Das Schicksal aktivierter T-Zellen wird durch eine Vielzahl NF-kappaB regulierter Ziel-Gene bestimmt, wobei aktivierende und deaktivierende Signale für die Ausbalancierung einer adäquaten T-Zell Antwort benötigt werden. Im Rahmen dieser Arbeit konnte gezeigt werden, dass die negativ-regulatorische Modulierung des Carma1-Bcl10-Malt1 (CBM)-Proteinkomplexes für die Steuerung der NF-kappaB Aktivität in T-Zellen von großer Bedeutung ist. Überraschenderweise ist die Bildung des CBM-Komplexes abhängig von IKKbeta, einer Kinase, die zuvor ausschließlich mit CBM-nachgelagerten Effektorfunktionen in Verbindung gebracht wurde. IKKbeta übernimmt eine duale Funktion bei der Regulation des CBM-Komplexes: Obwohl IKKbeta zunächst für die Bildung des CBM-Komplexes benötigt wird, führt die Phosphorylierung der CBM-Komplexkomponente Bcl10 durch IKKbeta bereits kurze Zeit nach Beginn der T-Zell Aktivierung zu einer Dämpfung der Signalübertragung. Biochemische Analysen zeigen, dass die Phosphorylierung von Bcl10 die Proteinaffinitäten innerhalb des CBM-Komplexes beeinflusst, wodurch es zu einer Umlagerung des Komplexes mit negativ-regulatorischem Effekt kommt. Weiterführende Experimente haben aufgedeckt, dass Bcl10 im Zuge anhaltender T-Zell Stimulation lysosomal degradiert wird. Die Degradation von Bcl10 führt zum Zerfall des CBM-Komplexes und unterbindet die weitere Signalübertragung trotz persistenter Stimulation. Die Tatsache, dass beide in dieser Arbeit identifizierten negativ-regulatorischen Mechanismen am CBM-Komplex angreifen, unterstreicht die Bedeutung dieses Komplexes für die Signalübertragung in T-Zellen. Weiterhin besteht aufgrund der präsentierten Daten Anlass zur Annahme, dass in aktivierten T-Zellen ein vielfältig positiv und negativ regulierter Multikomponentenkomplex gebildet wird, der eine nicht-hierarchische Signalübertragung unterstützt. / A multitude of NF-kappaB regulated target genes determines the fate of activated T cells, whereas activating and de-activating signals are crucial for balancing adequate T cell responses. The presented data illustrate that negative-regulatory modulation of the Carma1-Bcl10-Malt1 (CBM)-complex is of great importance for the control of NF-kappaB activity in T cells. Surprisingly IKKbeta, a kinase that so far was thought to be involved in CBM-downstream effector functions, is needed for CBM-complex formation. IKKbeta exhibits a dual function regulating the CBM-complex: while initially being essential for the formation of the CBM-complex, phosphorylation of the CBM-complex component Bcl10 by IKKbeta shortly after the onset of T cell activation leads to a damping of signal transduction. Biochemical analysis reveal that Bcl10 phosphorylation influences the intermolecular protein affinities of the CBM-complex components causing a remodeling of the complex with a negative-regulatory effect. Further experiments uncover that upon persistent T cell activation Bcl10 is degraded by the lysosome. Bcl10 degradation promotes the collapse of the CBM-complex and thereby interferes with ongoing signal transduction despite persistent stimulation. Considering the fact that both negative-regulatory processes affect CBM-complex activity underscores the important role of this complex in T cell signal transduction. Moreover, the presented data demonstrate that formation of a multi-component signaling complex in activated T cells facilitates versatile positive, negative and non-hierarchical regulation.

