Os avanços científicos das últimas décadas permitiram que a compreensão do câncer evoluísse de uma visão simplista, na qual o principal motor seria uma atividade celular hiperploriferativa, para uma visão mais complexa onde o estado fisiológico geral permite a gênese e progressão tumoral. Essa evolução permite o desenvolvimento de novas abordagens terapêuticas e traz novas esperanças para o tratamento de muitos tipos de cânceres ainda extremamente deletérios. Dentro desse novo panorama, terapias que estimulem a imunidade antitumoral têm se mostrado extremamente promissoras. Nesse trabalho, procuramos investigar os efeitos antitumorais desencadeados pela combinação da indução de morte celular e imunoestimulação. Para tanto, visamos à recuperação da via de p53 (pela transferência gênica do próprio p53 ou p19) associada à transferência gênica de IFNbeta. A transferência gênica foi mediada por vetores adenovirais do sorotipo 5. Nossas observações, em um modelo murino de carcinoma pulmonar, permitem concluir que esta linhagem é sensível a morte induzida pela transferência gênica de p19 e não p53. Porém, a transferência gênica intratumoral de IFNbeta se mostrou chave no controle do crescimento do tumor primário. Destacamos, entretanto, que a associação de IFNbeta com p19 produziu efeitos imunoprotetores superiores à transferência de IFNbeta ou p19 sozinhos. Tal efeito parece ser dependente da indução de fatores quimiotáxicos e conseqüente recrutamento de neutrófilos para o sítio tumoral. O efeito da transferência gênica combinada de ambos os genes IFNbeta e p19 se mostrou ainda mais promissor quando associado à cisplatina, induzindo uma notável redução no crescimento tumoral / Scientific advances from the last decades enabled the evolution of our knowledge of cancer from a simplistic vision, in which the main motor was an excessive cell proliferation, to a more complex one, where the general physiologic state enables tumorigenesis and tumor progression. This evolution enabled the development of new therapies and brings new hopes for the treatment of several types of cancers. In this context, therapies that induce an antitumor immunity are very promising. In this work, we are investigating the antitumor effects triggered by the combination of cell death induction and immunostimulation. To this end, we aimed to restore p53 pathway (by p53 or p19 gene transfer) associated with immunostimulation by IFNbeta gene transfer. The gene transfer was mediated by Adenovectors Serotype 5. Our observations in a murine model of lung cancer showed that this cell line is sensitive to cell death induced by p19 gene transfer, but not p53. Nevertheless, intratumoral gene transfer of IFNbeta, was crucial in controlling tumor growth. Moreover, p19 and IFNbeta association induced higher immunoprotecting effects than p19 or IFNbeta alone. This effect seems to be depending on the induction of chemotactic factors, and the recruitment of neutrophils to the tumor site. The effect of combined gene transfer of p19 and IFNbeta was even more promising when associated with Cisplatine, inducing a remarkable reduction in tumor growth

Philosophiae Doctor - PhD / Tuberculosis (TB) is an infectious disease that, despite all efforts devoted towards its eradication, remains a threat to many countries including South Africa. Current diagnostic assays do offer better performance than the conventional sputum smear microscopy and tuberculin skin tests. However, these assays have been proven to be affected by various factors including the condition of an individual's immune system and vaccination history. By far, electrochemical biosensors are amongst the currently investigated techniques to address the shortcomings associated with these diagnostics. / 2020-08-31

Les Cellules Dendritiques sont un groupe hétérogène de cellules présentatrices d’antigènes, importantes pour le contrôle des réponses innées et adaptatives. Les Cellules Dendritiques Plasmacytoïdes (pCD) en représentent une population unique, aux caractéristiques phénotypiques et fonctionnelles particulières, notamment par leur capacité à produire de grande quantité d’Interféron de type I (IFN). Cette signature IFN marque la physiopathologie du Lupus Erythémateux Systémique (LES), maladie auto-immune systémique. Les mécanismes à l’origine de cette production excessive d’IFN par les pCD restent incomplètement élucidés. Nous montrons, dans notre étude, chez l’homme comme dans un modèle murin que les plaquettes, activées dans le LES, participent à la production d’IFN via le CD40L. Cette production en excès d’IFN, a pour conséquence une maturation et activation d’autres Cellules Dendritiques (CD) entrainant l’activation inappropriée des lymphocytes T. Chez le sujet sain, cette activation inappropriée du système immunitaire adaptatif doit être strictement contrôlée afin d’assurer l’homéostasie du système immunitaire. Il a été montré précédemment que de nombreux lymphocytes aux caractéristiques phénotypiques