L’Adénovirus (AdV) entre dans la cellule hôte par endocytose puis s’échappe de l’endosome en lysant la membrane de ces vésicules, empêchant ainsi sa dégradation via les lysosomes. Or, les membranes endommagées sont reconnues comme des signaux de danger par le système immunitaire et peuvent déclencher une réponse antivirale, telle que l’expression d’interféron (IFN). Dans nos conditions expérimentales, nous avons montré que l’infection par l’AdV n’induit pas l’expression d’IFNβ et qu’au contraire, le virus semble inhiber cette réponse. En revanche, l’entrée du virus active TBK1 (Tank Binding Kinase 1) qui est une kinase clef de la voie IFN mais qui est également impliquée dans la régulation de l’autophagie, une voie de dégradation cellulaire. Notre laboratoire a précédemment montré que l’autophagie est activée lors de l’entrée de l’AdV, par la rupture de la membrane endosomale. Nous avons donc étudié le mécanisme d’activation et le rôle de TBK1 lors de l’infection par l’AdV. Nos résultats montrent que la rupture de la membrane endosomale induite par le virus est nécessaire pour l’activation de TBK1 et que cette kinase est recrutée spécifiquement sur les sites de dommage membranaire. De plus, nous avons montré que TBK1 est impliqué dans l’activation de l’autophagie induite par l’AdV. Cependant, contrairement à ce qui est décrit pour l’autophagie dirigée contre les bactéries, cette activation de TBK1 est indépendante de NDP52 et d’autres adaptateurs conventionnels de l’autophagie. En résumé, nos travaux montrent que l’AdV est capable de contrôler la réponse IFN et que les ruptures de membrane induites par le virus activent TBK1 et l’autophagie par un nouveau mécanisme. Nos données suggèrent un rôle conservé de TBK1 dans l’activation de l’autophagie sélective contre les agents pathogènes. / Adenoviruses enter host cells by endocytosis and then escape from the endosomal compartment by lysing the endosomal membrane, thereby preventing its degradation via lysosomes. However, damaged membranes are recognized as danger signals by the cell intrinsic immune system and trigger an antiviral response, such as expression of interferon (IFN). In our experimental conditions we have shown that adenovirus infection does not induce the expression of IFNβ. On the contrary, our data suggest that the virus appears to inhibit the IFNβ response. However, adenovirus entry activates TBK1 (Tank Binding Kinase 1), which is a key kinase of the IFN pathway but is also involved in the regulation of autophagy, a cellular degradation pathway. Our laboratory previously showed that autophagy is activated upon rupture of the endosomal membrane during adenovirus entry. We therefore studied the activation mechanism and the role of TBK1 during adenovirus infection. Our results show that virus-induced endosomal membrane rupture is required for activation of TBK1 and that this kinase is specifically recruited at membrane damage sites. In addition, we show that TBK1 is involved in the activation of autophagy induced by adenovirus. TBK1 activation is independent of NDP52 and other conventional autophagic adapters, which is in contrast to membrane damaging bacteria. Thus, autophagy targeting membrane penetrating adenoviruses differs from the one induced by bacteria. In summary our work shows that adenovirus is able to control the IFN response and that membrane rupture induced by adenoviruses activates TBK1 and autophagy by a novel mechanism. In contrast our data suggest a conserved role for TBK1 in driving selective autophagy against invading pathogens.