Human immunodeficiency virus (HIV-1) is the pathogenic agent of HIV infection thatprecedes the total breakdown of cellular immunity, a condition known as acquiredimmunodeficiency syndrome (AIDS). The pandemic nature of the disease has promptedintense research into its biology. Already, much is known about HIV-1 infection, lifecycle,and progression to aids. Systems biology enables the combination of complex data fromthese studies into a framework where their effect on the various levels of cellularorganization (i.e. Pathways, cells, tissues, organs and the whole body) could be studied insilico. In this thesis, first, we reviewed our knowledge of the HIV-1 Human InteractionDatabase. We examined its contents and identified processes that HIV-1 was not previouslyknown to interact with. Then, we attempted an in silico dynamic model of HIV-1 interaction. We built a model of HIV-1 interaction with the CD4 T cell activation pathway comprised of137 nodes (16 HIV-1, 121 human) and 336 interactions. The model reproduced expectedpatterns of T cell activation. Using interaction graph properties, we identified 26 host cellfactors, including MAPK1&3, Ikkb-Ikky-Ikka and PKA, which contribute to the net activationor inhibition of viral proteins. By following a logical Boolean formalism, we identified 9 hostcell factors essential to the functions of viral proteins in the activation pathway. This wasthe first attempt to model dynamic viral-host interaction relationships. Then, we organize HIV-1 interacting host genes into modules to represent cellular processesneeded by the virus. We combined HIV-1 interactions with host gene GO annotations toclassify host genes according to these needed cellular processes. We obtained 201 modulesand found the same set of viral proteins do not interact with host genes having similarmodules suggesting intelligence in its co-ordination of host processes. This work is one of agrowing list that explores coordination of HIV-1 interactions. But more importantly, it would bebeneficial to functionally downsize the large dynamic HIV-1 interaction network. Finally, in our discussion, we discuss our results and suggest possible ways in which our workon dynamic models could be improved. This work is opening up a new field of systems virologythat studies the effect of viruses on the host in terms of its temporal and spatial aspects.

No description available.

Helper T cells direct the immunological response to foreign pathogens and cancer. To become activated, helper T cells must recognize unique peptides presented on major histocompatibility complex II (pMHCII) by antigen presenting cells (APCs) with their T cell receptor (TCR). While much is known about helper T cell activation signaling cascades and the subsequent roles of helper T cell subsets, the initiation of helper T cell activation by the TCR and other co-receptors is less well understood. Specifically, the affinity of the TCR for its pMHCII can change helper T cell subset fate, proliferation, and alter the risk for activation induced cell death. High affinity TCRs are attractive targets for immunotherapies, but little is known about how helper T cells respond to high affinity TCRs. Here we describe high affinity TCR activation thresholds for both full length TCRs and chimeric antigen receptor TCRs both with and without the presence of the coreceptor CD4 and propose a mechanism whereby CD4 inhibits T cell activation via Lck sequestration and a CD4-independent method. Dysregulated helper T cells play critical roles in the development and perpetuation of systemic lupus erythematosus (SLE), a systemic autoimmune disease that causes widespread inflammation and organ damage throughout the body. Chronic inflammation in SLE affects the immune response to viruses and the risk of developing cancer. However, in SLE patients, it is unclear if viruses initiate the development of cancer directly or if the effects are non-interacting and concomitant. Here we describe the interactions between SLE, viruses, and cancer risk revealing that viruses and SLE do interact to increase the both the overall cancer risk and the risk for hematological malignancies. Due to vaccine efficacy, vaccine preventable diseases (VPDs) are no longer commonly experienced or understood by the public. Vaccines are a victim of their own success and according to the World Health Organization (WHO), vaccine hesitancy (VH) is one of the top threats to global health. VH is the refusal to accept vaccinations and the reasons for VH vary across time, place, and vaccine. Refuting VH is difficult as directly confronting false assumptions can cause individuals to become more entrenched in their position resulting in confirmation bias. Adults with VH attitudes are often motivated by concerns over personal liberty, harm, independence, and body purity. Here we describe the results of a VPD interview- and education-based intervention geared towards promoting positive vaccine attitudes for young adults and demonstrate that education focused on VPDs is more effective than vaccine safety.

