Dendritic cells (DC) constitute a heterogeneous leukocyte population having in common a unique capacity to induce primary T cell responses and are therefore most attractive candidates for immunomodulatory strategies. Two populations of blood DC (CD11c+ CD123dim and CD11c– CD123high) have been defined so far. However, their direct isolation for experimental purposes is hampered by their low frequency and by the lack of selective markers allowing large scale purification from blood. Here we describe the monoclonal antibody (mAb) M-DC8, which was generated by immunizing mice with highly enriched blood DC. This mAb specifically reacts with 0.2–1% of blood leukocytes and enables their direct isolation by a one-step immunomagnetic procedure from fresh mononuclear cells. These cells can be differentiated from T cells, B cells, NK cells and monocytes using lineage-specific antibodies. M-DC8+ cells express HLA class II molecules, CD33 and low levels of the costimulatory molecules CD86 and CD40. Upon in vitro culture M-DC8+ cells spontaneously mature into cells with the phenotype of highly stimulatory cells as documented by the upregulation of HLA-DR, CD86 and CD40; in parallel CD80 expression is induced. M-DC8+ cells display an outstanding capacity to present antigen. In particular, they proved to be excellent stimulators of autologous mixed leukocyte reaction and to activate T cells against primary antigens such as keyhole limpet hemocyanin. Furthermore, they induce differentiation of purified allogeneic cytotoxic T cells into alloantigen-specific cytotoxic effector cells. While the phenotypical analysis reveals similarities with the two known blood DC populations, the characteristic expression of Fc=γRIII (CD16) and the M-DC8 antigen clearly defines them as a novel population of blood DC. The mAb M-DC8 might thus be a valuable tool to determine circulating DC for diagnostic purposes and to isolate these cells for studies of antigen-specific T cell priming. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

L’immunothérapie a permis des avancées majeures dans la thérapie du cancer. Le traitement par des cellules T modifiées pour exprimer un récepteur chimérique d’antigène (CAR) a changé complètement la vision de la thérapie de la leucémie. L’efficacité de ce traitement sur des cancers résistants, a ouvert la voie à la thérapie cellulaire et génique dans ce contexte. Malgré les premiers résultats très positifs, il s’avère que l’épuisement cellulaire et la perte des cellules T thérapeutiques est un problème majeur pour maintenir l’efficacité de la thérapie CAR et prévenir les rechutes. Les travaux présentés dans cette thèse visent à permettre l’utilisation d’autres types cellulaires pour la thérapie CAR. L’hypothèse de travail est que les cellules NK ou les cellules souches hématopoïétiques (HSC) permettrait de dépasser les limites de la thérapie CAR utilisant les cellules T. Pour permettre l’utilisation des cellules NK, un des problèmes technique est la transduction par les vecteurs viraux. Les travaux présentés ici démontrent que l’utilisation de l’enveloppe BaEV permet une transduction efficace des NK avec un vecteur lentiviral. Par cette méthode nous avons pu générer de grandes quantités de cellules NK transduites avec un CAR, prouvant la possibilité d’utiliser les NK dans la thérapie CAR. L’utilisation des HSC dans la thérapie CAR, permettrait de produire des cellules CAR T en permanence pour renouveler les cellules T épuisées. Cependant, la surexpression d’un récepteur CAR sur toutes les cellules dérivant des HSC pourrait être un problème. Pour permettre l’utilisation des HSC, nous avons développé des promoteurs spécifiques courts restreignant l’expression du transgène à une population précise. Nous avons prouvé la spécificité d’un promoteur T et démontré la possibilité de l’utiliser dans le contexte de la thérapie CAR utilisant les HSC. Ces travaux sont une preuve de concept de l’utilisation d’autres cellules que les cellules T dans la thérapie CAR. / Immunotherapy has allowed major advances in cancer therapy. The treatment using modified T cells with a chimeric antigen receptor (CAR) completely changed the vision of leukemia therapy. The efficiency against resistant cancer paved the way to cellular and gene therapy in this context. Despite very positive results at first, the disappearance and exhaustion of therapeutic cells seems to be a major problem to maintain the efficiency of the CAR treatment and prevent relapses. The work of this thesis is to allow the use cell types other than T cells for CAR therapy. The hypothesis is that NK cells or hematopoietic stem cells (HSC) could overcome the limitation of CAR therapy using T cells. To allow the use of CAR NK cells, a major technical issue is the transduction by viral vectors. The work presented here shows the use of BaEV envelope to pseudotype vectors allows an efficient transduction of NK cells. Using this method, we were able to produce large amounts of CAR NK cells, showing the possibility to use NK cells in CAR therapy. The use of HSC in CAR therapy, could allow the permanent replenishment of the pool of CAR T cells once exhausted. Despite that advantage, the overexpression of a CAR receptor on all hematopoietic cells coming from those HSC could be an issue. To allow the use of HSC, we developed short specific promoters restraining the expression of the transgene to a precise population. We prove the specificity of a T cell promoter and demonstrated the possibility to use it in CAR therapy using HSC. This work is a proof of concept of the use of other population than T cells in CAR therapy.

