T cells are an essential component of the adaptive immune system, which evolved to facilitate development of long-term, effective protection against infectious diseases. Upon activation, T cells play an important role in clearing infections, and especially, in preventing establishment of subsequent infections with the same pathogen. Because this is such a powerful response, it must be tightly regulated. Our lab has long been interested in how signaling molecules regulate the function of T and B lymphocytes. Our prior studies stimulated an interest in the signaling adapter molecule, Tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3). Our group previously produced a T cell-conditional (CD4-Cre) TRAF3-/- mouse, which demonstrated that TRAF3 unexpectedly plays an important positive role in T cell functions, including providing help for B cell responses, protection from infectious pathogens, cytokine production and proliferation. After TCR engagement, TRAF3 associates with the T Cell Receptor (TCR)/CD28 complex. These data identified a new role for TRAF3 in T cell activation. There are three signals that are required for full T cell activation. The three types of receptors that deliver these signals are the TCR, co-stimulatory receptors and cytokine receptors. This dissertation explores the regulatory role of TRAF3 in the 3 signals required for T cellsactivation. In signal 1, TRAF3 enhances TCR signaling by regulating the localization of the TCR inhibitors, PTPase non-receptor type 22 (PTPN22) and the c-Src kinase (Csk). Our lab previously reported that recruitment of TRAF3 to the TCR complex requires co-stimulation of CD28, the primary receptor for signal 2. In this dissertation, we show that TRAF3 associates with the Linker of Activated T cells (LAT) complex, demonstrating preference for distinct LAT-associated proteins. For delivery of signal 3, T cells require stimulation of a cytokine receptor, such as IFNαR, for differentiation of a T cell to an effector cell. Upon IFN stimulation, TRAF3 inhibits IFNαR-induced early molecular events, which results in the regulation of both canonical and non-canonical IFNαR signaling pathways. The results presented in this dissertation highlight the dynamic roles of TRAF3 as a regulator of T cell activation, by regulating multiple T cell signaling pathways.

La voie JAK / STAT est une voie majeure de signalisation intracellulaire, activée par plusieurs cytokines, y compris par les interférons (IFNs). Mon laboratoire a précédemment établi que l'endocytose et le trafic du récepteur de l’IFN alpha/beta (IFNAR) jusqu’à l'endosome précoce, joue un rôle clé dans l’activation de la voie de signalisation JAK/STAT et dans les activités antivirales et antiprolifératives induites par les IFNs de type I. Le récepteur de l’IFN de type I se compose de deux sous-unités: IFNAR1 et IFNAR2. Durant le trafic du récepteur vers l'endosome précoce, les deux sous-unités prennent des itinéraires différents - IFNAR1 est ubiquitiné et dégradé au lysosome tandis que IFNAR2 est recyclé vers la membrane plasmique. Les mécanismes moléculaires permettant un trafic différent des IFNAR1 et IFNAR2 restent mal compris.J’ai tout d’abord étudié le trafic intracellulaire des sous-unités IFNAR1 et IFNAR2 et j’ai pu montrer que les protéines Rab4 et Rab11 sont nécessaires au recyclage d’IFNAR2 à la membrane plasmique. Par la suite, j’ai identifié le complexe rétromère endosomal - VPS26A/VPS29/VPS35 comme un partenaire d’interaction avec IFNAR2 nécessaire pour son recyclage. En outre, j’ai montré que le complexe rétromère contrôle la séparation spatiotemporelle des sous-unités IFNAR1 et IFNAR2 au niveau de l'endosome précoce. En effet, la déplétion du complexe rétromère entraine une prolongation de l’association endosomale de deux sous-unités et une prolongation de la signalisation JAK/STAT induite par l’IFN tant pour la réponse précoce (phosphorylation de STAT1) que la réponse à plus long terme (expression des gènes stimulés par l'IFN). Des résultats en cours de publication par l’équipe montrent un rôle d’ESCRT-0 dans l’initiation de la signalisation JAK/STAT. J’ai montré que le rôle de la machinerie ESCRT dans ce processus est limité au complexe ESCRT-0. Les sous-complexes en aval d’ESCRT-0, bien que nécessaires à la dégradation d’IFNAR1, ne sont pas impliqués dans l’activation de la voie JAK/STAT.En conclusion, ces résultats nous ont permis d’établir un modèle dans lequel le retromère joue un rôle clef dans la régulation spatio-temporelle du tri endosomal d’IFNAR et de la signalisation JAK/STAT au niveau de l’endosome précoce. Après la