Les cellules lymphoïdes innées de groupe 3 (ILC3) contribuent activement à l’homéostasie intestinale par leur production d’Interleukin-22 (IL-22). Ces ILC3 regroupent 2 sous-populations majeures, les LTi (« Lymphoid Tissue inducer »), caractérisées par l’expression du récepteur au chimiokine CCR6, et les ILC3 exprimant le facteur de transcription (FT) T-bet, qui comprennent une population positive pour le marqueur de surface NKp46, récepteur originalement utilisé pour identifier les ILC de groupe 1 (ILC1). Les ILC1 jouent un rôle prépondérant dans la réponse aux pathogènes intracellulaires et anti-tumorale. Jusqu’à présent, trois populations majeures composent les ILC1 : les lymphocytes cytotoxiques Natural Killer (NK ou ILC1b), qui dépendent largement du FT Eomes et expriment l’intégrine CD49b ; les ILC1 hépatiques et intestinaux, qui dépendent du FT T-bet et expriment CD49a (ILC1a) ; et une population CD49a+ et DX5+ indépendante du FT Nfil3 localisée dans les glandes salivaires ou l’utérus (ILC1ab). Mes travaux visent à comprendre la biologie des ILC exprimant NKp46, ainsi que les facteurs impliqués dans leur développement, leur maturation et leur fonction. La majeure partie de ma thèse se concentre sur les NKp46+ ILC3. Premièrement, nous démontrons un rôle majeur pour le récepteur aux chimiokine CXCR6 dans la localisation des NKp46+ ILC3 dans les villi de la lamina propria intestinale (Satoh-Takayama et al. 2014). Deuxièmement, j’ai mis en évidence que NKp46+ ILC3 pouvait perdre l’expression de NKp46 (Verrier et al. 2016). Déclenchée par le TGFβ, cette perte d’expression est associée à une plus forte capacité à produire de l’IL-22, mais aussi à l’acquisition de marqueurs identifiant les LTi (CCR6, MHC-II), démontrant ainsi la plasticité des NKp46+ ILC3. Enfin, en collaboration avec le groupe de Rachel Golub, nous avons confirmé le rôle présumé de la molécule Notch dans cette plasticité (Chea et al. 2016). Dans ce manuscrit, je discuterai du développement et de l’hétérogénéité des ILC3, ILC1a, ILC1b et ILC1ab. L’ensemble de mes résultats soutient une vision dynamique de la biologie des ILC reflétant l’adaptation de ces cellules effectrices face à leur environnement. En caractérisant les différents acteurs impliqués dans ce processus dynamique, mes travaux pourront servir au développement de thérapies visant à contrôler l’équilibre entre ces différentes populations dans divers pathologies comme le cancer, les infections virales, ou encore les maladies intestinales / Group 3 Innate Lymphoid cells (ILC3) actively maintain mucosal homeostasis through the production of Interleukin-22 (IL-22). ILC3 encompass 2 major populations, LTi (« Lymphoid Tissue inducer »), characterized by the expression of the chemokine receptor CCR6, and ILC3 that express the transcription factor T-bet, which include a population expressing the surface marker NKp46, a receptor originally used to identify group 1 ILC (ILC1). ILC1 plays a major role in the defense against intracellular pathogens and anti-tumoral responses. Three major ILC1 populations have been identified: the cytotoxic lymphocytes « Natural Killer » (NK or ILC1b), which largely rely for on the transcription factor Eomes their generation and express the integrin CD49b; hepatic and intestinal ILC1 that depends on the T-bet transcription factor and express CD49a (ILC1a); and a population that expresses CD49a and CD49b (ILC1ab) and populates the salivary gland and the uterus, which is independent of the transcription factor Nfil3. My work aimed to understand the biology of NKp46 expressing ILC, as well as factor involved in their development, maturation and function. The major part of my work focuses on NKp46+ ILC3. First, we demonstrate a major role for the chemokine receptor CXCR6 in their localisation in the lamina propria villi (Satoh-Takayama et al. 2014). Second, I showed that NKp46+ ILC3 could lose NKp46 expression (Verrier et al. 2016). Induced by TGFβ, this loss of expression was associated with higher IL-22 production and by the acquisition of markers identifying LTi (CCR6, MHC-II), demonstrating NKp46+ ILC3 plasticity. Finally, in collaboration with Rachel Golub’s group, we confirmed a putative role for Notch-signaling in this plasticity (Chea et al. 2016). In this manuscript, I will discuss the development and the heterogeneity of ILC3, ILC1a, ILC1b and ILC1ab. All the results I generated support a dynamic vision of ILC biology, which reflects how they adapt in response to environmental cues. By characterizing the different actors involved in this dynamic process, my work could be used to design therapies aiming at controlling the equilibrium between these different populations in diverse pathologies such as cancer, viral infection, or intestinal diseases

