Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Le syndrome de Gougerot-Sjögren (SGS) est une maladie auto-immune systémique. Il se caractérise principalement par une infiltration lymphocytaire des glandes salivaires (GS) et lacrymales responsable d’une sécheresse buccale et oculaire. Par ailleurs,les Toll-like récepteurs (TLR) endosomaux – notamment le TLR9 qui reconnait l’acide désoxyribonucléique (ADN) microbien mais aussi, dans certaines conditions, l’ADN du soi –s’avèrent être importants pour l’activation des lymphocytes B (LB) lors du lupus, une maladie proche du SGS. Nos travaux montrent que la stimulation du TLR9 chez les LB transitionnels,des LB immatures fraichement émigrés de la moelle osseuse, favorise leur différenciation selon la voie des LB de la zone marginale, et entraine la sécrétion d’auto-anticorps. L’analyse des LB infiltrant les GS lors du SGS révèle que ce phénomène pourrait être impliqué dans la physiopathologie de cette maladie. De plus, nous montrons que LL37, un peptide produit dans les GS, pourrait participer à l’activation du TLR9 des LB transitionnels. Enfin, nous avons mis en évidence une inattendue expression du TLR9 à la surface des LB. Si l’étude des conséquences fonctionnelles de cette localisation reste à poursuivre, elle semble avoir un effet négatif sur la stimulation du TLR9 endosomal. En conclusion, ces résultats suggèrent que leTLR9 puisse être une nouvelle cible thérapeutique lors du SGS. / Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease. It is mainly characterized by B cell and T cell infiltration in lacrimal and salivary glands (SG) responsible for eye and mouth dryness. In addition, endosomal Toll-like receptors (TLR) – especially TLR9 which recognizes microbial deoxyribonucleic acid (DNA) and also, under certain conditions, self DNA – are important for B cell activation during lupus, a disease close topSS. Our work shows that TLR9 stimulation on transitional B cells, immature B cells freshly emigrated from bone marrow, promotes their differentiation into marginal zone B cell pathway and drives to auto-antibodies production. Analysis of infiltrating B cells in pSS SG reveals that this phenomenon might be involved in the pathogenesis of the disease.Furthermore, we show that LL37, a peptide produced in the SG, could participate in TLR9activation of transitional B cells. Finally, we demonstrated an unexpected TLR9 expression on B cell surface. If the functional consequences of this localization remain to be more precisely evaluated, it seems that cell surface TLR9 has a negative effect on endosomal TLR9 stimulation. In conclusion, these results suggest TLR9 could be a new therapeutic target incase of pSS.

Le Syndrome Néphrotique à Lésions Glomérulaires Minimes (SNLGM) et la glomérulonéphrite extra membraneuse (GEM) sont deux podocytopathies primitives d'origine immunitaire associant des altérations immunes et des atteintes podocytaires à l'origine d'un syndrome néphrotique. Cependant, bien que l'origine dysimmunitaire soit confortée par de nombreux arguments cliniques, les mécanismes impliqués restent obscurs. Initialement identifié dans les lymphocytes T (LT) de patients en phase de poussée de SNLGM, le gène c-mip est également exprimé dans les podocytes de patients atteints de SNLGM et de GEM, alors qu'il est physiologiquement réprimé. Ainsi, les objectifs de ce travail étaient : 1) appréhender le rôle de c-mip dans le LT d'une part à travers l'étude d'un modèle murin transgénique (Tg), et 2) comprendre la fonction de c-mip au niveau du podocyte grâce au modèle expérimental de GEM humaine induit chez le rat.Le modèle murin Tg Lck-cmip surexprime spécifiquement c-mip dans les LT matures périphériques. Cette surexpression est à l'origine d'un phénotype lymphocytaire altéré marqué par une accumulation de LT naïf, et une inhibition de la synthèse de cytokines de type TH1 et TH2, après une activation T spécifique ex vivo. Cette régulation négative est associée à une accumulation des formes inactives des kinases de la famille des Src et un blocage du recrutement des lipids rafts nécessaire à la formation de la synapse immunologique. Ces résultats suggèrent donc que c-mip est un régulateur négatif de l'activation T impliqué dans la signalisation proximale lymphocytaire et pourrait être impliqué dans l'hyporéactivité lymphocytaire observée chez les patients atteints de SNLGM actif.L'étude de la néphrite de Heymann passive, un modèle expérimental de GEM humaine, montre que l'induction podocytaire de c-mip coïncide avec l'apparition de la protéinurie. Cette surexpression est associée d'une part, à une diminution des taux de synaptopodine qui engendre une diminution de l'activité RhoA, à l'origine d'une désorganisation du cytosquelette podocytaire, et d'autre part à une induction de DAPK (death-associated protein kinase) et ILK (Integrin Linked Kinase) impliquées dans des phénomènes pro-apoptotiques. La cyclosporine A en inhibant l'expression de c-mip restaure les taux de DAPK et ILK ainsi que l'activité RhoA. Ainsi dans le podocyte, c-mip semble impliquer dans les troubles de la signalisation podocytaire aboutissant à une protéinurie néphrotique.C-mip semble donc jouer un rôle crucial dans les perturbations podocytaires et lymphocytaires observées chez les patients atteints de podocytopathies primitives et représente à ce titre une cible thérapeutique.Mots clefs :C-mip, Syndrome Néphrotique à Lésions Glomérulaires Minimes, Glomerulonéphrite Extra Membraneuse, signalisation proximale, lymphocyte T, podocyte, cytosquelette / Minimal Change Nephrotic Syndrome (MCNS) and Membranous Nephropathy (MN) are two primitive immune podocytopathies associating immune alterations and podocyte damage ultimately leading to proteinuria. Although the immune origin of the disease is corroborated by numerous clinical data, the mechanisms involved are