Interferon Regulatory Factor -1 (IRF-1) is a transcription factor that acts as a tumour suppressor in cancer cells. The inactivation or deletion of IRF-1 either in one or both allele has been frequently reported in leukaemia and myelodysplasia (MDS). On the other hand nucleophosmin (NPM), a nucleo-cytoplasmic shuttling phosphoprotein is also known to be aberrant in some form of leukaemia. NPM was first proposed as a binding partner of IRF-1 in 1997 and suggested to inactivate IRF-1 by inhibiting its DNA binding ability. No further researches on the interaction between IRF-1 and NPM1 was reported prior to the start of my PhD. In the research presented here the interaction and mechanism by which IRF-1 might be inactivated by NPM was studied. Under the context of both NPM and IRF-1 being frequently associated with leukaemia and MDS, the study was done to determine the role of NPM under its naïve condition and a most frequent mutated condition (NPMc+), where the C-terminal of NPM was frequently mutated to give rise to a cytoplasmic NPM in certain leukaemia. In this current research, the direct interaction between IRF1 and NPM was further confirmed both in vitro as well as within the cells. Following this, the effect of this interaction in respect to the leukaemic condition having NPMc+ mutation was done, by comparing the end results on AML2 (leukaemic cells with intact wild type NPM) and AML3 (leukaemic cells having a single NPM allele mutated to form NPMc+) cells. In this research, overexpression of wild type NPM (NPMwt) was found to increase IRF-1 transcriptional activity. On further analysis, the DNA binding activity of IRF-1 due to the presence of NPMwt or NPMc+ was not always inhibited, instead it shows a change in binding specificity, where NPMwt bound IRF-1, lacks DNA binding ability and DNA bound IRF-1 has a reduced binding towards NPM. This is being studied further in terms of NPM overexpression and increased IRF-1 transcriptional activity, as the order of addition (order of interaction in vivo) plays a major role in activating or deactivating IRF-1. This along with the increased transcriptional activity of IRF-1 suggests a novel function of NPM, where it could act in favour of IRF- 1 activity. Additionally, the NPM induced change in IRF-1 localisation was confirmed by the cytoplasmic localised IRF-1 in NPMc+ expressing cells and nucleolar sequestration in NPMwt overexpressing cells. This gives a novel mechanism by which NPM regulates IRF-1. Further, the NPMc+ specific colocalisation of IRF-1 urges to study the other proteins that may have been re-localised in AML cells due to the NPMc+ specific interaction. A mass spectrometric analysis on the cellular distribution of total proteins were analysed between AML2 (cells with NPMwt) and AML3 (cells containing NPMc+). Specific proteins related to cancer have been identified to be differentially distributed rather than being a random translocation. With this being said, a peptide phage display technology coupled with next generation sequencing was done to identify NPMwt binding peptides that can be used in drug discovery process or as small molecule inhibitors or activators. Three different peptides were selected at the end of the study that bind very effectively to NPMwt. These peptide can either aid or restrict NPM activity and need to be validated and studied in the future.

Equine herpesvirus-1 (EHV-1) is one of the most important and prevalent viral pathogens of horses causing a major threat to the equine industry throughout most of the world. EHV-1 primarily causes respiratory disease but viral spread to distant organs enables the development of more severe sequelae; abortion and neurologic disease. In order to produce disease, EHV-1 has to overcome the innate barrier of the type-I interferon (IFN) system in host cells. However, the underlying mechanisms employed by EHV-1 to circumvent the type-I IFN response in host cells are not well understood. In this project study, using molecular techniques, we explored how EHV-1 is able to escape the type-I IFN response in host cells during infection. We also investigated whether EHV-4, a closely related but less pathogenic virus, has similar effects on type-I IFN as a clue to understanding how widespread IFN suppressive function is found among equine alphaherpesviruses. Our data showed that inhibition of the type-I IFN response in host cells is not a function of neuropathogenicity of EHV-1 strains. However, a reduced type-I IFN response correlated with pathogenicity as EHV-4, unlike EHV-1, was unable to down-regulate the type-I IFN response in equine endothelial cells (EECs). Investigation of the mechanisms employed by EHV-1 to suppress type-I IFN revealed that the virus sequentially prevented outside-in signaling events that lead to type-I IFN production. Specifically, EHV-1 blocked the expression of Toll-like receptors (TLR) 3 and TLR4 at 6 hours post-infection (hpi) and 12 hpi. EHV-1 also prevented the transcription of IRF7 and IRF9 at different time-points during infection. The virus also perturbed the JAK-STAT signaling pathway by negatively regulating the cellular levels of TYK2 and phosphorylation-mediated activation of STAT2 molecules. Immunofluorescence data revealed that during infection, EHV-1 was able to sequester STAT2 molecules from nuclear translocation. This may be a limiting step preventing the formation of interferon- stimulated gene factor 3 (ISGF3) whose nuclear translocation is required to transactivate interferon-stimulated genes (ISGs) including IRF7. Further investigation showed that unlike EHV-1, EHV-4 only interfered with phosphorylation-mediated activated STAT1 and STAT2 molecules at 3 and 6 hpi. EHV-4 was unable to block TLR3/4 and IRF7/9 mRNA expression at any time-point. Intriguingly, while viral late gene of EHV-1 mediates inhibition of STAT phosphorylation, our data showed that for EHV-4, a virus late gene did not mediate the inhibition of STAT phosphorylation. The findings from this study help illuminate how EHV-1 strategically interferes with limiting steps required for type-I IFN response in host cells to promote pathology. Our data also strengthen the hypothesis that the ability to shut off host factors required for type-I IFN production might be directly related to the degree of pathogenicity of the EHV subtypes.