Les lymphocytes B jouent un rôle central dans l’immunité humorale par leur capacité à présenter des antigènes aux lymphocytes T, à sécréter des cytokines et à se différencier en plasmocytes produisant des anticorps. Ces fonctions peuvent être induites par leur stimulation in vitro. Par leur aptitude à présenter des antigènes indépendamment de la spécificité du récepteur des lymphocytes B (BCR), les lymphocytes B peuvent être utilisés comme cellules présentatrices d’antigènes (antigen-presenting cells, APC) afin d’induire la réponse cellulaire des lymphocytes T CD8+ cytotoxiques spécifiques. L’immunité cellulaire est cruciale pour prévenir les infections contre certains virus et en immunothérapie du cancer. L’objectif général de ces travaux est d’étudier la biologie des lymphocytes B. Plus particulièrement, nous souhaitons comprendre et améliorer leur fonction de présentation d’antigène afin d’utiliser les lymphocytes B comme source alternative d’APC. Dans la première partie de ces travaux, notre attention s’est portée sur la compréhension du mécanisme de présentation croisée par le complexe majeur d’histocompatibilité de classe I (CMH-I) par lequel un épitope de la protéine gp100 du mélanome, inséré dans une nanoparticule pseudo-virale (VLP) composée de la protéine de surface du virus de la mosaïque de la papaye (PapMV), est présenté par les lymphocytes B. Cette VLP est une plateforme vaccinale capable de stimuler le système immunitaire sans l’aide d’adjuvant et facilite la présentation croisée de l’épitope inséré, de façon indépendante de l’activité du protéasome. Les résultats obtenus démontrent que l’apprêtement de l’épitope inséré dans la nanoparticule s’effectue selon une voie de présentation croisée vacuolaire qui dépend de l’activité de la cathepsine S, de l’acidification des lysosomes et requiert l’induction de l’autophagie. Ainsi, nous avons défini plus précisément le mécanisme de présentation croisée par lequel les lymphocytes B apprêtent et présentent un épitope inséré dans la VLP de PapMV. Par la suite, nous avons cherché à améliorer le protocole d’activation in vitro permettant d’amplifier et d’induire les fonctions de présentation d’antigènes des lymphocytes B, dans le but d’utiliser ces cellules pour activer les réponses cellulaires des lymphocytes T CD8+ cytotoxiques. Les stimulations in vitro des lymphocytes B par le CD40 ligand (CD40L) et l’interleukine (IL)-21 ou la combinaison de l’IL-4 et l’IL-21 au lieu de l’activation standard avec le CD40L et l’IL-4 ont été évaluées. Nos résultats ont approfondi nos connaissances de la biologie des lymphocytes B. Nous avons démontré que la stimulation des lymphocytes B avec le CD40L et l’IL-21 augmente leur prolifération, mais mène à leur différenciation en plasmocytes sécrétant des anticorps. Au contraire, la stimulation avec le CD40L et l’IL-4 induit efficacement leur fonction de présentation d’antigènes. La stimulation des lymphocytes B avec le CD40L et la combinaison de l’IL-4 et de l’IL-21 augmente leur prolifération, mène seulement faiblement à leur différenciation en cellules sécrétrices d’anticorps, mais induit efficacement leur fonction de présentation d’antigènes. Nous avons démontré pour la première fois que cette méthode permet de générer des APC puissantes capables d’induire la réponse des lymphocytes T CD8+ cytotoxiques in vitro. Nos résultats nous permettent de postuler que ces cellules pourraient être capables de mener à une réponse cellulaire in vivo. En tant qu’APC efficaces, les lymphocytes B pourraient être utilisés dans une stratégie vaccinale ou être employés comme APC afin d’améliorer les traitements d’immunothérapie du cancer par transfert adoptif de lymphocytes T. Ainsi, les travaux présentés dans cette thèse ont porté tant sur la compréhension des mécanismes de présentation croisée d’un épitope inséré dans la VLP de PapMV par les lymphocytes B, que sur l’amélioration de la méthode permettant d’induire leur fonction de présentation d’antigènes pour activer