still unknown. In previous works by the team, the c-mip protein was found expressed in T lymphocytes (TL) from patients with MCNS relapse, and in podocytes from MCNS and MN patients, while it is physiologically repressed. The aims of the present work were: firstly, to investigate the rôle of c-mip in TL by the study of Lck-cmip transgenic mice (Tg); secondly, to understand c-mip function in podocyte using a rat experimental model of human MN (Heymann nephritis).Transgenic mice overexpressed specifically c-mip in peripheral mature TL. This expression led to an altered TL phenotype characterized by accumulation of naïve LT associated with inhibition of TH1's and TH2's cytokines, after T-specific activation ex vivo. This negative regulation was correlated with an increase in the inactive forms of Src kinases and a blockage of the lipid raft clustering required for immunological synapse formation. These results suggest that (i) c-mip is a negative regulator of the proximal signaling events associated with TL activation involved in proximal signaling and (ii) it could be involved in the TL hyporeactivity described in SNLGM patients.In the study based on passive Heymann Nephritis, the experimental model of MN, the results highlight a correlation between podocyte expression of c-mip and proteinuria. This expression is associated, on the one hand, with a decrease in synaptopodin levels, which generate a decrease of RhoA activity resulting in podocyte cytoskeleton disorganization, and on the other hand with DAPK (Death Associated Protein Kinase) and ILK (Integrin Linked Kinase) induction, known to be involved in pro-apoptotic mechanisms. Cyclosporin A inhibited c-mip expression and restored the basal levels of DAPK, ILK and Rhoa activity. These results suggest that in podocyte, c-mip could be involved in proximal signaling alterations leading to nephrotic proteinuria.In conclusion, c-mip could play a crucial rôle in both the lymphocyte and podocyte alterations observed in patients suffering from primitive podocytopathies, strongly suggesting its potential as a therapeutic target in these disorders.Key words :C-mip, MCNS, MN, proximal signaling, Src kinase, T lymphocyte, podocyte, cytoskeleton

Résumé de la thèse:Le syndrome néphrotique idiopathique (SNI) est la forme la plus fréquente de néphropathies glomérulaires et résulte d'altérations touchant les podocytes. La progression de la maladie est associée à une déplétion podocytaire et l'apparition de glomérulosclérose. Malgré de nombreuses études moléculaires et des avancées scientifiques indiscutables sur les formes génétiques, la pathogénie du SNI reste une énigme.Nous avons trouvé que la protéine c-mip est spécifiquement induite dans les podocytes des patients atteints de SNI.Nous avons montré que les souris transgéniques c-mip développent une protéinurie néphrotique qui n'est associée ni à des lésions inflammatoires glomérulaires ou interstitielles, ni à des dépôts de complexes immuns circulants ou de complément. Les études in vitro et in vivo ont démontré que c-mip se lie à Fyn et bloque la liaison de Fyn avec la néphrine et N WASP. Il en résulte une inhibition de la voie de signalisation de la néphrine et l'incapacité de N-WASP à recruter Nck, ce qui altère l'organisation du cytosquelette podocytaire et contribue au développement de la protéinurie masssive.D'autre part, nous avons montré que Wt1 se lie au promoteur de c-mip et bloque sa transactivation. Au cours du SNI acquis, les résultats obtenus in vitro et dans les souris transgéniques suggèrent que c-mip inhibe la transcription du gène de Wt1 médiée par NF-κB, interagit avec Wt1 via son domaine LRR et favorise la dégradation de Wt1 par le protéasome.Nous avons également trouvé que c-mip interfère avec l'activation de la voie NF-κB en destabilisant la sous-unité RelA, tandis que la sous-unité p50 est préservée. Les résultats in vitro et dans le modèle murin suggèrent que c-mip est dotée de propriétés pro-apoptotiques.Ces travaux montrent que la protéine c-mip joue un rôle crucial dans la physiopathologie du SNI et constitue une cible thérapeutique de choix. / SummaryPodocyte damages are the initiating event in the pathogenesis of idiopathic nephrotic syndrome (INS). Progression of podocyte disease is associated with cellular depletion and appearance of glomerulosclerosis. The molecular pathophysiology of this disease remains an enigma.We showed that c-mip (c-maf inducing protein) is up-regulated in podocytes during the active phase of INS.We generated c-mip transgenic mice overexpressing c-mip specifically in podocytes. These mice developed morphological and biochemical alterations similar to INS. We demonstrated that c-mip switches off podocyte proximal signaling by preventing the interaction between Fyn and nephrin, resulting in the inhibition of nephrin phosphorylation in vitro and in vivo. Moreover, we found that the in vivo interactions of Fyn with Nck and N WASP are inhibited, which may account for disorganization of the cytoskeleton and the effacement of foot processes.We showed that, under physiological conditions, Wt1 inhibits the transcriptional induction of c-mip. Conversely, we demonstrated that, under pathological conditions, c-mip inhibits NF κB mediated-Wt-1 transcription, interacts in vitro and in vivo with Wt1 via its LRR domain, and targets Wt1 to proteasome degradation.We also observed that the induction of c-mip in patients with INS is correlated with a downregulation of RelA in podocytes. We showed that c-mip alters NF-κB signaling by destabilizing the RelA protein, while p50 is preserved. Morever, the results established in stably transfected podocytes and in transgenic mice suggest that c-mip is a proapoptotic protein.Collectively, these data postulate that c-mip functions as a negative regulator and plays a central role in podocytes disorders during INS.