Influenza is one of the most common diseases worldwide, yet the vaccines against influenza are only 35% effective at protecting against infection. Creating a more effective vaccine requires an understanding of the foundation and the factors that contribute to a strong and protective adaptive immune response. T-bet [TBX21] is a transcription factor that plays an instrumental role in the orchestration of the type 1 immune response, which is the specialized response used by the immune system for a cell-mediated response against intracellular pathogens, such as influenza. It has yet to be explored in an influenza setting on the role T-bet in the production of antibodies. The aim of this study is to understand T-bet's role in production of antibody isotypes and identify whether expression of T-bet is more important for antibody production in T cells or B cells. We expected T-bet knockout (KO) mice to have IgG2a and that T-bet expression would be more important in T cells for antibody production. An enzyme-linked immunosorbent assay (ELISA) was used to measure the amount of virus-specific antibody in T-bet KO versus wild type (WT) mice infected with influenza. The results show that the T-bet KO and WT mice have relatively the same amount of IgG and IgG1, but the T-bet KO have a significantly lower level of IgG2a, confirming T-bet's importance for its production. To distinguish the importance of T-bet expression while T-bet expression in T cells was constant, a model was developed to allow us to control expression of T-bet in B cells. The results however were inconclusive, and the experiment will have to be repeated to make a firm conclusion on the roles of lymphocytes in the control of IgG isotypes. Overall, these results indicate that the manipulation of T-bet expression can be used as a vector to control IgG antibody levels, which holds potential for the improvement of vaccines.

Self-reactive CD4+ helper T cells (Th) are key causal agents in the pathogenesis of many autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE) is a CD4+T cell model of the demyelinating autoimmune disease multiple sclerosis (MS). It has been shown that EAE is caused by CD4+ T-cells that produce pro-inflammatory cytokines IFN-γ (Th1) and IL-17 (Th17). As such, understanding how these Th cells are generated and controlled is essential. There is debate as to whether Th1 and Th17 cells act independently in EAE or if there is plasticity between these two subtypes, and whether the capacity to switch from Th1 to Th17 confers pathogenic capacity. T-bet was first described as the master transcription factor for Th1 cells, and is thought to have a critical role in EAE even though IFN-γ, the Th1 archetypal cytokine, has been shown to be redundant. More recent work has shown that T-bet is expressed in multiple immune cell types, and it remains unclear in what cells the expression of T-bet is required for EAE. Considerable efforts have been put into understanding the role of T-bet in EAE pathogenesis, with a view to modulate cells expressing T-bet for therapy. The hypothesis of this work was that T-bet has multifaceted roles in EAE, in initiating and directing an immune response in innate antigen presenting cells such as dendritic cells (DC) as well as programming pathogenic effector CD4+ T cell (Teff) response to antigen. T-bet-/- mice were studied using different models of EAE to dissect the role of T-bet in disease pathogenesis. Active immunisation of C57BL/6 mice with the immunodominant peptide from myelin oligodendrocyte glycoprotein (MOG35-55) showed that T-bet-/- mice developed EAE with an IL-17 dominated profile and critically, T-bet-/- mice were able to produce GM-CSF which has recently been described as a key cytokine for EAE. T-bet-/- cells were not able to transfer EAE in a model of passive transfer EAE, where CD4+ T cells were polarised towards a Th1 profile in vitro. Illustrating that T-bet is required in CD4+ T cells for Th1 mediated EAE. DC driven EAE showed that T-bet-/- DC were able to activate CD4+ T cells in vitro and cause EAE upon co-transfer into host mice with transgenic CD4+ T cells. Thus, it has been shown that T-bet is not required in EAE. This work represents a step further towards understanding the disease mechanisms involved in EAE and suggests T-bet is not an appropriate therapeutic target for the treatment of MS.