Le lymphome angioimmunoblastique à cellules T (AITL) est un lymphome périphérique à cellules T agressif dont les symptômes sont la lymphadénopathie et l'hypergammaglobulinémie. Actuellement, les patients atteints du AITL ont des options de thérapeutiques limitées et des résultats cliniques défavorables, avec un taux de survie sur 5 ans d'environ 30%. Les cellules tumorales du AITL proviennent de cellules T CD4+ appelées cellules T auxiliaires folliculaires (Tfh). Les cellules Tfh sont essentielles dans le centre germinatif (GC), où elles facilitent l'expansion et la différentiation des cellules B en plasmocytes. Cette fonction d'aide est soutenue par de nombreuses protéines dérivées des cellules Tfh et des programmes de transcription qui pourraient aussi fonctionner dans les cellules tumorales du AITL. Par conséquent, la perturbation des principaux mécanismes de signalisation soutenant l'identité des cellules Tfh et leurs interactions avec les cellules B pourrait inhiber la croissance du AITL. Des études ont démontré que les cellules hyperactives de type Tfh provoquent une accumulation de cellules immunitaires telles que les cellules B, les plasmocytes et les macrophages dans les tumeurs. Cependant, le microenvironnement du AITL n'a pas été bien étudié et il n'a pas été vérifié si certaines cellules immunitaires pourraient être utilisées pour arrêter la croissance de la tumeur. Bien que l’on trouve des cellules Tfh circulantes dans l’AITL humain, le taux de propagation peut varier d’un patient à l’autre. Ainsi, une possibilité est la présence de mécanismes de surveillance immunitaire s'opposant à la progression de la tumeur. En accord avec cette hypothèse, un signal positif pour la phagocytose nommé SLAMF7 (contrebalancé par la voie inhibitrice CD47-SIRPα) est exprimé dans un sous-ensemble de patients atteints du AITL. Toutefois, la corrélation entre les différents niveaux d'expression du SLAMF7 et l'amélioration des résultats pour les patients n'a pas été étudiée. En utilisant des souris Roquinsan/+, qui développent spontanément l’AITL, nous avons étudié le rôle des mécanismes de signalisation immunitaire dans les cellules tumorales de type Tfh et du microenvironnement tumoral. Nous avons cherché à inhiber les protéines et les voies de signalisation typiques des cellules Tfh dans les tumeurs afin d'évaluer la valeur thérapeutique potentielle. Nous avons aussi étudié le rôle de la phagocytose dépendante des macrophages dans le contexte SLAMF7 et comment la modulation de la signalisation de CD47-SIRPα peut améliorer l'efficacité de la phagocytose des cellules tumorales. Notre hypothèse centrale est qu'en supprimant les programmes fondamentaux des cellules Tfh ou en favorisant l'élimination phagocytaire des cellules tumorales de type Tfh, nous pouvons favoriser la régression de la tumeur. Nous avons démontré que les tumeurs AITL nécessitent des protéines d’identité des cellules Tfh essentielles telles que le facteur de transcription Bcl6 et la protéine adaptatrice SAP, ainsi que la communication entre les cellules T et B (T-B). Même en l'absence de GC classiques, les cellules tumorales de type Tfh ont apporté un soutien aux cellules B. Cela est démontré par des titres élevés d'IgG et l'accumulation de cellules précurseurs des plasmocytes dans les tumeurs. Nous avons trouvé des preuves de l'opposition entre la surveillance immunitaire et l'évasion au sein des tumeurs de type AITL, car les cellules Tfh augmentent l’expression de la molécule inhibitrice CD47 tandis que les macrophages stimulent le niveau de SLAMF7. Les cellules de type AITL ont été phagocytées plus efficacement in vitro quand la signalisation du CD47 était bloquée. En résumé, nous démontrons que les voies de signalisation importantes pour l'identité des cellules Tfh et la communication entre les cellules T et B sont essentielles pour la progression de l’AITL et suggèrent qu’une surveillance immunitaire continue par les macrophages peut influencer l’évolution de la maladie. Des études futures pourraient explorer la possibilité de combiner des inhibiteurs de l'activité des cellules Tfh ou T-B avec des médicaments qui stimulent l'activité phagocytaire antitumorale pour améliorer l'efficacité thérapeutique du traitement. / Angioimmunoblastic T cell lymphoma (AITL) is an aggressive peripheral T cell lymphoma manifesting with symptoms such as generalized lymphadenopathy and hypergammaglobulinemia. Currently, AITL patients have limited treatment options and poor clinical outcomes with a 5-year survival rate around 30%. AITL tumor cells derive from a subset of CD4+ T cell, the T follicular helper (Tfh) cell. Tfh cells are essential in germinal centers (GC), where they facilitate B cell expansion and differentiation into plasma cells. This helper function is supported by numerous Tfh cell-derived proteins and transcriptional programs which may still be operational in AITL tumor