The discovery that cationic lipids can independently stimulate the immune system has generated interest in their potential as vaccine adjuvants. Here, we show that the cationic lipid R-DOTAP can independently stimulate type 1 interferon production in dendritic cells in both primary culture and immortalized cell culture. Levels of type 1 interferon production are cell line-dependent and limited in vitro by lipid-induced cell death. We show that cationic lipids can independently activate TLR-7 and TLR-9, suggesting a mechanism for type 1 interferon induction. This TLR-stimulatory activity is not restricted to R-DOTAP and can be extended to other similar cationic lipids in a lipid-specific and TLR-specific manner.

Transient ablation of regulatory T cells has been shown to be effective at hindering tumor growth and metastasis in murine breast cancer model. Based on our lab’s previous work, we have demonstrated that NK cells and CD8+ cytotoxic T cells were not required for the protective effect of Treg cell ablation. However, we also reported that CD4+ helper T cells and IFN-γ were required for the protective effect of Treg cell ablation. Furthermore, we observed that CD11B+ cells responded to Treg ablation therapy by up-regulating target genes of IFN-γ. Therefore, this study aimed to investigate the connection between the myeloid cell compartment and IFN-γ signaling after regulatory T cell ablation therapy. Through a combination of conditional knockout mouse models, cellular fate mapping experiments, adoptive transfers, and co-injection experiments, we demonstrated that tumor-associated macrophages (TAMs), derived from the bone marrow via CCR2/CCL2 axis, were responsible for the therapeutic effect of regulatory T cell ablation. In addition, we determined that IFN-γ signaling was required for the TAMs to mediate the protective phenotype seen after regulatory T cell ablation. Furthermore, based on our findings, we developed a genetic signature based on TAMs from treated or untreated tumors that had a predictive value for patient survival. Thus, our findings indicated a strong connection between IFN-γ release, classical activation of TAMs, and depletion of regulatory T cells, and taken together, our data could offer potential clinical strategies to mimic regulatory T cell ablation.

The Adames strain of Punta Toro virus (PTV-A) causes acute hepatic disease in hamsters and mice similar to that seen in natural Rift Valley fever virus (RVFV) infection, while the Balliet strain (PTV-B) is apathogenic. The ability of PTV-A to suppress the interferon (IFN) response has been demonstrated in hamsters and is thought to be a contributing factor to PTV-A's pathogenicity in hamsters. PTV-B is not assumed to exhibit this IFN-antagonistic activity, as it stimulates production of significantly higher IFN-β levels. To elucidate the role of IFN in resistance of mice to PTV-B infection, we utilized mice deficient in a critical IFN signaling protein, STAT-1. We found that these mice were drastically more susceptible to PTV-B, which caused 100% lethality compared to 0% in their wild-type counterparts. STAT-1 deficient mice were also more susceptible to PTV-A, as these mice succumbed to infection significantly earlier than wild-type mice (p=0.0058). We sought to determine whether PTV-A's IFN-antagonistic mechanism is functional in mice by examining expression of IFN-β in primary macrophages infected with either strain. We found that IFN-β protein concentration is higher in samples taken from PTV-B-infected cells. We employed quantitative PCR arrays specific to IFN signaling and response pathways to evaluate changes in gene expression throughout the course of infection with either virus strain. We found several genes with differentially regulated expression between PTV-A- and PTV-B-infected macrophages, including Ifnβ1 and multiple Ifnα subtypes. Also, several genes coding for inflammatory and chemotactic molecules, Cxcl11, Cxcl10, Cxcl9, Vcam1, and Il6, demonstrated increased expression in PTV-B samples compared to PTV-A. Of particular interest, Isg20, a 3'-5' exonuclease with specificity for single-stranded RNA, was stimulated ~2-fold higher by PTV-B, and Iigp1, from the family of GTPases associated with host defense against intracellular pathogens, was stimulated ~2.7-fold higher by PTV-B. The individual functions of each of these genes in mouse resistance to PTV-B could be a focus of future studies to better understand essential host defense mechanisms to phleboviral infection.