séparation rétromère-dépendante des sous-unités du récepteur et la terminaison de la signalisation JAK/STAT, la dégradation lysosomale d’IFNAR1 est assurée par la machinerie ESCRT en aval d’ESCRT-0. / The JAK/STAT pathway is a major intracellular signaling pathway that is activated by several cytokines including interferons (IFNs). My laboratory has previously established that endocytosis and trafficking of the IFN alpha/beta receptor (IFNAR) to the early endosome is key for the activation of JAK/STAT signaling and for the antiviral and antiproliferative activities induced by type I IFNs. The functional type I IFN receptor - IFNAR - consists of two subunits: IFNAR1 and IFNAR2. Upon endocytosis to the early endosome, both subunits take different trafficking routes – IFNAR1 is ubiquitinated and degraded in the lysosome, whereas IFNAR2 recycles back to the plasma membrane. The molecular mechanisms behind the separation of IFNAR1 and IFNAR2, as well as their distinct trafficking routes remain poorly understood.First, I studied the intracellular trafficking of IFNAR1 and IFNAR2 subunits and showed the requirement for Rab4 and Rab11 in IFNAR2 recycling to the plasma membrane. Next, I identified the endosomal retromer complex – VPS26A/VPS29/VPS35 as an IFNAR2 interacting partner, required for IFNAR2 recycling. Additionally, I was able to show that retromer controls the spatiotemporal separation of IFNAR1 and IFNAR2 subunits at the early endosome. Indeed, retromer depletion resulted in prolonged endosomal association of both subunits and prolonged activation of IFN-induced JAK/STAT signaling, at both early (STAT1 phosphorylation) and longer time response (IFN-stimulated gene expression). Unpublished results of our team indicate the role of ESCRT-0 in initiation of the IFN-mediated JAK/STAT signaling. I found that role of the ESCRT machinery in this process is limited to the ESCRT-0. ESCRT subcomplexes downstream of ESCRT-0, although required for IFNAR1 degradation, are not involved in activation of the JAK/STAT pathway.In conclusion, these results permit us to draw a model in which the retromer is a key spatiotemporal regulator of IFNAR endosomal sorting and the JAK/STAT signaling at level of the early endosome. Once retromer-mediated subunits separation is accomplished and JAK/STAT signaling is terminated, ESRCT machinery downstream to ESCRT-0 mediates IFNAR1 lysosomal degradation.

Pathogenesis of autoimmune and auto-inflammatory diseases is induced by auto-aggressive helper T (Th) cells (i.e. Th1 and Th17 cells), and can be controlled by regulatory T cells (Tregs) characterized by expression of the transcription factor Foxp3. Thus, development of autoimmunity is regulated by the balance of Tregs and Th1/Th17 cells. Prostaglandin E₂ (PGE₂) is a bioactive lipid mediator with immune-modulatory potential that acts through 4 receptors (EP1-4). It has been shown that PGE₂ facilitates Th1 and Th17 cell development and expansion, therefore promoting autoimmune inflammation. However, the role of PGE₂ in Treg development and function is largely unclear. The aim of this PhD was to test the hypothesis that PGE₂ regulates Treg development, function and subsequent immune response. I observed that in vivo inhibition of endogenous PGE₂ biosynthesis using a COX inhibitor resulted in increased Foxp3+ Tregs in various lymphoid organs. This response was prevented by addition of an EP4 agonist. PGE₂-EP4 signalling particularly inhibits RORγt+ Tregs in the intestine. This was not observed in either antibiotic-treated mice or MyD88/TRIF double-knockout mice, suggesting gut commensal microbiota involvement. In addition, PGE₂ has a role in microbiota-dependent regulation of intestinal CD11c+MHCII+CD11b+CD103- mononuclear phagocytes (MNPs) which drive intestinal Treg expansion through production of type 1 interferons. Consistent with these in vivo observations, gut microbial metabolites from indomethacin treated mice enhanced in vitro RORγt+ Treg differentiation in the dendritic cell- T cell co-culture system. Adoptive transfer of caecal microbiota from COX inhibitor- treated mice into naïve mice also provided protective benefits in a chemical (DSS)-induced colitis disease model. In summary, this work has demonstrated that PGE₂ affects intestinal Tregs, indicating a novel mechanism for interaction of PGE₂, the adaptive immune system and the gut microbiota in homeostasis within this environment. These findings increase our understanding of the role of PGE₂ in development of inflammatory bowel disease and offer potential therapeutic strategies for treating this disease.