cells. Therefore, disrupting key signaling mechanisms sustaining Tfh cell identity and their ability to interact with B cells could inhibit AITL tumor growth. Studies have demonstrated that these hyperactive Tfh-like cells lead to the accumulation of immune cell subsets such as B cells, plasma cells, and macrophages within tumor lymph nodes. Nevertheless, the AITL tumor microenvironment itself has not been well-studied and whether some immune cells could be harnessed to impede tumor growth has not been tested. In human AITL, although circulating Tfh cells have been reported, the rate of tumor spreading can vary between patients. As such, one possibility is the presence of immune surveillance mechanisms opposing tumor progression. In line with this idea, SLAMF7, a positive signal for macrophage-mediated phagocytosis (counterbalanced by the inhibitory CD47-SIRPα pathway), is expressed in a subset of AITL patients. Despite this, whether differing levels of SLAMF7 expression correlates with improved patient outcomes has not been investigated. Using Roquinsan/+ mice, a spontaneous AITL-like mouse model, we addressed the role of immune signaling mechanisms within Tfh-like tumor cells and the surrounding tumor microenvironment that would promote tumor regression. First, we aimed to inhibit signature Tfh cell proteins and downstream signaling pathways in developed AITL-like tumors to evaluate potential therapeutic value. Second, we investigated the role of macrophage-mediated phagocytosis in the context of SLAMF7 and how modulating CD47-SIRPα signaling may enhance the efficiency of AITL tumor cell engulfment. Our central hypothesis is that by removing fundamental Tfh cell supporting programs from tumor cells or by promoting the phagocytic removal of Tfh-like tumor cells we can favour tumor regression and impair future growth. Through this work, we demonstrated that AITL-like tumors continuously require critical Tfh cell identity proteins such as transcription factor Bcl6 and adaptor protein SAP, as well as T cell-B cell (T-B) crosstalk. Importantly, despite the absence of conventional GCs, Tfh-like tumor cells provided functional support to B cells as evidenced by elevated IgG titers and accumulation of plasma cell precursors in tumors. We also found evidence of opposition between immune surveillance and evasion within AITL-like tumors as Tfh-like cells upregulated inhibitory CD47 levels while macrophages increased expression of prophagocytic SLAMF7. Moreover, AITL-like tumor cells were more efficiently phagocytosed in vitro when CD47 signaling was blocked. Taken together, we demonstrate that pathways important for Tfh cell identity and T-B communication are critical for AITL-like disease progression and suggest that ongoing macrophage-mediated immune surveillance may influence disease outcomes. Future studies may explore combining inhibitors of Tfh cell activity or T-B crosstalk along with drugs which boost antitumor phagocytic activity to further improve the therapeutic efficacy of treatment.

Les lymphocytes T orchestrent la réponse immunitaire adaptative afin de nous protéger contre les pathogènes. Les lymphocytes T sont dotés d’un récepteur de surface, le récepteur de cellules T (TCR), qui transmet le signal de stimulation vers l’intérieur de la cellule afin d’amorcer la cascade d’activation des cellules T. Le TCR est un complexe multimérique composé des dimères TCRαβ, CDεγ, CD3εδ et CD3ζζ. Les chaînes TCRαβ reconnaissent les antigènes pathogéniques tandis que les chaînes CD3 initient la cascade de signalisation des cellules T par la phosphorylation de leurs chaînes cytoplasmiques. Il est toujours incompris comment le signal d’activation du TCR est transmis des chaînes TCRαβ jusqu’aux domaines cytoplasmiques des chaînes CD3. Chez les lymphocytes T au repos, les chaînes CD3ε et CD3ζ sont associées au feuillet interne de la membrane plasmique (MP). Le domaine cytoplasmique de CD3ε et CD3ζ est riche en acides aminés basiques, ce qui permet leur association électrostatique avec les phospholipides acides de la MP. La phosphatidylsérine (PS) est le phospholipide acide le plus abondant de la MP. La PS est redistribuée exclusivement à la face cytoplasmique de la MP. Lors de l’activation des lymphocytes T, les chaînes CD3ε et CD3ζ des TCRs doivent se détacher de la PS pour leur phosphorylation. La dissociation membranaire d’un grand nombre de chaînes CD3 est essentielle à l’amplification de l’activation des lymphocytes T. Un mécanisme de dissociation des chaînes CD3ε et CD3ζ des TCRs proposé dans la littérature est par l’élévation intracellulaire de calcium. Un influx robuste de calcium est généré suivant la stimulation des cellules T. En plus d’être essentiel à l’activation efficace des cellules T, il a été proposé que le calcium neutralise les phospholipides acides de la MP afin de dissocier les chaînes CD3. Le calcium est également un