de type mémoire-effecteur (TEM) peuplent les tissus périphériques, notamment le tissu cutané. Ces TEM sont capables de s’activer et proliférer localement en réponse à un stimulus. Les Cellules de Langerhans (LC) sont des cellules dendritiques résidant au niveau cutané dans l’épiderme. Leur fonction est à ce jour l’objet d’une controverse entre une fonction immuno-stimulante (modèle humain) et une fonction immuno-régulatrice (modèle murin). Nous démontrons dans cette étude que les LC, à l’état basal, chez l’homme, induisent la prolifération de Lymphocytes T régulateurs (Treg) au niveau cutané, capables de bloquer la stimulation inappropriée des TEM cutanés. Cependant en présence d’un stimulus infectieux, les LC induisent préférentiellement la prolifération des TEM en limitant celle des Treg. Les LC semblent être à la fois immuno-régulatrices ou stimulantes en fonction du contexte biologique auquel elles sont confrontées. / Dendritic Cell (DC) are a heterogeneous group of antigen-presenting leukocytes that are important in activation of both the innate and adaptative arms of the immune system. Plasmacytoid Dendritic Cells (pDC) represent a unique population, characterized by their ability to produce large amounts of type I Interferon (IFN). This « IFN signature » is a prominent feature of Systemic Lupus disease (SLE). Mechanisms leading to the excessive production of type I IFN remain largely unknown. Here, in our present study, we demonstrate that platelets are activated in SLE patients by circulating immune complexes and represent a major reservoir of CD40L. Activated platelets potentiate the production of type I IFN by pCD through a CD40L/CD40 interaction. Excessive production of type I IFN by pCD leads to DC activation and maturation and inappropriate activation of auto-reactive T cells.Under steady state condition, inappropriate activation of the immune system must be tightly controlled. It has been previously shown that normal adult human skin contains a large number of resident T cells (TRM) expressing the phenotype of Effector Memory T cells (TEM). These TEMTRM are specific for antigens previously encountered through skin and can be activated and proliferate under specific stimulation. Langerhans Cells (LC) are a group of skin resident DC living in epidermis. There is currently substantial controversy regarding the physiologic role of LC with regard to immunoregulation versus immunostimulation. Here we show that under steady state condition, LC induce the proliferation of a small subset of TRM. These proliferating TRM express the phenotype of TREG and are functional. However this stimulation of TREG could be reversed in the presence of foreign antigen in a dose-dependant fashion, as the addition of a pathogen to LC and TRM led to diminished TREG proliferation and increased TEM proliferation. These findings establish a novel immunological role for LC in human skin, allowing for the constitutive maintenance of tolerance, while also permitting the stimulation of resident immune memory in response to infectious challenge

Le but de ce travail a été d’étudier l’implication de la réponse immunitaire innée au cours du lupus érythémateux systémique (LES) et de la polyarthrite rhumatoïde (PR). Dans le LES humain, nous montrons que les plaquettes sont activées par les complexes immuns circulants (CICs) et que le CD154 dérivé des plaquettes activées participe avec les CICs à la sécrétion aberrante par les cellules dendritiques d’interféron alpha, cytokine fondamentale dans la physiopathologie du LES. Dans deux modèles murins nous démontrons qu’inhiber l’activation plaquettaire améliore significativement les paramètres lupiques et la survie suggérant que l’activation plaquettaire pourrait constituer une nouvelle cible thérapeutique. Les cytokines inflammatoires tiennent un rôle central dans la physiopathologie de la PR mais les déterminants de leur production restent mal connus. Des polymorphismes du facteur de transcription interferon regulatory factor 5 (IRF5) sont associés au risque de développer une PR. Dans un modèle murin, nous montrons que l’inactivation du gène codant pour IRF5 améliore la maladie. IRF5 étant impliqué dans la signalisation des TLRs, nous mettons alors en évidence in vivo que l’inactivation des voies de signalisation des Toll-like récepteurs (TLRs) TLR3 et TLR7, récepteurs pour les ARN, améliore la maladie. Nous démontrons in vitro un effet synergique dépendant d’IRF5 de ces TLRs pour la production de cytokines inflammatoires. Enfin les souris invalidées pour la signalisation combinée des TLR3 et TLR7 sont spectaculairement épargnées par la maladie suggérant ainsi un rôle clé pour les ligands ARN endogènes dans la pathogénie de la PR. Ces données démontrent l’importance des mécanismes immunitaires innés dans la physiopathologie des maladies auto-immunes systémiques, apportant ainsi de nouvelles voies de recherche thérapeutiques. / The aim of the present sudy was to investigate the involvement