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Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required. In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.

Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required. In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.:Inhaltsverzeichnis I Abbildungsverzeichnis III Tabellenverzeichnis IV Bibliographische Beschreibung V Abkürzungsverzeichnis VII 1 Einleitung 1 1.1 CIK-Zellen (CIK) 3 1.1.1 Merkmale von CIK-Zellen 3 1.1.2 Wirkungsmechanismen von CIK-Zellen 3 1.1.3 Studienlage 4 1.1.4 Bisherige Ansätze zur Verbesserung der Kultivierungsbedingungen 6 1.2 Mesenchymale Stammzellen (MSC) 7 1.2.1 Allgemein 7 1.2.2 Differenzierung von MSC 7 1.2.3 Heterogenität und Einflussfaktoren der MSC - Identitätsproblematik 8 1.2.4 Charakterisierung von MSC 9 1.2.5 Therapeutische Einsatzmöglichkeiten von MSC 11 2 Zielformulierung 15 3 Material und Methoden 16 3.1 Tiere 16 3.2 Materialien 17 3.2.1 Materialien für Zellkultur 17 3.2.2 Materialien für FACS-Analyse 18 3.2.3 Materialien für Zytotoxizitätsassay 19 3.2.4 Materialien für CFU-F-Assay 20 3.3 Methoden 21 3.3.1 Statistische Auswertung 21 3.3.2 Zellkultur 22 3.3.3 FACS (Fluorescence Activated Cell Sorting) 26 3.3.4 Markierung der MSC mit DiD 28 3.3.5 Zytotoxizitätsassay (LDH-Freisetzungsassay) 29 3.3.6 CFU-F-Assay 32 4 Ergebnisse 34 4.1 Beeinflussung der Wachstumskurve 34 4.1.1 Der Wachstumskurvenverlauf von CIK-Zellen (Kontrollen) 34 4.1.2 Der Wachstumskurvenverlauf von CIK-Zellen in der Kokultur mit MSC 35 4.1.3 Der Wachstumskurvenverlauf in MSC-konditioniertem Medium 37 4.1.4 Der Wachstumskurvenverlauf bei Restimulierung an Tag 14 38 4.2 Beeinflussung des Oberflächenphänotyps 40 4.2.1 Der Oberflächenphänotyp von CIK-Zellen 40 4.2.2 Vergleich Oberflächenphänotyp Kontrollen mit kokultivierten CIK 43 4.3 Beeinflussung der Vitalität 46 4.4 Beeinflussung der Zytotoxizität 48 4.5 Identifizierung der MSC 49 4.5.1 Adhärenz an Plastikoberflächen 50 4.5.2 Fibroblastenähnliche Wachstumsmorphologie 50 4.5.3 Wachstum in Colony-Forming-Units 51 4.5.4 Der Oberflächenphänotyp von MSC 53 4.6 Schicksal der MSC in der Kokultur 54 4.6.1 Der Oberflächenphänotyp der adhärenten Zellen nach Kokultur 54 4.6.2 Kokultur mit DiD gelabelten MSC 57 5 Diskussion 59 5.1 Beeinflussung der Wachstumskurve 60 5.1.1 Mechanismen der Beeinflussung des Wachstumskurvenverlaufs 60 5.1.2 Fehlerbetrachtung 68 5.2 Identifizierung der CIK sowie Beeinflussung von Phänotyp und Vitalität 69 5.3 Beeinflussung der Zytotoxizität 70 5.3.1 Vergleich Zytotoxizität Kontrollen mit Kokulturen 70 5.3.2 Fehlerbetrachtung 71 5.4 Identifizierung der MSC 72 6 Schlussfolgerung 75 7 Ausblick 77 8 Zusammenfassung 79 Literaturverzeichnis 83 Danksagung I

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.

Dans le travail portant sur l'immunorégulation de la réponse immunitaire dans le modèle expérimental d'asthme, nous avons montré que les basophiles étaient activés par des ARN double brin poly(A:U) et que cette stimulation exacerbait les réponses d'asthme. Nous avons ensuite étudié la modulation de l'asthme en utilisant des agonistes de la voie TLR/IL1R. Les résultats obtenus ont montré que le R848, agoniste de TLR7 qui induit les réponses immunitaires antivirales de type Th1, et l'IL-33 qui favorise les réponses Th2, activaient les cellules NKT dont la production rapide et modulée des cytokines pourrait avoir un effet modulateur dans l'asthme. Nous avons montré que les cellules NKT avaient une fonction régulatrice sur le développement et l'activité des cellules Th17. Enfin, nous avons décrit les effets protecteurs et suppresseurs induits par le R848 dans le modèle d'asthme et montré que ces effets suppresseurs étaient dépendants des cellules T régulatrices et du TGF-β.

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.

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.