co-facteur dans l’activité de plusieurs enzymes, comme la scramblase lipidique TMEM16F. TMEM16F redistribue la PS à la MP suivant l’élévation du calcium intracellulaire, ce qui résulte en la réduction de la PS au feuillet interne. Nous avons donc émis l’hypothèse que le calcium régule la dissociation des chaînes CD3 par l’activation de TMEM16F. Notre étude démontre que la redistribution calcium-dépendante de la PS par TMEM16F est essentielle à la dissociation membranaire de CD3ε dans la lignée de cellules T Jurkat. La réduction de l’expression de TMEM16F par ARN interférant (shTMEM16F) empêche la dissociation massive des chaînes CD3ε suivant la stimulation des cellules T. De plus, les cellules shTMEM16F démontrent une diminution de la phosphorylation des molécules de signalisation des cellules T. En contraste, l’expression d’une forme constitutivement active de TMEM16F augmente la redistribution de PS à la MP, la dissociation membranaire des chaînes CD3ε et la phosphorylation des molécules de signalisation. Notre étude démontre que la redistribution de la PS par la scramblase calcium-dépendante TMEM16F régule la dissociation membranaire des chaînes CD3 du TCR afin d’amplifier l’activation des cellules T. Enfin, nous avons confirmé les défauts d’activation dans des cellules T murines primaires exprimant shTMEM16F lors d’une réponse immunitaire. En conclusion, notre étude démontre le rôle de la régulation de la PS dans l’activation des cellules T. Nous avons démontré que nous pouvons modifier le niveau d’activation des cellules T en modulant la PS à la MP. Nos résultats ont ainsi plusieurs implications pour la conception et l’amélioration des immunothérapies basées sur les cellules T. / T lymphocytes protect us against pathogens by orchestrating the adaptive immune response. T lymphocytes possess a specific surface receptor, the T cell receptor (TCR), which conveys the stimulation signal towards the cytoplasm for the initiation of the T cell activation cascade. The TCR is a multimeric complex composed of the TCRαβ, CDεγ, CD3εδ and CD3ζζ dimers. The TCRαβ chains recognize the pathogenic antigens while the CD3 chains initiate the T cells signaling cascade through the phosphorylation of their cytoplasmic tails. It is not yet understood how the TCR activating signal is transmitted through the membrane from the TCRαβ chains towards the cytoplasmic tails of the CD3 chains. In resting T cells, the CD3ε and CD3ζ chains are associated to the inner leaflet of the plasma membrane (PM). The cytoplasmic tails of CD3ε and CD3ζ are rich in basic amino acids, which allow electrostatic association with acidic phospholipids at the PM. Phosphatidylserine (PS) is the most abundant acidic phospholipid and is exclusively distributed towards the cytoplasmic PM leaflet. During T cell activation, the CD3ε and CD3ζ cytoplasmic tails have to dissociate from PS for their phosphorylation. The membrane dissociation of a large number of CD3 chains is essential for the amplification of T cell activation. A mechanism of CD3ε and CD3ζ chain dissociation that has been proposed in the literature is through intracellular calcium elevation. A robust calcium influx is generated following T cell stimulation. In addition to its essential role in regulating T cell activation, it has been proposed that calcium ions neutralize the PM acidic phospholipids for CD3 chain dissociation. Calcium is also an essential cofactor for the activity of many enzymes, such as the phospholipid scramblase TMEM16F. TMEM16F redistributes PS at the PM following intracellular calcium mobilization, resulting in a reduction of inner leaflet PS. We propose that calcium regulates CD3 chain dissociation through TMEM16F activity. Our study demonstrates that calcium-dependent PS redistribution by TMEM16F is required for CD3ε membrane dissociation in the Jurkat T cell line. Reduction of TMEM16F expression by shRNA targeting (shTMEM16F) prevents massive CD3ε chain dissociation following 8 T cell stimulation. The shTMEM16F cells show a reduction in the phosphorylation of TCR-proximal signaling molecules. In contrast, expression of a constitutively active mutant of TMEM16F increases PS redistribution, CD3ε chain dissociation and phosphorylation of TCR-proximal signaling molecules. Our study demonstrates that PS redistribution by the calcium-dependent TMEM16F scramblase regulates CD3 chain dissociation for the amplification of T cell activation. In addition, we have confirmed T cell activation defects in shTMEM16F murine primary T cells during an immune response. In conclusion, our study demonstrates the role of PS regulation by TMEM16F in T cell activation. We showed that we could modify the level of T cell activation by modulating the concentration of PS at the inner leaflet of the PM. Our results thus have important implications for the development and improvement of immune receptor-based cancer immunotherapies.