Die Impfung mittels Antigen-beladener dendritischer Zellen [DZ] ist mittlerweile eine gut etablierte Technik, die dann zum Einsatz kommt, wenn Standard-Impftechniken versagen, vor Krankheiten zu schützen beziehungsweise diese zu heilen. Die Effizienz dieser Technik konnte bereits für diverse Infektionskrankheiten und Krebserkrankungen in experimentellen Tiermodellen sowie am Menschen gezeigt werden. Hierbei ist die Möglichkeit zur wohldefinierten Manipulation und Antigenbeladung der DZ ein großer Vorteil gegenüber den konventionellen Ansätzen. Jedoch ist vor allem bei der Anwendung im klinischen Bereich die Präparation, Herstellung und Manipulation dieser autologen DZ mit einem erheblichen technischen, zeitlichen sowie finanziellen Aufwand verbunden. Hinsichtlich einer Präventivimpfung gegen eine pandemische Infektionskrankheit, die in hauptsächlich unterentwickelten Ländern vorkommt, wird dieser Aufwand sicherlich ein Hindernis darstellen. Daher muss für solche Fälle ein maßgeschneiderter Impfstoff entwickelt werden, der sich am Vorbild des effektiven DZ-basierten Impfstoffs orientiert. Für die Impfung gegen die Leishmania Parasiten besteht so ein DZ-basierter Impfstoff bereits. Dessen Wirkung, eine T-Zell Antwort vom Typ Th1 zu induzieren, wurde bereits in mehreren Veröffentlichungen demonstriert. Zusätzlich hat aber eine unserer Studien gezeigt, dass das typische Th1-bezogene Zytokin IL-12 zur Differenzierung naiver T-Zellen nicht von den injizierten DZ bereitgestellt werden muss, sondern von der geimpften Maus. Dies gab erste Hinweise auf eine stärkere Beteiligung des Wirts-Immunsystems als zuvor angenommen. Daher sollte hier vertieft der Mechanismus dieser DZ-basierten Impfung untersucht werden, wobei modifizierte Impfstoff-Ansätze zum Einsatz kommen sollten. Dabei wurden die Fragen nach der vom Impfstoff transportierten Information und dem Empfänger dieser Information berücksichtigt. Das aktuelle Paradigma zur DZ-basierten Impfung besagt, dass transferierte DZ im direkten Kontakt mittels dreier Signale T-Zellen stimulieren und aktivieren. Dafür müssen diese DZ mit dem entsprechenden Antigen beladen und aktiviert worden sein um das Antigen-Peptide mittels MHC Molekül im Kontext der Co-Stimulation präsentieren zu können. Jedoch zeigt diese Studie hier, dass weder eine Aktivierung der DZ noch die Präsentation des Antigens mittels passender MHC Moleküle notwendig ist für die Induktion einer protektiven Immunantwort gegen Leishmania Parasiten. Aufgeschlossene, mit Antigen beladene DZ müssen nicht vor dem Transfer mit CpG ODN aktiviert worden sein, um entsprechende Immunität zu verleihen. Ebenso hat der MHC Typ in diesem Falle auch keinen Einfluss auf die Effektivität des Impfstoffs. Da im Weiteren aufgeschlossene mit Leishmania-Antigen beladene Makrophagen nach Impfung die gleiche Wirkung erzielen, wie vorangegangene DZ-basierte Impfstoffe, können keine DZ spezifischen Mechanismen Schlüsselkomponenten der Induktion einer protektiven Immunität sein. Darüber hinaus konnte gezeigt werden, dass die DZ der geimpften Mäuse, eine maßgebliche Rolle bei der Verarbeitung transferierter Signale spielen. Suspensionen aufgeschlossener DZ stellen eine Kombination aus freigesetzten löslichen Molekülen sowie Membranvesikeln dar, die sich nach dem Aufschluss gebildet haben. Nach Auftrennung dieser beiden Fraktionen konnte gezeigt werden, dass ausschließlich die Membran-Fraktion nach Verimpfung eine geeignete Immunantwort zum Schutz vor Leishmania Parasiten induzieren kann. Als Vorteil dieser Aufreinigung erweist sich zudem die stabile Lagermöglichkeit bei -80°C. Somit ist klar gezeigt, dass die Immunität-verleihende Einheit dieser Impfstoffvarianten in der Membran-Fraktion liegt. Verfolgt man die Induktion Th1-zugehöriger Zytokine in in vivo Experimenten so ergibt sich im Falle der Gesamtsuspension aufgeschlossener, mit Leishmania-Antigen beladener DZ ein klares Bild. Diese Suspension erzeugt das volle Spektrum der DZ-basierten Impfung gegen Leishmania Parasiten. Es kann sowohl Produktion von IL-12 und IL-2 als auch eine antigenspezifische T-Zell Proliferation nach Stimulation von Splenozyten mit der entsprechenden Suspension verzeichnet werden. Außerdem produzieren Splenozyten von entsprechend geimpften Mäusen nach Stimulation mit Leishmania-Antigen erhebliche Mengen des entscheidenden Zytokins IFNγ. Obwohl jedoch die Verimpfung aufgereinigter Membranvesikel dieses Ansatzes im Tierversuch zu biologisch sowie statistisch signifikanten Ergebnissen führt, lassen sich die entsprechend Th1-bezogenen Zytokine im in vivo Ansatz nur in geringen Maße nachweisen. Ob dies jedoch für einen in vivo unbemerkten Aktivitätsverlust des Vakzins oder für andere lymphatische Organe als Ort der