By cross-linking B7-DC on dendritic cells (DC) the human IgM antibody (B7-DC XAb) shifts polarized immune responses from Th2 to Th1 in an antigen-specific manner. The molecular determinants governing the ability of DC to reprogram the polarity of T cell recall responses are not yet known. In addition to the expected role of T-bet expressed by T cells in regulating Th1 responses, we find using in vitro assays and an established in vivo model of allergic airway inflammation that T-bet expression by DC is also required for the polarity shift promoted by B7-DC XAb. T-bet expression by both T cells and DC is critically important for B7-DC XAb-induced down-regulation of IL-4, up-regulation of IFN-γ and suppression of allergic airway inflammation. Moreover, retroviral reconstitution of T-bet expression in T-bet-deficient DC rescued their ability to modulate both naive and memory T-cell responses from Th2 to Th1. Our observations further our understanding of the critical mediators controlling the ability of DC to modify the responses of previously activated T cells and reveal the interesting use of the same transcription factor to regulate the inductive phenotype of DC and the inducible phenotype of T cells.

No description available.

Le cancer de la vessie représente un vrai problème de santé publique, avec une surveillance et suivi clinique à long terme en raison de l’importance des fréquences de récidives. La chimiothérapie reste souvent inefficace. L’objectif de cette thèse est donc la recherche de marqueurs sérologiques et moléculaires à valeur pronostique dans le cancer de la vessie qui peuvent servir à prédire la maladie. D’abord, nous avons étudié trois facteurs de transcriptions des lymphocytes T activées qui sont T-bet, GATA-3 et Bcl-6. Nous avons montré une surexpression de T-bet chez les malades à stade invasif et de haut grade, cependant, la surexpression de GATA-3 et Bcl-6 a été corrélée au stade superficiel et de bas grade. La survie a été corrélée avec le groupe des malades sans histoires de récidive ou progression et avec la surexpression de Bcl-6 et GATA-3. Cependant les malades qui expriment fortement T-bet répondent mieux au BCG. Ensuite, nous avons visé la détection de FasL et TRAIL solubles dans le sérum des malades atteints du cancer vésicale. Nous avons montré une surexpression de sFasL et sTRAIL chez les malades à stade superficiel et de bas grade. Le rôle anti-tumoral de ces cytokines a été confirmé sur deux lignées du cancer de la vessie montrant que le traitement avec le sérum riche en sFasL ou en sTRAIL diminue la viabilité cellulaire in vitro. A la fin de cette thèse, nous avons testé l’activation p-Akt dans la tumeur vésicale. Nous avons montré une surexpression de p-Akt au sein des tumeurs comparées au tissu sain adjacent, et au sein des malades à stade invasif et de haut grade. Akt semble être un marqueur de progression tumorale dans le cancer de la vessie. / Bladder cancer is the first most common urogenital cancer in men in Tunisia, with a high recurrence rate. Patients with muscle-invasive disease develop metastasis. The need for expensive continuous surveillance. In this thesis we try to search some candidate biomarkers. Their use for cancer staging and personalization of therapy at the time of diagnosis in order to identify a better treatment could improve patient care. The aim of this first part of our study was to investigate the clinical significance of three immune cell-related transcription factors, T-bet, GATA-3 and Bcl-6 in Tunisian patients with bladder cancer. We found that T-bet level was significantly higher in invasive carcinoma with high- grade. However, T-bet is predictive of response to BCG. On the contrary, the expression of GATA-3 and Bcl-6 was significantly higher in non-invasive carcinoma with low grade. We furthermore studied the effect of activation of soluble FasL and TRAIL molecule in bladder cancer. We demonstrate that the mean serum level of sFasL was higher in patients than in normal donors. sFasL was only higher than in sera of healthy donors where patients had superficial stage and low- and medium-grade cancer. sTRAIL was significantly lower in sera from patients with invasive and high-grade bladder carcinoma than in controls. Finally, we demonstrate that p-Akt levels in patients with invasive carcinoma and high-grade bladder cancer were significantly elevated compared to patients with non-invasive and low grade bladder cancer. Altogether, our results suggest that Akt activation can provide useful prognostic information.