les lymphocytes T CD8+ cytotoxiques. Ces travaux de recherche fondamentale ont permis de contribuer à des avancées sur les connaissances de la biologie des lymphocytes B. Ils offrent de nouvelles pistes de réflexion quant aux utilisations biotechnologiques des lymphocytes B comme source alternative d’APC pour des applications de recherche fondamentale et clinique telles que la vaccination et les traitements d’immunothérapie du cancer. / B lymphocytes are central to humoral immunity due to their ability to present antigens to T cells, secrete cytokines and to differentiate into antibody-producing plasma cells. These functions can be induced by their in vitro stimulation. Being able to present antigens independently of the specificity of their B cell receptor (BCR), B cells can be used as antigen-presenting cells (APC) to induce specific cytotoxic CD8+ T cell cellular responses. Cellular immunity is crucial to prevent infections against viruses and in cancer immunotherapy. The main aim of this thesis is to study B cell biology. Specifically, we aim to deepen our understanding of their antigen presentation function and improve this function to use B cells as an alternative source of APC. First, we focused on deciphering the class I major histocompatibility complex (MHC-I) cross-presentation mechanism by which an epitope from gp100 melanoma protein, inserted in a virus-like particle (VLP) made of the coat protein of the papaya mosaic virus (PapMV), is presented by B cells. This VLP is a vaccine platform able to stimulate the immune system with no adjuvant and mediate a proteasome independent cross-presentation of the inserted epitope. Our results show that the inserted epitope is processed through a vacuolar pathway dependent on cathepsin S activity, lysosome acidification and requires the induction of autophagy. Thus, we provide a more detailed characterization of the mechanism used by B cells to process and cross-present an epitope inserted in PapMV VLP. Secondly, we aimed to improve the in vitro activation protocol used to expand B cells and induce their antigen presentation functions to use these cells to trigger cytotoxic CD8+ T cell cellular responses. We evaluated the in vitro stimulation of B cells with CD40 ligand (CD40L) and interleukin (IL)-21 or the combination of IL-4 and IL-21 instead of the standard activation method based on CD40L and IL-4. Our results deepen our knowledge of B cell biology. We showed that stimulating B cells with CD40L and IL-21 increases their proliferation but leads to their differentiation in antibody-producing plasma cells. In comparison, the stimulation with CD40L and IL-4 efficiently induces their antigen presentation function. The stimulation of B lymphocytes with CD40L and the combination of IL-4 and IL-21 increases their proliferation, weakly leads to their differentiation in antibody-secreting cells but is very efficient in inducing their antigen presentation function. We show for the first time that this method can generate potent APC able to induce cytotoxic CD8+ T cell responses in vitro. Our results allow us to hypothesize that these cells could be capable of triggering cellular immunity in vivo. As efficient APC, B cells could be used in a vaccination strategy or be employed as APC to improve cancer immunotherapy treatments such as adoptive cell transfer of T lymphocytes. Thus, the work presented in this thesis provides a deeper understanding of the antigen cross-presentation pathway by which B cells process and present an epitope inserted in PapMV VLP. It also reports an improved method to induce antigen presentation function of B cells to stimulate cytotoxic CD8+ T cells. This research work constitutes a leap forward in fundamental B cell research by increasing our knowledge of B cell biology. It also brings new opportunities regarding biotechnological uses of B cells as an alternative source of APC for fundamental and clinical applications such as vaccination and cancer immunotherapy treatments.