Regulatorische Foxp3+CD4+ T-Zellen sind essentiell für das Gleichgewicht des intestinalen Immunsystems. Eine Einschränkung ihrer Suppressionsfunktion wird bei Patienten mit Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX)-Syndrom beobachtet und führt im Tiermodell zu lymphoproliferativen Erkrankungen und intestinalen Entzündungen. Von entscheidender Bedeutung für Homöostase und Suppressionsfunktion regulatorischer T-Zellen ist das Signalmolekül Interleukin-2 (IL-2). Im Gegensatz zu Effektor-T-Zellen exprimieren Foxp3+CD4+ T-Zellen den hochaffinen IL-2-Rezeptor αβγ konstitutiv. IL-2 wird von regulatorischen T-Zellen nicht in relevanten Mengen exprimiert. Sie sind somit auf von anderen Zellen sezerniertes IL-2 angewiesen. In der vorliegenden Arbeit wird gezeigt, dass im Tiermodell regulatorische Foxp3+CD4+ T-Zellen durch Applikation eines IL-2/anti-IL-2-Antikörperkomplex nicht nur in mesenterialen Lymphknoten und Milz, sondern auch lokal in der Lamina propria mucosae des Kolons der Versuchstiere expandiert werden. Als relevante Quelle von IL-2 in situ könnten aktivierte proliferierende T-Zellen dienen. Um dies näher zu untersuchen, wurde die Proteinexpression proliferierender Einzelzellen mittels Matrix assisted laser desorption/ionisation-Time of flight-Massenspektrometrie-Imaging (MALDI-Imaging) analysiert. Es gelang die Identifikation präferentiell in lymphoiden Geweben exprimierter Peptidmassen. Obwohl die Einzelzellanalyse mittels MALDI-Imaging prinzipiell möglich erscheint, ist ein Nachweis von Zytokinen wie IL-2 derzeit aufgrund fehlender Sensitivität im Proteinmassebereich zwischen 10kDa und 20kDa nicht möglich. Die therapeutischen Möglichkeiten der Expansion regulatorischer Foxp3+ T-Zellen durch stabile IL-2-Rezeptor-Agonisten und die Rolle von IL-2 für die intestinale Immunregulation sollten weiter untersucht werden.:Bibliographische Beschreibung 3 Inhaltsverzeichnis 4 Abkürzungsverzeichnis 7 1. Einleitung 9 1.1. Störung der Barrierefunktion des intestinalen Immunsystems als Ursache chronisch entzündlicher Darmerkrankungen 9 1.2. Foxp3+ regulatorische T-Zellen 10 1.3. Die Rolle von Interleukin-2 für regulatorische T-Zellen 11 1.4. Signaltransduktion in regulatorischen T-Zellen als Grundlage ihrer selektiven Expansion und Induktion 12 1.5. Möglichkeiten der präferentiellen Expansion regulatorischer T-Zellen 15 1.5.1. Expansion regulatorischer T-Zellen durch Agonisten des hochaffinen IL-2-Rezeptors 15 1.5.2. Induktion regulatorischer T-Zellen durch TGF-β 16 1.5.3. Expansion regulatorischer T-Zellen durch Rapamycin (Sirolimus) 17 1.5.4. Expansion regulatorischer T-Zellen durch UVB-Bestrahlung bzw. Vitamin D-Rezeptor-Agonisten 18 1.5.5. Expansion regulatorischer T-Zellen durch Histon-Deacetylaseinhibitoren 19 1.6. Suppression von Effektor-T-Zellen durch regulatorische T-Zellen 20 1.6.1. Zellkontaktabhängige Mechanismen 20 1.6.2. Zellkontaktunabhängige Mechanismen 22 1.7. Matrix assisted laser desorption ionisation-Time of flight-Massenspektromie (MALDI-TOF-MS): Bedeutung und Funktion 23 1.8. Zielstellung 25 2. Materialien und Methoden 26 2.1. Versuchstiere 26 2.2. IL-2/IgG2b-Fusionsprotein-Vorexperiment 26 2.2.1. Induktion von 2,4,6-Trinitrobenzensulfonsäure (TNBS)-Kolitis 26 2.2.2. Durchführung des IL-2/IgG2b-Fusionsprotein-Vorexperimentes 26 2.3. Durchführung des IL-2/anti-IL-2-Antikörperkomplex-Experiments 27 2.4. Durchflusszytometrie 27 2.5. Histologische Färbungen 28 2.5.1. Probenvorbereitung 28 2.5.2. Hämatoxylin/Eosin (HE) Färbung 29 2.5.3. Immunfluoreszenz-Färbungen 29 2.5.4. Ki67-Schnellfärbung 30 2.5.5. Mikroskopie und Photographie 30 2.6. Histologische Auswertungen 31 2.6.1. Kolitis-Score 31 2.6.2. Bildanalyse 31 2.6.3. Validierung der automatischen Bildanalyse mittels CellProfiler 33 2.7. MALDI-Imaging 35 2.7.1. Probenvorbereitung für MALDI-Imaging 35 2.7.2. Analyse der Peptidexpression in Gewebeschnitten mittels MALDI-Imaging 36 2.8. Statistische Auswertungen 36 2.8.1. Statistische Tests 36 2.8.2. Berechnung der zu erwartenden Zahl von Kontakten zwischen Ki67+ und Foxp3+ Zellen 36 3. Ergebnisse 38 3.1. Design des IL-2/anti-IL-2-Antikörperkomplex Experimentes 38 3.2. Mit IL-2/anti-IL-2-Antikörperkomplex behandelte Tiere zeigen Splenomegalie und Lymphadenomegalie 40 3.3. Behandlung mit einem IL-2/anti-IL-2-Antikörperkomplex führt zur präferentiellen Expansion regulatorischer T-Zellen in mesenterialen Lymphknoten und Milz 41 3.4. Behandlung mit IL-2/anti-IL-2-Antikörperkomplex führt nicht zu Kolitis 43 3.5. Behandlung mit IL-2/anti-IL-2-Antikörperkomplex führt zur präferentiellen Expansion regulatorischer T-Zellen in der Lamina propria mucosae 45 3.6. IL-2/anti-IL-2-Antikörperkomplex steigert die Proliferation regulatorischer T-Zellen in der Lamina propria mucosae und lymphoiden Follikeln des Kolons 47 3.7. Regulatorische T-Zellen sind nach Behandlung mit IL-2/anti-IL-2-Antikörperkomplex weiter mit proliferierenden Zellen assoziiert. 