of the innate immune system in the pathogenesis of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), two models of systemic autoimmune diseases. We show that in SLE patients, platelets are activated by circulating immune complexes. Activated platelet derived CD154 enhances interferon alpha production by immune complex-stimulated dendritic cells in a CD154/CD40 dependent manner. In lupus prone mice, we show that targeting platelet activation improves all measures of disease and overall survival suggesting that this process may provide a new therapeutic target. Proinflammatory cytokines play a central role in the pathogenesis of RA. Gain-of-function polymorphisms in the transcription factor interferon regulatory factor 5 (IRF5) are associated with an increased of developping RA. We now show that IRF5 deficiency substantially reduces disease in a mouse model of RA. As IRF5 participates in TLR signaling, we evaluated TLR requirements in this model and found a significant réduction in disease severity in mice deficient in signaling through the RNA-sensing TLR3 and TLR7. In vitro studies show that TLR3 and TLR7 synergize for proinflammatory cytokine production and that this synergy is markedly diminished in the absence of IRF5. Notably, mice with combined deficiency of TLR3 and TLR7 signaling developed minimal disease indicating an important role for endogenous RNA ligands in disease pathogenesis. Our results bring new elements in the comprehension of the role of innate immunity in the pathogenesis of systemic autoimmune diseases and provide new therapeutic targets.

Après 30 années de recherche, de nombreux obstacles s'opposent encore à la conception d'un vaccin contre le SIDA. En effet, il n'existe pas de consensus sur les corrélats immunitaires de protection qu'il devra induire ni sur les mécanismes à l'origine de la progression vers le SIDA chez les individus infectés. Dans un premier temps, nous avons cherché à concevoir un virus hybride structuralement semblable au VIH-1 et capable de se répliquer exclusivement dans le cytoplasme des cellules infectées. Dans cet objectif, nous avons développé des nouveaux vecteurs bi et tri-cistroniques dérivés du poliovirus et contenant les séquences des gènes gag et/ou env du VIH-1. Nous avons montré que ces réplicons permettaient l'expression des protéines structurales du VIH-1 sous leur forme mature. Dans un second temps, nous avons mis en évidence une corrélation indiquant que, plus la composition nucléotidique (% A/T/G/C) d'un lentivirus diverge de celle de son hôte, plus la probabilité qu'il soit pathogène est élevée. Nous avons montré que l'optimisation artificielle de la composition nucléotidique de séquences d'ARN lentivirales diminuait leur capacité d'induction d'interféron (IFN-I) après transfection. Nous avons ensuite synthétisé un virus de l'immunodéficience simienne (VIS) dont la séquence a été artificiellement optimisée à la composition nucléotidique moyenne du macaque. Ce virus présente une capacité d'induction d'IFN-I in vitro réduite par rapport au VIS sauvage. Ces données indiquent pour la première fois un lien entre la composition nucléotidique du génome des lentivirus et la progression vers le SIDA. Elles suggèrent de nouvelles stratégies vaccinales d'atténuation du VIH-1. / After over thirty years of AIDS epidemic, we still need to identify immunological correlates of protection against AIDS and we do not properly understand how HIV causes AIDS in infected individuals. In order to reproduce the protective capacity of live attenuated viruses, we first aimed at generating a hybrid virus structurally similar to HIV-1 and able to replicate exclusively in the cytoplasm of infected cells. We developed new polioviral pluricistronic vectors that contain HIV-1 packaging sequences, gag gene and/or env gene. We then showed that the use of these replicons was compatible with the production of processed and mature HIV structural proteins. Secondly, we investigated the consequences of the lentivirus nucleotide composition (% A/T/G/C) bias on their pathogenicity. We found a correlation, indicating that AIDS results from infection by primate lentiviruses having the most divergent nucleotide composition compared to their hosts, whereas less divergent lentiviruses cause non-pathogenic infections. A strong type I interferon (IFN-I) response during the chronic phase of infection is a typical feature of lentiviral pathogenic infection. We showed that nucleotide optimization of lentiviral RNA sequences dramatically reduce their in vitro capacity to induce IFN-I. We synthesized a simian immunodeficiency virus (SIV), whose genome sequence was artificially optimized to the macaque average nucleotide composition. This virus showed a reduced capacity to stimulate IFN-I in vitro than wt SIV. These data indicate for the first time a link between the nucleotide composition of lentiviruses and their pathogenicity. They suggest new vaccine attenuation strategies against AIDS.