<p>  </p> <p>Metastatic breast cancer (MBC) is an extremely recalcitrant disease capable of overcoming targeted therapies and evading immune surveillance via the engagement of complicated signaling networks. Resistance to targeted therapies and therapeutic failure of immune checkpoint blockade (ICB) are two major challenges in treating MBC. To survive in the dynamic tumor microenvironment (TME) during metastatic progression, shared signaling nodes are required for MBC cells to regulate the signaling networks efficiently, which are potential multifunctional therapeutic targets. SH2 containing protein tyrosine phosphatase-2 (SHP2) is a druggable oncogenic phosphatase that is a key shared node in both tumor cells and immune cells. How tumor-cell autonomous SHP2 manages its signaling inputs and outputs to facilitate the growth of tumor cells, drug resistance, immunosuppression, and the limited response of ICB in MBC is not fully understood. Herein, we used inducible genetic depletion and two distinct types of pharmacological inhibitors to investigate anti-tumor effects with immune reprogramming during SHP2 targeting. </p> <p>We first focus on the signaling inputs and outputs of SHP2. We find that phosphorylation of SHP2 at Y542 predicts the survival rates of breast cancer patients and their immune profiles. Phosphorylation of SHP2 at Y542 is elevated with differential activation mechanisms under a growth-factor-induced and extracellular matrix (ECM)-rich culture environment. Phosphorylation of SHP2 at Y542 is also elevated in HER2 positive MBC cells upon acquired resistance to the HER2 kinase inhibitor, neratinib. The resistant cells can be targeted by SHP2 inhibitors. SHP2 inhibitors block ERK1/2 and AKT signaling and readily prevented MBC cell growth induced by multiple growth factors. Inhibition of SHP2 also blocks these signaling events generated from the ECM signaling. In fact, the inhibitory effects of SHP2 blockade are actually enhanced in the ECM-rich culture environment. We utilize the <em>in vitro</em> T-cell killing assays and demonstrate that pretreatment of tumor cells with FGF2 and PDGF reduces the cytotoxicity of CD8+ T cells in a SHP2-dependent manner. Both growth factors and ECM-rich culture environment transcriptionally induce PD-L1 via SHP2. SHP2 inhibition balances MAPK signaling and STAT1 signaling, which prevents growth factor-mediated suppression of INF-γ-induced expression of MHC class I. </p> <p>Next, we evaluate the efficacy of SHP2 inhibitors. Blockade of SHP2 in the adjuvant setting decreased pulmonary metastasis <em>in vivo</em> and extended the survival of systemic tumor-bearing mice. Tumor-cell autonomous depletion of SHP2 reduces pulmonary metastasis and relieves exhaustion markers on CD8+ and CD4+ cells. Meanwhile, both systemic SHP2 inhibition and tumor-cell autonomous SHP2 depletion reduce tumor-infiltrated CD4+ T cells and M2-polarized tumor associated macrophages. </p> <p>Finally, we investigate potential combination therapies with SHP2 inhibitors. The combination of SHP2 inhibitors and FGFR-targeted kinase inhibitors synergistically blocks the growth of MBC cells. Pharmacological inhibition SHP2 sensitizes MBC cells growing in the lung to α-PD-L1 antibody treatment via relieving T cell exhaustion induced by ICB. </p> <p>Overall, our findings support the conclusion that MBC cells are capable of simultaneously engaging several survival pathways and immune-suppressive mechanisms via SHP2 in response to multiple growth factors and ECM signaling. Inhibition of SHP2, potentially in combination with other targeted agents and ICB, holds promise for the therapeutic management of MBC.</p>

<p>  </p> <p>The phosphatases of regenerating liver are a subfamily of protein tyrosine phosphatases that consist of PRL1, PRL2 and PRL3. The overexpression of PRLs promote cell proliferation, migration and invasion and contribute to tumorigenesis and metastasis to aggravate survival outcome. Although there is increasing interest in understanding the implication of these phosphatases in tumor development, currently, limited knowledge is available about their mechanism of action and the efficacy of PRL inhibition in <em>in vivo</em> tumor models, the tumor extrinsic role of PRLs that allow them to impact tumor development, as well as <em>in vivo</em> physiological function of PRLs that could implicate them in diseases other than cancer. The work presented here aims to address these limitations.</p> <p><br></p>