T-Zell Instruktion spricht, ist noch unbekannt und muss noch geklärt werden. / Dendritic cell-based vaccination is a well established technique for preventive and therapeutic instruction of the immune system where conservative vaccine formulations fail to cure or prevent diseases, respectively. Efficiency of this technique already was demonstrated in infectious diseases as well as for cancer in animal or human studies. Well controlled manipulation and antigen-loading of immature DC is most beneficial to this technique. But, time-consuming and cost-extensive procedures for preparation of DC precursors, expansion and stimulation of DC and inpatient administration are big disadvantages regarding vaccine development for pandemic infectious diseases that occur mainly in underdeveloped countries. Therefore vaccines are needed that are pathogen-tailored and able to induce equal immune responses as their DC-based vaccine models. For vaccination against Leishmania parasites such a DC-based vaccine is feasible and its efficacy to induce protective Th1-based immune responses was already demonstrated in several animal studies. But, one of our own studies indicated supportive activity of host cells exceeding the allocation of T cells to become activated by transferred DC. IL-12, an important cytokine for the induction of Th1-related immune responses, has to be produced by host cells. Therefore, the aim of this study was to investigate the mechanism of BMDC-based vaccination with regard to simplification of the vaccine formulation. Key questions that have been addressed are: Which cells process the information that is transferred by the injected DC and what are the key components of this information? Further more, it was looked at whether altered vaccine formulations are able to induce protective immunity and whether they share equal molecular mechanisms. The current paradigm of BMDC-based vaccination proposes direct interaction of transferred BMDC with host T cells. These BMDC have to be antigen-loaded for stimulation via antigen-peptide-MHC molecule-complexes and they have to be activated for proper co-stimulation of T cells. Here, this study demonstrates that neither activation for co-stimulation nor direct interaction with adequate MHC molecules is needed for the induction of protective immunity against infection with Leishmania-parasites. Disrupted antigen-loaded BMDC are able to induce protective immunity in BALB/c mice without pre-stimulation via CpG ODN. Beyond, if BMDC were used with a different MHC-background than recipient mice then the vaccine still would be efficient in terms of reduction of footpad swelling and parasite load in draining lymph nodes. Even more, DC-specific features are no key component that leads to protective immunity as vaccination with disrupted antigen-loaded MΦ shows equal properties than before mentioned vaccine formulations. Further more, it was found that host DC play a major role in transforming the incoming signal, received from transferred antigen-loaded DC, into Th1-related stimuli and Leishmania-antigen-specific T cell activation. Suspensions of disrupted antigen-loaded DC resemble a combination of laid off soluble molecules together with exosome-like vesicles that formed after disruption of membranes. Here it was shown that separation of the membranous and soluble fractions and subsequent transfer into BALB/c mice will lead to protection of these mice against infection with L. major promastigotes only if the membranous fraction is used as vaccine. More, this vaccine formulation takes advantage of easy storage at -80°C with no need of fresh production. This clearly demonstrates that the immunity-inducing principle of disrupted DC-based vaccination lies within the membrane enclosed fraction. On a molecular level, disrupted antigen-loaded DC induce Th1-related cytokines during vaccination and as response on pathogen encounter. In vivo assays revealed IL-12 production and antigen-specific T cell proliferation among splenocytes that were stimulated with disrupted antigen-loaded DC. Splenocytes of accordingly vaccinated mice produce tremendous amounts of IFNγ after stimulation with Leishmania parasites. In summary, disrupted antigen-loaded BMDC fulfil all characteristics of DC-based vaccination against Leishmania major. But, while purification of membranes of antigen-loaded DC and subsequent transfer to BALB/c mice leads to control of the disease in the animal model, only slight levels of Th1-related cytokines are seen in the in vivo assays. Whether this points towards a loss of vaccine activity on unseen levels or unknown sites where Th1-related immunity is induced by both, complete solution and purified membranes, still has to be determined.