50 3.8. MALDI-Imaging als Möglichkeit der Proteinexpressionsanalyse in situ 52 3.8.1. Vergleich der Proteinexpression in verschiedenen Geweben von Ileum und Zäkum mit der Expression in Thymus und mesenterialem Lymphknoten 55 3.8.2. MALDI-Imaging nach Schnellfärbung Ki67+ Zellen mit Streptavidin 63 3.8.3. Analyse der Massenspektren von Einzelzellen mittels MALDI-Imaging 66 4. Diskussion 68 4.1. Applikation von IL-2/anti-IL-2-Antikörperkomplex hat keine fatalen Nebenwirkungen 68 4.2. IL-2/anti-IL-2-Antikörperkomplex führt zur präferentiellen Expansion regulatorischer T-Zellen in mesenterialen Lymphknoten, Milz und Kolon 70 4.3. IL-10 als wichtiger Vermittler der Suppressionsaktivität durch IL-2/anti-IL-2-Antikörperkomplex expandierter regulatorischer T-Zellen 71 4.4. Expansion regulatorischer T-Zellen beim Menschen: Voraussetzungen und Chancen 72 4.5. Regulatorische T-Zellen akkumulieren an proliferierenden Zellen 73 4.6. Nachweis spezifischer Massen in Gewebe und Einzelzellen mittels MALDI-Imaging 74 5. Zusammenfassung 80 Literaturverzeichnis 83 Publikationen 90 Erklärung über die eigenständige Abfassung der Arbeit 97 Lebenslauf 98 Danksagungen 99

T lymphocytes are distinguished by the expression of αβ TCR or γδ TCR on their cell surface. The kinetic differences in the effector functions classifies γδ T cells as innate-like lymphocytes and αβ T cells as adaptive lymphocytes. Although distinct, αβ and γδ T cell lineages produce a common array of cytokines to mount an effective immune response against a pathogen. The production of cytokine IL-17 is a shared characteristic between the γδ T (Tγδ17) cells and the CD4 T (Th17) cells. γδ T cells develop into Tγδ17 cells in the thymus whereas CD4 T cells differentiate into Th17 cells in response to antigens in the peripheral lymphoid tissues. γδ T cells exported from the thymus, as pre-made effectors, are the early IL-17 producers compared with the late IL-17 producing Th17 cells. In this thesis we describe how TGFβ-SMAD2 dependent pathway selectively regulates Th17 cell differentiation but not Tγδ17 cells generation. We further illustrate the requirement of WNT-HMG box transcription factor (TF) signaling for the thymic programming of Tγδ17 cells. Cytokine TGFβ in co-operation with IL-6 induces the differentiation of Th17 cells. Conversely, TGFβ signaling also regulates the differentiation and maintenance of CD4+FOXP3+ regulatory T cells. The mechanism by which TGFβ signals synergize with IL-6 to generate inflammatory versus immunosuppressive T cell subsets is unclear. TGFβ signaling activates receptor SMADs, SMAD2 and SMAD3, which associate with a variety of nuclear factors to regulate gene transcription. Defining relative contributions of distinct SMAD molecules for CD4 T cell differentiation is critical for mapping the versatile intracellular TGFβ signaling pathways that tailor TGFβ activities to the state of host interaction with pathogens. We show here that SMAD2 is essential for Th17 cell differentiation and that it acts in part by modulating the expression of IL-6R on T cells. While mice lacking SMAD2 specifically in T cells do not develop spontaneous lymphoproliferative autoimmunity, Smad2-/- T cells are impaired in their response to TGFβ in vitro and in vivo and they are more pathogenic than controls when transferred into lymphopenic mice. These results demonstrate that SMAD2 is essential for TGFβ signaling in CD4+ T effector cell differentiation and that it possesses functional capabilities distinct from SMAD3. Although SMAD2 is essential for the differentiation of Th17 cells, TGFβ signaling via SMAD2 is not required for the thymic programming of innate Tγδ17 cells. Among different γδ T cells, Vγ2+ (V2) γδ T cells are the major IL-17 producing subsets. We demonstrate that Sry-high mobility group (HMG) box TFs regulate the development of V2 Tγδ17 cells. We show that the HMG box TF, SOX13 functions in a positive loop for the intrathymic generation of V2 Tγδ17 cells. SOX13 regulates the programming of Tγδ17 cells by controlling the expression of B-lymphoid kinase (BLK) in developing immature V2 γδ T cells. BLK is an Src-family kinase expressed by all Tγδ17 cells. Furthermore, we show another HMG box TF, TCF1, the nuclear effector of canonical WNT signaling, is the primary negative regulator of IL-17 production by all γδ T cells. We propose that the antagonism of SOX13 and TCF1 determines the generation of IL-17 producing γδ T cells. We also show that extrinsic cues from αβ T cells do not affect the generation of IL-17 producing γδ T cells. Using OP9-DL1 culture system, we demonstrate that the progenitors of V2 Tγδ17 cells are the c-Kit+ early thymic precursors.