Multiple Sklerose (MS) ist eine heterogene Krankheit des Zentralnervensystems, deren pathologische Mechanismen noch nicht vollständig aufgeklärt sind. Die gegenwärtige Hypothese ist, daß pro-inflammatorische T-Zellen entscheidend an der Pathogenese der MS beteiligt sind. Man geht davon aus, daß eine Fehlregulation der T-Zell-Kontrolle, möglicherweise bedingt durch ein Ungleichgewicht an Apoptose-regulierenden Molekülen, dabei eine Rolle spielt. Tatsächlich zielen therapeutische Strategien darauf ab, T-Zell-Aktivierung, Proliferation und Produktion von Zytokinen zu verringern, oder T-Zell-Eliminierung zu fördern. Diese Arbeit sollte zum einen die Bedeutung regulatorischer Faktoren klären, die für das überleben der T-Zellen von MS-Patienten verantwortlich sind. Zum anderen sollten die antiproliferative oder Apoptose-fördende Wirkung potentiell therapeutisch wirksamer Moleküle untersucht werden. Eine eingeschränkte Regulation der autoreaktiven T-Zellen durch Apoptose in der Peripherie und im ZNS trägt möglicherweise zur Pathophysiologie der MS bei. Als Schlüsselfaktoren der Regulation von Apoptose wurden Mitglieder der Bcl-2-Familie in MS-Patienten und Probanden untersucht. Diese Faktoren wurden in Relation zu der Suszeptibilität der T-Zellen gegenüber aktivierungsinduziertem Zelltod (sog. Activation-induced cell death oder AICD) überprüft. Um die in-vivo-Elimination der Antigen-reaktiven T-Zellen nachzuahmen, wurde ein in-vitro-Modell des AICD mit repetitiver T-Zell-Stimulation verwendet. Tatsächlich zeigten polyklonale T-Zellen von MS-Patienten eine verringerte Suszeptibilität für AICD, nachgewiesen sowohl durch verminderte Caspaseaktivtät (p=0.013) als auch durch DNA-Fragmentierung (p=0.0071). Weiter wurden höhere Spiegel des Proteins Bcl-XL in den Immunzellen von MS-Patienten mit Immunoblotting gemessen (p=0.014). Eine inverse Korrelation zwischen der Expression an Bcl-XL und der Empfindlichkeit der T-Zellen gegenüber AICD steht in Übereinstimmung mit vorhergehenden Daten bezüglich der Bedeutung dieses Proteins für die Apoptose-Resistenz von T-Zellen. Es wurde bereits gezeigt, daß dieses Molekül die Ausprägung der experimentell-autoimmun Enzephalomyelitis, des Tiermodells der MS, verstärkt. Zusammen mit den erhöhten Bcl-XL-Werten bei MS-Patienten, ergeben sich nun Perspektiven für einen therapeutischen Ansatz. Abgesehen von dem Konzept die apoptotische Eliminierung von T-Zellen zu unterstützen, streben gegenwärtige therapeutische Strategien an, die Aktivierung und weitere Proliferation der schädlichen T-Zellen zu hemmen. Basierend auf klinischer Erfahrung mit eher unselektiven Therapien, ist es ein therapeutisches Ziel, neue immunomodulatorische Substanzen mit besserer Selektivität zu finden, um das Nutzen/Risiko-Verhältnis zu maximieren. Aus diesem Grund wurden zwei unterschiedliche Substanzen untersucht die beide den Zellzyklus beeinflussen. Als erster Kandidat wurde der kürzlich entdeckte Todesligand TRAIL (engl.: TNF-related apoptosis inducing ligand) aus der TNF/NGF-Familie untersucht, da diesem bereits T-Zell-regulatorische Funktionen zugeschrieben worden waren, humane Antigen-spezifische T-Zellen jedoch resistent gegenüber TRAIL-induzierter Apoptose sind. Der zweite Kandidat mit potenziell therapeutischer Wirkung bei MS ist Atorvastatin, ein HMG-CoA-Reduktase-Hemmer, der bereits als Lipidsenker bei Patienten eingesetzt wird. Um die Hypothese zu überprüfen, daß diese Substanzen T-Zell-Rezeptor-Signale beeinflussen können, wurden humane Antigen-spezifische T-Zell-Linien von MS-Patienten und gesunden Probanden eingesetzt. Diese wurden hinsichtlich T-Helfer-Phänotyp und Peptid-Spezifität charakterisiert. Eine Behandlung mit TRAIL führte zur Hemmung der Proliferation in unterschiedlichem Ausmaß (6.2% - 63.8%). Atorvastatin hemmte in Abhängigkeit von der Dosis ebenso die Proliferation