Tick-borne encephalitis virus is the cause of much morbidity and sometimes a fatal infection. A vaccine based on formaldehyde inactivated virus is currently the only available way of preventing disease. This vaccine gives a high rate of seroconversion but there are reports of vaccination breakthrough, even in people who have demonstrated a neutralizing antibody response. The T cell response to inactivated TBE vaccine is largely unknown, but could be of importance for the effect of the vaccine. This study characterizes aspects of the T cell response by investigating the expression of two transcription factors, T-bet and GATA-3 with RT-PCR. T-bet is expressed in CD4+ T cells of the Th1 type, while GATA-3 is expressed in CD4+ T cells of the Th2 type. Our data show that vaccination with inactivated TBE vaccine leads to increase in expression of the T-bet gene when cells of vaccinated subjects are cultured with TBE virus. In contrast, the expression of GATA-3 remains unaffected by vaccination. Thus, this study suggests that the inactivated TBE vaccine leads to a Th1-like immune response in humans.

Hepatitis C virus (HCV) induces a high rate of chronic infection via dysregulation of host immunity. We have previously shown that T-cell immunoglobulin and mucin domain protein-3 (Tim-3) is up-regulated on monocyte/macrophages (M/Mφ) during chronic HCV infection; little is known, however, about the transcription factor that controls its expression in these cells. In this study, we investigated the role of transcription factor, T-box expressed in T cells (T-bet), in Tim-3 expression in M/Mφ in the setting of HCV infection. We demonstrate that T-bet is constitutively expressed in resting CD14+ M/Mφ in the peripheral blood. M/Mφ from chronically HCV-infected individuals exhibit a significant increase in T-bet expression that positively correlates with an increased level of Tim-3 expression. Up-regulation of T-bet is also observed in CD14+ M/Mφ incubated with HCV+ Huh7.5 cells, as well as in primary M/Mφ or monocytic THP-1 cells exposed to HCV core protein in vitro, which is reversible by blocking HCV core/gC1qR interactions. Moreover, the HCV core-induced up-regulation of T-bet and Tim-3 expression in M/Mφ can be abrogated by incubating the cells with SP600125 – an inhibitor for the c-Jun N-terminal kinase (JNK) signalling pathway. Importantly, silencing T-bet gene expression decreases Tim-3 expression and enhances interleukin-12 secretion as well as signal transducer and activator of transcription 1 phosphorylation. These data suggest that T-bet, induced by the HCV core/gC1qR interaction, enhances Tim-3 expression via the JNK pathway, leading to dampened M/Mφ function during HCV infection. These findings reveal a novel mechanism for Tim-3 regulation via T-bet during HCV infection, providing new targets to combat this global epidemic viral disease.