La Broncho-Pneumopathie chronique obstructive (BPCO) est un problème majeur en santé publique puisque ce sera la 3ème cause de mortalité en 2020. Il s'agit d'une maladie inflammatoire chronique du poumon se traduisant par une obstruction progressive des bronches, partiellement ou non réversible, incluant bronchite chronique, hypersécrétion de mucus et emphysème. L'atteinte ne se limite pas au poumon et affecte d'autres organes dont le tube digestif en favorisant la maladie de Crohn. Le premier facteur de risque impliqué dans le développement de cette maladie est l’exposition à la fumée de cigarette qui induit un stress oxydatif au sein du poumon responsable d'une inflammation chronique et du développement de la BPCO. L'interleukine-17 joue un rôle essentiel dans ce processus en contrôlant l'inflammation et l'altération de la fonction respiratoire. L'objectif de cette thèse est de comprendre les facteurs contrôlant cette réponse IL-17 afin de proposer ensuite de nouvelles voies thérapeutiques. Dans la première partie, nous nous sommes focalisés sur le stress oxydatif et à son impact sur les cellules de l’immunité innée. Ensuite, nous abordons le rôle d'un facteur immunorégulateur, l'IL-10, et à son interférence avec le microbiote digestif. Cela nous a amené à nous intéresser aux lésions digestives associées au tabagisme. Un modèle murin d’exposition chronique à la fumée de cigarette a été développé afin de reproduire la physiopathologie de la BPCO. Concernant l'impact du stress oxydatif, nous avons étudié le rôle des cellules iNKT (cellules ayant un puissant potentiel dans l’immunorégulation et dans l’inflammation) qui sont activées par ce type de stress. Les cellules iNKT sont rapidement recrutées et activées au sein du poumon suite à l’exposition à la fumée de cigarette. En utilisant des souris déficientes, nous avons montré l’importance de ces cellules dans la physiopathologie de la BPCO. Cette pathogénicité est dépendante de la production de l’interleukine-17 par ces cellules et est initiée par le stress oxydatif sur les cellules épithéliales pulmonaires et les cellules dendritiques qui activent les cellules iNKT.Dans la seconde partie du projet, l’IL-10 intervient notamment dans le contrôle de l'inflammation afin d’éviter le développement de réponses immunologiques exacerbées dans certains contextes. Dans les poumons exposés à la fumée de cigarette, la production d’IL-10 est augmentée et la déficience pour cette cytokine entraîne une augmentation de la réponse Th17 et du déclin de la fonction pulmonaire. Une dysbiose (altération du microbiote) est observée avec la fumée de cigarette et la déficience à l'IL-10. De plus, une déplétion des bactéries Gram+ par antibiothérapie permet de limiter le développement de la réponse IL-17 et de l'atteinte pulmonaire soulignant l'importance du microbiote. En conclusion, nous avons identifié le stress oxydatif et l'IL-10 comme facteurs intervenant dans la réponse IL-17 associé à la BPCO. Ce travail souligne également le rôle du microbiome comme un organe à part entière et la modulation de ce dernier pourrait aboutir à l'identification de nouvelles voies thérapeutiques. / Chronic Obstructive Pulmonary Disease (COPD) is a major health problem which is going to become the third leading cause of death worldwide by 2020. COPD is characterized by a chronic inflammation of the airways causing progressive bronchial obstruction, with no completely reversible airflow limitation, including chronic bronchitis, mucus hypersecretion and emphysema. The pathology is not limited to the airways and can affect others organs including the gastro-intestinal tract promoting Crohn disease. Cigarette smoke exposure is the most important risk factor for developing COPD. Exposure to cigarette smoke induces a strong burden of reactives oxygen species and this oxidative stress is responsible for a chronic inflammation and the development of COPD. Interleukin (IL)-17 plays a critical role in controlling process of inflammation and lung function decline.The aim of this thesis is the understanding which factors are controlling the IL-17 response in order to propose new therapeutic approaches.In the first part, we focused on oxidative stress and its impact on innate immune cells. Then we addressed the role of an immunoregulatory factor, the interleukin (IL)-10, and its interference with intestinal microbiota. This part lead us to study intestinal damages linked to cigarette smoking.To mimic the physiopathology of COPD, we set up a mouse model of chronic exposure to cigarette smoke. Concerning the impact of oxidative stress, we investigated the role of iNKT cells (cells with a crucial potent role in immunoregulation and inflammation) activated by this type of stress. iNKT cells rapidly accumulate and be activated within the lungs of cigarette smoke exposed mice. Using deficient mice, we demonstrated that these cells strongly contribute to the COPD pathogenesis. This pathogenicity is iNKT cells-produced IL-17 dependant and initiated by the effect of oxidative stress on airway epithelial cells and dendritic cells activating iNKT.In the second part of the work, IL-10 interferes notably in the inflammation control in order to avoid exacerbated immunological responses development in some contexts. In cigarette smoke exposed lungs, IL-10 production is up-regulated and the deficiency for this cytokine leads to an increased Th17 response and to lung function decline. An altered microbiota (named dysbiosis) is observed with cigarette smoke exposure and IL-10 deficiency. Moreover, Gram+ bacteria depletion using antibiotics is able to limit the IL-17 response development and the lung function decline highlighting the crucial role for microbiota.To conclude, we identified two factors, oxidative stress and IL-10, implicated in IL-17 response linked to COPD. This work also underlines the role of microbioma as a whole organ and the modulation of this microbioma could result in new therapeutic ways identification in COPD.

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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.