γ production and lymphocyte proliferation is increased and decreased, respectively, only during active infection and does not correlate with treatment response. The T helper 1/T helper 2 (Th1/Th2) hypothesis could not be confirmed in the context of tuberculosis but instead constitutive IL-4 production may play a role as a growth factor.

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.