Antigen-spezifischer T-Zellen. Beide Substanzen wirkten antiproliferativ unabhängig von der Antigenpräsentation, aufgrund ihrer Fähigkeit, die Proliferation in Abwesenheit von professionellen Antigen-präsentierenden Zellen zu vermindern. Diese Eigenschaft weißt auf einen direkten Einfluß auf die T-Zell-Funktion hin. Die TRAIL-induzierte Hypoproliferation war assoziiert mit einer Herunterregulation der Zyklin-abhängigen Kinase CDK4 (engl.: cyclin dependent kinase 4), einem Schlüsselenzym für die nach T-Zell-Rezeptor-Stimulation einsetzende Transition von der G1- zur S-Phase des Zellzyklus. Inkubation mit Atorvastatin induzierte ebenso eine Verminderung von CDK4, begleitet von einer Erhöhung von p27Kip1. Die Atorvastatin-vermittelte Proliferations- und Zellzyklus-Blockade konnte durch Mevalonat rückgängig gemacht werden. Mevalonat ist ein Zwischenprodukt des HMG-CoA-Reduktaseweges. Atorvastatin scheint demnach einen direkten Einfluß auf diese Enzymkaskade zu haben, der wichtig für die Isoprenylierung von GTPase-Proteinen der Rho-Familie ist. T-Zell-Rezeptor-Stimulation führt zur Freisetzung von Kalzium aus intrazellulären Speichern und nachfolgend zur Öffnung transmembranöser Kalzium-Kanäle (sog. calcium release-activated calcium oder CRAC-Kanäle), die eine für die T-Zellaktivierung notwendige und anhaltende Erhöhung der intrazellulären Kalzium-Konzentration hervorruft. Nach Behandlung mit TRAIL wurde eine konzentrationsabhängige Inhibition des Einstroms extrazellulärer Kalzium-Ionen durch die CRAC-Kanäle beobachtet. Dies wurde mit löslichem TRAIL-Rezeptor-Fusionsprotein, einem TRAIL-Antagonisten, rückgängig gemacht. Die Blockade von Kalzium-abhängigen Aktivierungssignalen stellt damit möglicherweise einen primären immunregulatorischen Mechanismus für diese Todesliganden dar. Jedoch wurde keine Auswirkung von Atorvastatin auf die T-Zellaktivierung beobachtet, da der Einstrom von extrazellulärem Kalzium nicht beeinflußt wurde. Während Studien zum TRAIL-vermittelten Einfluß auf die T-Zell-Aktivierung und dem Zellzyklus erst in der präklinischen Phase sind, werden Statine, die ebenfalls den Zellzyklus beeinflussen, bereits in der Therapie anderer Erkrankungen angewand. Darüber hinaus werden derzeit bereits klinische Studien mit Statinen zur MS-Therapie durchgeführt. Weitere Untersuchungen zu den detaillierten Mechanismen antiproliferativer Substanzen mit potenziellem therapeutischen Effekt in der MS ermöglichen die Entwicklung von selektiveren immunomodulatorischen Therapien mit höherem therapeutischen Nutzen für MS-Patienten. / Multiple sclerosis (MS) is a heterogeneous disease of the central nervous system whose pathological mechanisms are far from completely understood. The current hypothesis is that pro-inflammatory T cells are orchestrating the pathogenesis of this condition. It is considered that a dysregulation in T cell control to be involved, with an imbalance in apoptosis-regulating molecules possibly playing a role. In fact, therapeutic strategies aim to reduce T cell activation, proliferation and cytokine production or to promote T cell elimination. The focus of this thesis was to identify the role of regulatory molecules for T cell survival in the immune pathogenesis of MS, and to investigate antiproliferative or apoptosis-promoting effects on T cells by potential therapeutic molecules. A limitation in the apoptotic regulation of autoreactive T cells in the periphery and in the CNS may contribute to the pathophysiology of MS. As key regulators of apoptosis, members of the Bcl-2 family were investigated in both MS patients and controls. These factors were examined in relation to the susceptibility of T cells, from both groups, towards activation-induced cell death (AICD). To mimic the in vivo elimination of antigen-reactive T cells, an in vitro model of AICD involving repetitive T cell receptor mediated stimulation was utilized. In fact, polyclonal T cells from MS patients showed a decreased susceptibility to undergo AICD as shown by both caspase activity (p=0.013) and DNA fragmentation (p=0.0071) assays. Furthermore, Bcl-XL protein levels, as measured by immunoblotting, were increased in the peripheral