Differenzierungsentscheidungen von Zellen werden durch die Integration mehrerer Stimuli bestimmt. Die Differenzierung von Helfer-T-Zellen (Th-Zellen) ist hierfür ein gut untersuchtes Beispiel: reife Th-Zellen entwickeln sich beim Kontakt mit einem für sie spezifischen Antigen zu einem spezialisierten Subtyp, der von den in ihrer Umgebung vorhandenen Zytokinen abhängt und exprimieren dann einen spezifischen Mastertranskriptionsfaktor. Die häufigsten Th-Zell-Subtypen sind T-bet-exprimierende Th1-Zellen und GATA-3-exprimierende Th2-Zellen. Neuere Entdeckungen bezüglich der Plastizität von Th-Zell-Subtypen sowie die Existenz von T-bet+GATA-3+ Hybrid-Phänotypen haben die detaillierte Untersuchung vom Differenzierungsprozessen von Th-Zellen mit komplexer Zytokinsignale motiviert. Dazu haben wir systematisch die Zytokine IFN-g, IL-12 und IL-4 während der primären Differenzierung Th-Zellen titriert und Signaltransduktion und Zielgenexpression quantifiziert. Der Umfang und die Komplexität der Daten machten eine systematische Analyse notwendig, um involvierte Mechanismen genau zu identifizieren. Lineare Regressionsanalyse wurde verwendet, um die Netzwerktopologie zu extrahieren, wobei schon bekannte und zahlreiche neue Interaktionen vorausgesagt wurden. Die prognostizierte Netzwerktopologie wurde dann verwendet, um ein mechanistisches, mathematisches Modell der Zytokinsignalintegration zu entwickeln. Diese Methode hat ein hochgradig vernetztes regulatorisches Netzwerk inferiert. Bisher nicht beschriebene Funktionen von STAT-Proteine, die die Neuverkabelung des Netzwerkes während der Differenzierung vermitteln, wurden vorhergesagt. Ausgewählte neue Interaktionen wurden in gezielten genetischen Experimenten bestätigt. Während gegenseitige Inhibitionsmotive oft als kanonische digitale Schalter interpretiert werden, funktioniert das Th-Zell-Netwerk als ein Rheostat, der Variationen der Zytokinsignale in graduelle Expressionsänderungen der Mastertranskriptionsfaktoren übersetzt. Unsere Arbeit erklärt mechanistisch das beobachtete Kontinuum von Th-Zelldifferenzierungszuständen entlang der Th1-Th2-Achse und beschreibt eine quantitative Methode für die datenbasierte Inferenz zellulärer Netzwerke der Signalintegration. / Cell-fate decisions are governed by the integration of multiple stimuli. Th cell differentiation is a well-studied example thereof: mature Th cells differentiate into a specialised subtype upon encounter with their cognate antigen depending on the polarising cytokines present in their environment and start expressing specific master transcription factors. The most common Th cell subtypes are T-bet-expressing Th1 cells and GATA-3-expressing Th2 cells. Recent discoveries concerning the plasticity of Th cell subtypes as well as the existence of stable T-bet+GATA-3+ hybrid Th1/2 phenotypes have stimulated the detailed study of the differentiation process under different assumptions than the hitherto valid paradigm of single master transcription factor expression by using complex cytokine signals as inputs. Here, we developed a data-based approach for inferring the molecular network underlying the differentiation of T-bet- and/or GATA-3 expressing lymphocytes. We performed systematic titrations of the polarising cytokines IFN-g, IL-12 and IL-4 during primary differentiation of Th cells and quantified signal transduction as well as target-gene expression. The size and complexity of the dataset made a systematic analysis necessary to identify the mechanisms involved. To extract the network topology, we used linear regression analysis, retrieving known regulatory mechanisms and predicting numerous novel ones. This network topology was used to develop a mechanistic mathematical model of cytokine signal integration. This approach inferred a highly connected regulatory network. Previously undescribed functions of STAT proteins mediating network rewiring during differentiation were predicted. Selected new interactions were confirmed by experiments using gene-deficient cells. Importantly, while mutual-inhibition motifs are often considered canonical digital switches, the inferred Th-cell network acts as a rheostat, generating a continuum of differentiated states along the Th1-Th2 axis. This work explains the observed Th1-Th2 cell fate continuum mechanistically and provides a quantitative framework for the data-based inference of cellular signal integration networks.