Transcriptional deregulation has emerged as one of the leading causes in various human diseases. More than fifty percent of cancers arise due to frequent mutations in genes regulating transcription. Higher-order assembly via protein-protein interactions is one common mechanism of transcriptional regulation. Despite their critical role in regulating gene transcription and therapeutic relevance, detailed mechanistic understanding of these assemblies remains scarce. The primary focus of this thesis is to uncover important structural principles underlying the assembly and stability of multi-domain protein assemblies by characterizing components of the IFNβ enhanceosome and the CtBP-mediated repression complex. Using a combination of biochemical and structural analyses, I showed that the transcriptional activator C-terminal binding protein 2 (CtBP2) is a tetramer by solving the 3.6Å cryoEM structure of CtBP2. I highlighted the types of interactions that stabilize the homo-tetramer and showed the relevance of the tetramer in CtBP2 transcriptional activity. Second, I used an integrative approach to investigate the structural features leading to activation of interferon regulator factor 3 (IRF3) and its interaction with DNA. Although this work mostly focused on components of the CtBP2-mediated complex and IFNβ enhanceosome, the principles described here can be applied to other complexes. Therefore, these studies provide an overall understanding on how other macromolecular complexes regulate gene transcription. Furthermore, our structural-based analyses will provide a basis for designing drugs that can regulate gene transcription in cancer and immunological disorders.

Jak3, a member of the Janus family of tyrosine kinases, is essential for signaling via the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. These Jak3-dependent cytokines primarily activate STAT5 and are critical for lymphoid generation and differentiation. Using naïve CD4+ T cells from Jak3-deficient mice and wild type CD4+ T cells treated with a pharmacological inhibitor of Jak3, we report that Jak3-dependent cytokine signals are not required for the proliferation of naïve CD4+ T cells. This is illustrated by the similar percentage of divided cells, comparable cell divisions, intact cell cycle progression and unaffected regulation of cell cycle proteins in the absence of Jak3. In contrast to proliferation, differentiation of naïve CD4+ T cells into Th1 effector cells requires Jak3-dependent cytokine signals. In the absence of Jak3, naïve CD4+ T cells proliferate robustly, but produce little IFN-γ after Th1 polarization in vitro. This defect is not due to reduced activation of STAT1 or STAT4, nor to impaired up-regulation of the transcription factor T-bet. Instead, we find that T-bet binding to the Ifng promoter is greatly diminished in the absence of Jak3-dependent signals, correlating with a decrease in Ifng promoter accessibility and histone acetylation. These data indicate that while Jak3-dependent signals are dispensable for naïve CD4+ T cell proliferation, Jak3 regulates epigenetic modification and chromatin remodeling of the Ifng locus during Th1 differentiation.