immune cells of MS patients (p=0.014). An inverse correlation observed between Bcl-XL levels and susceptibility of T cells to undergo AICD is in line with previous data on the significance of this anti-apoptotic protein in T cell resistance. Since this molecule has already been shown to aggravate the outcome of experimental autoimmune encephalitis, the animal model for MS, the observation of elevated Bcl-XL levels in patients offers perspectives towards therapeutic manipulation in MS. Apart from promoting apoptotic elimination, current therapeutic strategies aim at inhibiting activation and further proliferation of potentially harmful T cells. Based on clinical experience with rather non-selective therapies that promote T cell elimination, a therapeutic goal is to identify newer immunomodulatory substances with better selectivity in order to maximize the therapy's benefit to risk ratio. Thus, two different substances, both interfering with cell cycle regulation, were investigated. The first candidate was the recently discovered member of the TNF/NGF family of death ligands, TNF-related apoptosis inducing ligand (TRAIL) since it has been reported to have immunoregulatory functions and since human antigen-specific T cells were shown to be resistant towards apoptosis induction by this ligand. The second candidate drug with potential in MS therapy is atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme (HMG-CoA) reductase inhibitor and lipid-lowering drug, already indicated for anomalies in lipid metabolism. In order to prove the hypothesis that these substances interfere with T cell receptor signaling, human antigen-specific T cell lines from both MS patients and controls, characterized with regards to T helper differentiation and peptide specificity, were employed. Exogenous treatment of TRAIL resulted in an inhibition in proliferation, albeit to varying degrees (6.2% - 63.8% inhibition). Atorvastatin also inhibited proliferation of antigen-specific T cell lines in a dose-dependent manner. Both compounds induced hypoproliferation independently of antigen presentation, as shown by their ability to block T cell proliferation in response to direct T cell receptor engagement, thus indicating a direct influence on T cell function. The growth inhibition by TRAIL was associated with a downregulation of the cell cycle regulator CDK4, indicative of an inhibition of cell cycle progression at the G1/S transition. Incubating T cells with atorvastatin also induced a downregulation of CDK4 expression, which was accompanied by an upregulation of p27Kip1 expression. The atorvastatin-mediated inhibition in proliferation and cell cycle progression could be reversed by mevalonate, an intermediate product of the HMG-CoA reductase pathway, suggesting a direct involvement of atorvastatin in this pathway, necessary for the isoprenylation of small GTPase proteins of the Rho family. Utilizing a thapsigargin model of calcium influx to activate the same calcium-release activated calcium (CRAC) channels as T cell receptor-stimulation by antigen, an inhibition in calcium influx could be observed on pre-incubating T cells with TRAIL. Co-incubating with human recombinant TRAIL receptor 2 fusion protein, a competitive antagonist for TRAIL, reversed this inhibition. A direct influence on calcium influx is indicative of an influence of TRAIL on the activation status of human T cells. Therefore, TRAIL directly inhibits activation of these cells via blockade of calcium influx. However, no impact of atorvastatin on early T cell activation was observed, since calcium influx was unaffected. While TRAIL-mediated interference with T cell activation and further cell cycle progression is still in the pre-clinical phase, statins, which have also been shown here to interfere with the T cell cycle, are already employed in the clinic for other ailments. In fact, clinical trials are currently being undertaken with this group of drugs for MS. Further studies on detailed mechanisms of antiproliferative substances effective in MS will allow the development of highly selective immunomodulatory agents with increased beneficial profile as MS therapy.