Abstract not available

The mosquito-borne Usutu virus (USUV) is a zoonotic flavivirus and an emerging pathogen. So far therapeutical options or vaccines are not available in human and veterinary medicine. The bioenergetic profile based on extracellular flux analysis revealed an USUV infection-associated significant increase in basal and stressed glycolysis on Vero and with a tendency for basal glycolysis on the avian cell line TME-R derived from Eurasian blackbirds. On both cell lines this was accompanied by a significant drop in the metabolic potential of glycolysis. Moreover, glycolysis contributed to production of virus progeny, as inhibition of glycolysis with 2-deoxy-D-glucose reduced virus yield on Vero by one log10 step. Additionally, the increase in glycolysis observed on Vero cells after USUV infection was lost after the addition of exogenous type I interferon (IFN) b. To further explore the contribution of the IFN response pathway to the impact of USUV on cellular metabolism, USUV infection was characterized on human A549 respiratory cells with a knockout of the type I IFN receptor, either solely or together with the receptor of type III IFN. Notably, only the double knockout of types I and III IFN receptor increased permissiveness to USUV and supported viral replication together with an alteration of the glycolytic activity, namely an increase in basal glycolysis to an extent that a further increase after injection of metabolic stressors during extracellular flux analysis was not noted. This study provides evidence for glycolysis as a possible target for therapeutic intervention of USUV replication. Moreover, presented data highlight type I and type III IFN system as a determinant for human host cell permissiveness and for the infection-associated impact on glycolysis.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the immune-mediated destruction of insulin-producing beta-cells of pancreatic islets, culminating in critical insulin deficiency. Both genetic and environmental factors likely orchestrate an immune-mediated functional loss of beta cell mass, leading to the clinical manifestation of disease and lifelong dependence on insulin therapy. Additional evidence suggests the role of innate and adaptive immune mechanisms leading to inflammation in beta cells mediated by proinflammatory cytokines and chemokines, activation of beta-cell-reactive T cells,and failure of immune tolerance. Viral infections have been proposed as causal determinants or initiating triggers for T1D but remain unproven. Understanding the relationship between viral infections and the development of T1D is essential for T1D prevention. Importantly, virus-induced innate immune responses, particularly type I interferon (IFN-I, IFN-a/b), have been implicated in the initiation of islet autoimmunity and development of T1D. The goal of my thesis project is to investigate how the IFN-I signaling pathway affects the development of T1D using the LEW.1WR1 rat model of autoimmune diabetes. My hypothesis is that disrupting IFN-Isignaling via functional deficiency of the IFN-I interferon receptor (IFNAR) prevents or delays the development of virus-induced diabetes.For this purpose, I generated IFNAR subunit 1(IFNAR1)-deficient LEW.1WR1 rats using CRISPR-Cas9 genome editing and confirmed the functional disruption of IFNAR1. The absence of IFNAR1 results in a significant delay in onset and frequency of diabetes following poly I:C challenge and reduces the incidence of insulitis after poly I:C treatment. The frequency of diabetes induced by Kilham rat virus (KRV) is also reduced in IFNAR1-deficient LEW.1WR1 rats. Furthermore, I observe a decrease in CD8+T cells in spleens from KRV-infected IFNAR1-deficient rats relative to that in KRV-infected wild-type rats. While splenic regulatory T cells are depleted in WT rats during KRV-infection, no such decrease is observed in KRV-infected IFNAR1-deficient rats. A comprehensive bulk RNA-seq analysis reveals a decrease of interferon-stimulated genes and inflammatory gene expression in IFNAR1-deficient rats relative to wild-type rats following KRV challenge. Collectively, the results from these studies provided mechanistic insights into the essential role of virus-induced, IFN-I-initiated innate immune responses in the early phase of autoimmune diabetes pathogenesis.