The past two decades have seen an explosive increase of obesity rates worldwide, with more than one billion adults overweight and 300 million of them obese. Obesity and its associated complications have become leading causes of morbidity and mortality in the United States and major contributing factors to the rising costs of national health care. The pathophysiology of obesity and type 2 diabetes in rodents and humans is characterized by low-grade inflammation and chronic activation of immune pathways in adipose tissue and liver. The CD40 receptor and its ligand, CD40L, initiate immune cell signaling promoting inflammation, but conflicting data on CD40L-null mice confound its role in obesity-associated insulin resistance. A clear understanding of how CD40 and its ligand communicate to regulate and sustain the inflammatory environment of obesity is lacking. Here we demonstrate that CD40 receptor deficient mice on a high-fat diet display the expected decrease in hepatic cytokine levels, but paradoxically exhibit liver steatosis, insulin resistance and glucose intolerance compared with their age-matched wild-type controls. Hyperinsulinemic-euglycemic clamp studies also demonstrated insulin resistance in glucose utilization by the CD40-null mice compared with wild-type mice. In contrast to liver, visceral adipose tissue in CD40 deficient animals harbors elevated cytokine levels and infiltration of inflammatory cells, particularly macrophages and CD8+ effector T cells. In addition, ex vivo explants of epididymal adipose tissue from CD40-null mice display elevated basal and isoproterenol-stimulated lipolysis, suggesting a potential increase of lipid efflux from visceral fat to the liver. These findings reveal that 1) CD40-null mice represent an unusual model of hepatic steatosis with reduced hepatic inflammation, and 2) CD40 unexpectedly functions in adipose tissue to attenuate the chronic inflammation associated with obesity, thereby protecting against hepatic steatosis.

Immunologic self-tolerance is maintained by both central and peripheral mechanisms. Furthermore, regulation of mature lymphocyte responses is governed by inhibitory as well as stimulatory signals. TCR recognition of cognate peptide bound to MHC molecules provides the initial stimulus leading to T lymphocyte activation and determines the antigen specificity of any subsequent response. However, lymphocytes must discriminate between foreign and self antigens presented by self-MHC molecules to maintain self tolerance and avoid pathological autoimmunity. Consequently, TCR ligation alone is reported to result in abortive activation, T cell anergy, apoptosis, and tolerance. Under normal physiological conditions, costimulatory signals modify lymphocyte responsiveness to TCR ligation to prevent autoimmunity while enabling robust responses to foreign antigen. Members of the CD28/B7 superfamily provide the critical secondary signals essential for normal immune cell function. CD28 is an essential positive costimulatory molecule with critical functions in thymic development, lineage commitment, and regulation of peripheral lymphocyte responses to antigenic stimuli. CD28 ligation by APC-expressed B7 molecules alters proximal signaling events subsequent to MHC/TCR interactions, and initiates unique signaling pathways that alter mRNA stability and gene transcription. Furthermore, CD28 signaling is required for regulatory T cell development and function. Thus, CD28 has a central role in both potentiating lymphocyte activation mediated by TCR engagement and regulating peripheral tolerance. In contrast, Ctla-4 mediates an inhibitory signal upon binding B7 molecules on an antigen-presenting cell. Its importance in governing lymphocyte responses is manifested in the fatal lymphoproliferative disorder seen in Ctla-4-/- mice. The lymphocyte proliferation is polyclonal, antigen and CD28 dependent, and arises from defects in peripheral CD4+T cell regulation. The high percentage of peripheral T lymphocytes expressing activation markers is accompanied by lymphocyte infiltration into numerous non-lymphoid tissues and results in death by 3-4 weeks. While still controversial, Ctla-4 signaling has been reported to be essential for induction of peripheral T lymphocyte tolerance in vivo and in some model systems is proposed to regulate both T lymphocyte anergy induction and the immune suppressive effects of some regulatory T cells in the prevention of autoimmunity. Signaling pathways activated by TCR ligation and CD28 costimulation have been extensively characterized. In contrast, the mechanisms mediating Ctla-4 maintenance of tolerance remain largely unknown. Ctla-4 gene expression is tightly controlled during T cell development and activation, and its intracellular localization and expression on the cell surface is regulated by numerous pathways and intermediates. While a tailless Ctla-4 mutant is capable of inhibiting T cell activation, recent studies have shown that a ligand independent form of Ctla-4 is also capable of providing an inhibitory signal to T lymphocytes. In conjunction with the strictly controlled expression kinetics and the perfect amino acid homology between the intracellular domains of mouse and human Ctla-4, this data suggests that Ctla-4 may participate in the modulation or initiation of intracellular signaling pathways. Positive and negative costimulatory receptors on the T cell modify lymphocyte responses by altering both quantitative and qualitative aspects of the lymphocyte response including threshold of activation, cytokine secretion, and memory responses. Positive costimulation augments T cell responses, in part, by downregulating the expression of genes that actively maintain the quiescent phenotype. This study was initiated to determine the role of Ctla-4 ligation in modifying the global gene expression profile of stimulated T cells and to determine if the Ctla-4 mediated maintenance of T cell tolerance was achieved, in part, by altering the transcription of quiescence genes necessary for the prevention of T cell activation subsequent to TCR and CD28 stimulation. Previous studies investigating the influence of Ctla-4 ligation on transcriptional profiles of activated lymphocytes detected only quantitative alterations in the transcriptional regulation initiated by