Le facteur de transcription T-bet joue un rôle central dans la régulation de la différenciation des lymphocytes T. La protéine tyrosine kinase, la glycogène synthase kinase 3 (GSK-3), inhibe l'activation des lymphocytes T et contrôle l'expression de leurs récepteurs inhibiteurs PD-1 et LAG- 3. Bien que l'inhibition de GSK-3 puisse augmenter l'expression de T-bet, l'interrelation entre T-bet et GSK-3 dans l'immunité tumorale est inconnue. Dans cette étude, nous montrons que les souris knock-out T-bet (Tbet - / -) sont compromises dans leur capacité à contrôler la croissance des cellules tumorales du mélanome B16. Cependant, l'injection d'une petite molécule inhibitrice (SMI) de GSK-3 inverse cette condition compromise entraînant le contrôle de la croissance tumorale similaire à celle observée chez les souris de type sauvage. Un examen de Tbet - / - a montré une perte de cellules dendritiques (DC) et de cellules leucocytes polymorphonucléaires (PMN) potentiellement suppressives et de lymphocytes tumoraux T (TILs) CD4 + accompagnée d'une augmentation de cellules T CD8 +. L'analyse viSNE (avancé tSNE) a en outre montré une réduction de la population effectrice expérimentée à l'antigène dans les TILs CD8 + chez Tbet -/-. Cette population est marquée par la réduction de CD44. L'inhibition de GSK-3 n'a montré aucun effet sur la perte de DC, TILs CD4 +, PMN et les TILs CD8 + ainsi que l’expression de Granzyme B (GZMB) sur les cellules T CD8 +. La seule exception était une augmentation mineure néanmoins statistiquement significative du facteur de transcription Eomesdermin (Eomes) dans les TILs CD8 +. L'étude démontre un effet compensatoire inattendu de l'inhibition de GSK-3 sur la perte de T-bet. Il reste à élucider la nature complète du parcours de cette compensation. / The transcription factor T-bet plays a central role in regulating T-cell differentiation, while the protein tyrosine kinase, glycogen synthase kinase 3 (GSK-3) inhibits T-cell activation and controls the expression of inhibitory receptors PD-1 and LAG-3 on T-cells. Although GSK-3 inhibition can increase T-bet expression, the inter-relationship between T-bet and GSK-3 in tumor immunity is unknown. In this study, we show that T-bet knock-out (Tbet-/-) mice are compromised in their ability to control the growth of the B16 melanoma tumor cells. However, the injection of a small molecule inhibitor (SMI) of GSK-3 reverses this compromised condition resulting in the control of tumor growth similar to that seen in wild type mice. An examination of Tbet-/- showed a loss of dendritic cells (DC) and potentially suppressive polymorphonuclear leucocytes (PMN) and CD4+ cell tumor infiltrating lymphocytes (TILs) accompanied by an increase in CD8+ cells. viSNE analysis (advanced tSNE- t-Distributed Stochastic Neighbor Embedding) further showed a reduction of antigen experienced effector marker CD44 in CD8+ TILs in Tbet-/-. GSK-3 inhibition showed no effect on the loss of DCs, CD4+ TILs or the presence of PMNs or CD8+ T-cells or the loss of Granzyme B (GZMB) on CD8+ cells. The one exception was a minor but statistically significant increase in the transcription factor Eomesodermin (Eomes) in CD8+ TILs. The study demonstrates an unexpected compensatory effect of GSK-3 inhibition on the loss of T-bet. The full nature of the pathway that accounts for this compensation remains to be elucidated.