While TGF-β is able to regulate miRNA expression in numerous cell types, TGF-β-dependent changes in the miRNA profile of CD8+ T cells had not been studied before. Considering that TGF-β suppresses CD8+ T cell effector functions in numerous ways, we wondered whether induction of immune-regulatory miRNAs could add to the known transcriptional effects of TGF-β on immune effector molecules. In this study, we used miRNA arrays, deep sequencing and qRT-PCR to identify miRNAs that are modulated by TGF-β in human CD8+ T cells. Having found that the TGF-β-dependent downregulation of NKG2D surface expression in NK cells and CD8+ T cells does not go along with a corresponding reduction in mRNA levels, this pathway appeared to be a possible target of TGF-β-inducible miRNAs. However, this hypothesis could not be confirmed by miRNA reporter assays. Instead, we observed that DAP10 transcription is suppressed by TGF-β which in turn negatively affects NKG2D surface expression. In spite of promising preliminary experiments, technical difficulties associated with the transfection of primary NK cells and NK cell lines unfortunately precluded the final proof of this hypothesis. Instead, we focused on the TGF-β-induced changes in the miRNome of CD8+ T cells and confirmed the induction of the miR-23a cluster members, namely miR-23a, miR-27a and miR-24 by three different techniques. Searching for potential targets of these miRNAs which could contribute to the immunosuppressive action of TGF-β in T cells, we identified and confirmed a previously unknown regulation of IFN-γ mRNA by miR-27a and miR-24. Newly generated miRNA reporter constructs further revealed that LAMP1 mRNA is a target of miR-23a. Upon modulation of the miR-23a cluster in CD8+ T cells by the respective miRNA antagomirs and mimics, significant changes in IFN-γ expression confirmed the functional relevance of our findings. Effects on CD107a/LAMP1 expression were, in contrast, rather minimal. Still, overexpression of the miR-23a cluster attenuated the cytotoxic activity of antigen-specific CD8+ T cells. Taken together, these functional data reveal that the miR-23a cluster not only is induced by TGF-β, but also exerts a suppressive effect on CD8+ T-cell effector functions, even in the absence of TGF-β signaling. / Obwohl bekannt war, dass TGF- die miRNA Expression in zahlreichen Zelltypen moduliert, waren TGF- abhängige Veränderung des miRNA Profils in CD8+ T Zellen noch nicht untersucht worden. Da TGF-β die Effektorfunktionen von CD8+ T Zellen aber in vielfältiger Weise inhibiert, fragten wir uns, ob die transkriptionellen Effekte, die TGF-β bekanntermaßen auf Immuneffektormoleküle ausübt, noch durch die Induktion immunregulatorischer miRNAs ergänzt werden. Daher nutzten wir miRNA Arrays, Genomsequenzierungstechniken und Echtzeit-PCR um miRNAs zu identifizieren, welche in humane CD8+ T Zellen von TGF- moduliert werden. Die Beobachtung, dass die TGF--abhängige Herunterregulation der NKG2D Oberflächenexpression in Natürlichen Killerzellen und CD8+ T Zellen nicht mit einer entsprechenden Verringerung der mRNA Menge einhergeht, ließ zudem vermuten, dass dieser Signalweg über miRNAs reguliert werden könnte. Nach verschiedenen miRNA Reporterassays musste diese Hpothese jedoch verworfen werden. Stattdessen zeigte sich, dass TGF- die Transkription von DAP10 inhibiert was wiederum die Oberflächenexpression von NKG2D limitieren sollte. Trotz viel versprechender initialer Experimente scheiterte der letzgültige Beweis dieser Hypothese aber an der ungenügenden Transfizierbarkeit von primären NK Zellen sowie von NK Zelllinien. Daher konzentrierten wir uns im Weiteren auf die durch TGF- induzierten Veränderungen im miRNom von CD8+ T Zellen und konnten mit drei verschiedenen Techniken die Induktion des miR-23a Clusters (mit den einzelnen miRNAs miR-23a, miR-27a und miR-24) bestätigen. Auf der Suche nach potentiellen immunregulatorisch relevanten Zielgenen dieser miRNAs konnten wir erstmals eine Regulation von IFN- durch miR-27a und miR-24 nachweisen. Zu diesem Zweck generierte miRNA Reporterkonstrukte zeigten zudem, dass LAMP1 durch miR-23a reguliert wird. Nach Modulation des miR-23a Clusters durch die entsprechenden miRNA Antagomir und Surrogat-Konstrukte konnten wir auch in CD8+ T Zellen signifikante Veränderungen der IFN-γ Expression nachweisen und somit die funktionelle Relevanz unserer Befunde bestätigen. Die Effekte auf die Expression von CD107a/LAMP1 waren hingegen nur minimal. Trotzdem führte die Überexpression des miR-23a Clusters zu einer Verringerung der zytotoxischen Aktivität von antigenspezifischen CD8+ T Zellen. Zusammen genommen belegen diese funktionellen Untersuchungen, dass das miRNA-23a Cluster, welches durch TGF-β induziert wird, zur Hemung der Effektorfunktionen in CD8+ T Zellen beiträgt, und zwar sowohl in Gegenwart als auch in Abwesenheit von TGF-β.

Abstract not available

Megakaryocytes (Mks) are unique bone marrow cells, which produce platelets. Dysregulated Mk development can lead to abnormal platelet number and the production of functionally defective platelets, causing bleeding, thrombotic events, and leukaemia. Understanding the molecular mechanisms driving megakaryopoiesis may yield insights into the molecular genetics and cellular pathophysiology of a diversity of disorders. The primary aim of this thesis was to gain insight into the molecular events required for normal Mk development. As transcription factors and cytokines play a central role in driving Mk development, both of these processes were investigated. Fli-1 and GATA-1 are key transcription factors regulating Mk-gene expression, alone and co-operatively. To understand the mechanism of transcriptional synergy exerted by Fli-1 and GATA-1, in vitro assays were carried out investigating the interactions between Fli-1, GATA-1 and DNA that mediate synergy. A novel mechanism of synergy was identified, where Fli-1 DNA binding is not required, although an interaction between Fli-1 and GATA-1, and GATA-1 DNA binding is required. Importantly, the results demonstrate that Fli-1 DNA binding is not essential for promoting Mk-gene expression in primary murine bone marrow cells. Thrombopoietin (TPO) is the primary cytokine responsible for Mk and platelet development. Identifying novel TPO gene-targets may provide invaluable information to aid the understanding of the complex and unique processes required for Mk development. Using microarray technology, IFI16 was identified as a TPO-responsive gene that has not previously been studied in the Mk lineage. This work demonstrated that IFI16 is expressed in CD34+ HSC-derived Mks, and that the Jak/STAT pathway is essential for the activation of IFI16 by both TPO and IFN-??. Of biological significance, IFI16 was found to regulate both the proliferation and differentiation of primary Mks, suggesting that IFI16 may control the balance between these two essential processes. In conclusion, the data in this thesis presents a novel mechanism through which Fli-1 and GATA-1 regulate the synergistic activation of Mk genes. The identification and functional characterisation of a novel TPO-inducible gene, IFI16, involved in regulating the proliferation and differentiation of Mks is also described. These findings have implications for several congenital and malignant conditions affecting Mk and platelet development, and possibly a mechanism for IFN-induced thrombocytopaenia.