CD28 signaling. In contrast, our data suggests that quantitative effects of Ctla-4 ligation that differentially influence pathways acting downstream of stimulatory receptors results in a stable and qualitatively unique phenotype detectable at the level of the transcriptome. Thus, the cumulative effect of Ctla-4 signaling is unique and not constrained to reversing alterations in expression initiated by CD28. In addition, Ctla-4 ligation can be shown to influence T lymphocyte responsiveness and the resulting global expression profile within 4 hours after stimulation and prior to detectable Ctla-4 surface expression. In a subpopulation of T cells, TCR stimulation activates pathways that result in commitment to activation with 2-6 hours. In contrast, CD28 signaling must be maintained for 12-16 hours to ensure maximal responses at the population level. The period of sensitivity to Ctla-4 inhibition of activation is more constrained and does not extend beyond 12 hours. Together, these data support a potential role for Ctla-4 in modification of the early transcriptional response and may explain various alterations in phenotype resulting from Ctla-4 ligation that have been reported in secondary responses. Identification of genes involved in lymphocyte activation, maintenance of selftolerance, and attenuation of immune responses opens the door to therapeutic manipulation of the pathways implicated. CD28 costimulation results in general amplification of TCR-initiated transcriptional responses, and specifically alters the expression profile of a subset of genes. In contrast, Ctla-4 ligation directly and specifically alters the expression of a select group of genes when ligated, and results in minimal suppression of the global CD28-mediated costimulatory transcriptional response. Ctla-4 regulated genes comprise a heterogeneous family, but include known quiescence factors, transcriptional regulators, and various determinants of cell cycle progression and senescence. The role of Ctla-4 in maintaining self-tolerance indicates that targeted manipulation of these gene products presents a novel therapeutic opportunity, and suggests that the mechanisms involved in Ctla-4-mediated maintenance of peripheral T cell tolerance and regulation of immune responsiveness is more nuanced than previously thought. In addition, this study provides the most comprehensive description of global gene expression during primary lymphocyte activation yet available. The integration of statistical and bioinfomatics analyses with large scale data mining tools identifies genes not previously characterized in lymphocytes and can direct future work by predicting potentially interacting gene products and pathways.

T cells play a central role in cellular-mediated immunity and must become activated to participate as effector cells in the immune response. The activation process is highly intricate and involves stimulation of a number of downstream signaling pathways enabling T cells to proliferate and produce cytokines that are vital for proper effector function. This increase in protein production and protein folding activity adds to the normal physiological strain on cellular machinery. One cellular compartment that has generated a mechanism to mitigate the stress induced by increased protein production is the endoplasmic reticulum (ER). In general, an increase in cellular production of proteins that overwhelms a cell’s protein folding capability can alter ER homeostasis and lead to ER stress. To counteract this stress, an adaptive cellular mechanism known as the ER stress response (ERSR) is initiated. The ERSR allows a cell to cope with normal physiological stress within the ER caused by increased protein translation. In this dissertation, we show that in vitro and in vivoT cell activation involving T cell receptor (TCR) ligation in the presence of costimulation initiates the physiological ERSR. Interestingly, the ERSR was also activated in T cells exposed only to TCR ligation, a treatment known to induce the ‘non-responsive’ states of anergy and tolerance. We further identified a key component of the downstream TCR signaling pathway, protein kinase C (PKC), as an initiator of physiological ERSR signaling, thus revealing a previously unknown role for this serine/threonine protein kinase in T cells. Therefore, induction of the physiological ERSR through PKC signaling may be an important ‘preparatory’ mechanism initiated during the early activation phase of T cells. If ER stress is persistent and ER homeostasis is not reestablished, physiological ER stress becomes pathological and initiates cellular death pathways through ER stress-induced apoptotic signaling. We further present data demonstrating that absence of functional Gimap5, a putative GTPase implicated to play a role in TCR signaling and maintenance of overall T cell homeostasis, leads to pathological ER stress and apoptosis. Using the BioBreeding diabetes-prone (BBDP) rat, a model for type 1 diabetes (T1D), we link pathological ER stress and ER stress-induced apoptotic signaling to the observed T cell lymphopenic phenotype of the animal. By depleting the ER stress apoptotic factor CHOP with siRNA, we were able to protect Gimap5-/-BBDP rat T cells from ER stress-induced death. These findings indicate a direct relationship between Gimap5 and maintenance of ER homeostasis for T cell survival. Overall, our findings suggest that the ERSR is activated by physiological and pathological conditions that disrupt T cell homeostasis. TCR signaling that leads to PKC activation initiates a physiological ERSR, perhaps in preparation for a T cell response to antigen. In addition, we also describe an example of pathological ERSR induction in T cells. Namely, we report that the absence of functional Gimap5 protein in T cells causes CHOP-dependent ER stress-induced apoptosis, perhaps initiated by deregulation of TCR signaling. This indicates a dual role for TCR signaling and regulation in the initiation of both the physiological and pathological ERSR. Future research that provides insights into the molecular mechanisms that govern ERSR induction in TCR signaling and regulation may lead to development of therapeutic modalities for treatment of immune-mediated diseases such as T1D.

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