Contexte : La sclérose en plaques (SP) est une maladie chronique pouvant engendrer de nombreuses conséquences sur la qualité de vie des patients. Objectifs : L’objectif général de cette thèse est la modélisation des aspects temporels de la SP et de son traitement. Les objectifs spécifiques découlant de cette thèse sont : 1) Estimer l’effet à long terme des médicaments immunomodulateurs sur la progression et l’activité de la SP;  2) Décrire, au niveau populationnel, l’évolution de l’utilisation des soins associés à la SP et estimer l’effet de l’âge au diagnostic sur l’utilisation des soins associés à la SP ;  3) Évaluer la capacité de la méthode de trajectoire basée sur les groupes à détecter le nombre de trajectoires, la forme des trajectoires et le nombre de sujets dans chaque classe de trajectoire; 4) Proposer une nouvelle méthode pour visualiser les trajectoires longitudinales de dose et appliquer cette méthode pour un immunomodulateur utilisé dans le traitement de la SP. Méthodes : Les analyses pour les objectifs 1 et 4 ont été effectuées à partir des données de patients ayant eu un diagnostic de SP et traités à la clinique de SP du Centre hospitalier de l’Université de Montréal (CHUM). L’étude a été constituée à partir des informations colligées lors du suivi clinique des patients entre septembre 1977 et décembre 2016. Les caractéristiques socio-démographiques, l’histoire reproductive chez les femmes, la progression de la maladie, les comorbidités et médicaments ont été colligés à chaque visite médicale. Pour l’objectif 2, la cohorte de naissance québécoise sur l’immunité et la santé a été utilisée. Les individus faisant partie de cette cohorte sont nés entre 1970 et 1974 et le suivi a été réalisé jusqu’en 2014 en utilisant les bases de données médico-administratives et démographiques. Les informations sociodémographiques et celles concernant l’utilisation des services de santé pour la SP ont été obtenues pour chaque individu. Pour l’objectif 3, les données ont été générées en utilisant des scénarios basés sur la littérature. Les analyses effectuées incluent l’utilisation des modèles structurels marginaux (objectif 1), des modèles additifs généralisés (objectif 2) et des méthodes de trajectoires basées sur les groupes (objectifs 3 et 4). Résultats : Les résultats ont montré que l’utilisation de l’interféron bêta et de l’acétate de glatiramère est associée à une diminution du taux de progression d’incapacités et des poussées. Nous avons également constaté certaines différences au niveau des taux de visites chez un neurologue et chez un omnipraticien entre les personnes diagnostiquées avec la SP avant l’âge de 29 ans et celles diagnostiquées plus tard. De plus, nous avons montré l’importance d’utiliser plusieurs critères statistiques afin d’évaluer l’adéquation des groupes de trajectoires. Conclusions : L’utilisation de plusieurs variables représentant les issues dans les deux premiers objectifs nous a permis d’avoir une vue globale du traitement, des soins et de la progression de la SP. Cette thèse fait également des recommandations pour une bonne utilisation des méthodes de trajectoires et propose une alternative graphique pour visualiser les trajectoires de dose de médicaments. / Background: Multiple sclerosis (MS) is a chronic disease that can impact on the quality of life of patients in multiple ways. Objectives: The main goal of this thesis is the modelling of the temporal aspects of MS and its treatment. The specific goals of the thesis are: 1) To estimate the long-term effect of immunomodulatory drugs on the progression and the disease activity of MS; 2) To describe, at the population level, the evolution of health services related to MS and to estimate the effect of age at diagnosis on the use of health services for MS; 3) To assess the propensity of group-based trajectory modelling to detect the number of trajectories, the shape of trajectories and the number of subjects in each group of trajectories; 4) To propose a new graphical representation to visualize longitudinal patterns of medication dose and apply it to an immunomodulator used for treating MS patients. Methods: The analysis of objectives 1 and 4 were carried out using data from patients with a diagnosis of MS treated at the Montreal MS clinic, CHUM. The study was based on information collected during the clinical follow-up of patients between September 1975 and December 2016. The socio-demographic characteristics, reproductive history in women, disease progression, comorbidities and medications were collected at each medical visit. For objective 2, the Québec Birth Cohort on Immunity and Health was used. The individuals in this cohort were born between 1970 and 1974 and follow-up was carried out until 2014 using administrative health and demographic databases. Socio-demographic variables and information regarding the use of health services for MS were obtained for everyone. For objective 3, data were generated based on scenario from the literature. The analyses carried out include the use of marginal structural models (objective 1), generalized additive models (objective 2) and group-based trajectory methods (objectives 3 and 4). Results: The results showed that the use of interferon-beta and glatiramer acetate is associated with a decrease in the rate of progression of disability and relapses. We also found some differences in neurologist and general practitioner visit rates between people diagnosed with MS before age 29 and those diagnosed later. In addition, we have shown the importance of using several statistical criteria in order to assess the adequacy of groups of trajectories. Conclusions: The use of several variables for the outcomes in the first two objectives gave us a holistic view of the treatment, care and progression of MS. This thesis also makes recommendations for the good use of the trajectory modelling and proposes a graphic alternative to visualize patterns of medication dose.

Human cytomegalovirus (HCMV, Herpesviridae) can cause severe complications in the infected individuals undergoing hematopoietic stem cell transplantation. Nowadays, these patients are treated using antivirotics or HCMV-specific T cells derived from the seropositive graft donor. This study explored the possibility of redirecting HCMV-non-specific T cells from a seronegative donor towards HCMV-infected cells via chimeric antigen receptor (CAR), i.e. artificially designed T cell receptor. Viral glycoprotein B (gB) has been selected as a target for this receptor. Published sequence of a single chain variable fragment of a human antibody was used for the design of the CAR against gB (gBCAR). After the verification of production and surface localization in cell lines, gBCAR was being introduced into human T cells via lentiviral vectors. Human fetal lung fibroblasts (LEP) infected with HCMV were used as target cells after the expression of gB at their surface was demonstrated. gBCAR functionality was evaluated by the incubation of modified T cells with infected cells and subsequent analysis of media for IFNγ concentration, which was significantly higher in the setting of gBCAR T cells incubated with HCMV-LEP than in the control incubations. The results obtained show the specificity of gBCAR against...

Human immunodeficiency virus type 1 (HIV-1) is a lentivirus that causes acquired immunodeficiency syndrome (AIDS). Since the first cases of AIDS were described in 1981, HIV-1 has become one of the most serious public health threats in the world. There are approximately 38 million people worldwide currently living with HIV-1. 28 million of these people have access to antiretroviral therapy (ART) that is highly effective in reducing viral load to undetectable levels, thereby curbing the risk of viral transmission and preventing progression to AIDS. Despite their effectiveness in suppressing HIV-1 viremia, systemic inflammation remains as a hallmark of HIV-1 infection in vivo. This persistent immune activation is often associated with non-AIDS related complications, including elevated risk of neurocognitive and cardiovascular disorders. Several different mechanisms may contribute to this chronic immune activation and inflammation in people living with HIV-1 on ART. One of the contributing factors might be HIV-1 RNA expressed from the provirus. Even though ART potently suppresses HIV-1 replication, it fails to eradicate proviruses established prior to initiation of ART. Ongoing activation of CD4+ T cells and macrophages by HIV-1 proviral transcripts might contribute to the persistent inflammation that remains even after HIV-1 suppression by ART. Previously, our laboratory has shown that induction of innate immune signaling after HIV-1 challenge of primary human dendritic cells (DCs), macrophages, or CD4+ T cells requires integration, transcription from the nascent provirus, and nuclear export of intron-containing HIV-1 RNA through the Rev-CRM1 pathway. However, these studies failed to identify the innate immune sensor of intron-containing HIV-1 RNA. Here we conducted a targeted loss-of-function screen, using shRNA-expressing lentivectors in human DCs to identify this innate immune receptor. Of the twenty-one candidate genes targeted for knockdown by shRNA, the innate immune response to HIV-1 was inhibited only by knockdown of IFIH1, MAVS, and XPO1. The effect of IFIH1 and MAVS knockdowns on HIV-1-induced immune activation was confirmed in macrophages, and rescue of the knockdown with non-targetable coding sequence showed that IFIH1 protein was required. IFIH1 mutants that are defective for interaction with MAVS blocked activation, demonstrating that MAVS acts downstream of IFIH1 in this system. Since both IFIH1 and DDX58 signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation; the IFIH1-specific inhibitor Nipah virus V protein blocked the activation by HIV-1. RNA-Seq showed that IFIH1-knockdown in DCs globally disrupted the induction of IFN-1-stimulated genes. Altogether, results presented in this thesis reveal that IFIH1 is required for innate immune activation by intron-containing RNA from the HIV-1 provirus, and potentially contributes to chronic inflammation in people living with HIV-1.