La régulation transcriptionnelle des eucaryotes supérieurs est un processus hautement contrôlé du point de vue spatial et temporel lors du développement, ou en réaction à l’environnement. La transcription ciblée des gènes codant requiert l’assemblage d’un complexe de pré-initiation (PIC) aux promoteurs comprenant l’ARN Polymérase (Pol) II et les facteurs généraux de transcription (GTFs) et dépend de la médiation d’un signal par les facteurs activateurs de transcription (TFs). Les années récentes ont montré que la transition de l’initiation vers l’élongation productive de la transcription représente une étape clé de la régulation de l’expression des gènes. Ce processus est également contrôlé par la structure de la chromatine, les modifications d’histones et par la présence d’éléments cis-régulateurs tels que les ‘enhancers’ ou les ‘silencers’. Au cours de ma thèse, nous avons entrepris de décrypter les mécanismes de régulation transcriptionnelle impliqués dans les étapes du développement lymphocytaire. Nous avons essentiellement travaillé sur des thymocytes primaires murins isolés au stade de différenciation double positif (DP, CD4+/CD8+) pour lequel de nombreuses séquences de type ‘enhancers’ ont été caractérisées dans la littérature scientifique. Nous avons également utilisé des lymphocytes B humains (Raji) immortalisés pour certaines des expériences impliquant des manipulation génétiques complexes permettant l’étude de mutants du domaine carboxy-terminal (CTD) de Pol II. En couplant des approches d’analyse à l’échelle du génome au séquençage à haut-débit, nous avons établi des cartographies fines de la localisation de Pol II, des GTFs, des TFs,de modifications d’histones (ChIP-Seq) et de nucléosomes (MNase-seq) ainsi que la caractérisation de populations variées d’ARN par RNA-seq. Nos principaux résultats ont révélé (i) l’assemblage du PIC et la transcription des enhancers tissus-spécifiques, (ii) l’existence de plateforme d’initiation de la transcription (TIPs) aux enhancers et aux promoteurs tissus-spécifique, (ii) que le contenu en GC représente l’un des principaux éléments promoteurs mammifères en permettant une ouverture transcription-indépendante de la chromatine, (iv) l’importance d’une nouvelle modification post-traductionnelle du domaine CTD de Pol II pour la progression de l’enzyme en élongation et finalement (v) que la modification de l’histone H3 sur le résidu K36 methylé corrèle avec l’épissage des transcrits Pol II. Globalement, les résultats les plus important de ce manuscrit consistent dans la mise en évidence de la transcription des enhancers comme caractérisant l’expression des gènes tissus-spécifiques et dans l’importance des ilots CpG comme éléments promoteurs mammifères permettant la formation d’une structure ouverte de la chromatine. / Transcriptional regulation in higher eukaryotes resembles a tightly controlled temporal and spatial process, as exemplified during development or an organism’s response to environmental stimuli. Directed transcription requires the assembly of the preinitiation complex (PIC) at the promoter of protein-coding genes, including RNA Polymerase (Pol) II and the general transcription factors (GTFs), mediated by activating transcription factors (TFs). Several rate-limiting steps further control the progression of Pol II initiation to productive elongation of the gene. This process is further controlled by chromatin structure, histone modifications as well as cis-regulatory elements, such as enhancers or silencers. We set out to decipher some of these regulatory mechanisms during the tightly controlled process of lymphocyte development. Our work primarily made use of primary mouse thymocytes in CD4+/CD8+ double positive (DP, CD4+/CD8+) stage during T-cell development. To our advantage, many developmentally important cis-regulatory regions are well characterized in this cell population. For genetic manipulations, we made use of the Raji B-cell lymphoma cell-line. Using high throughput genome-wide approaches based on next generation sequencing (NGS), we performed both localization studies of Pol II, GTFs, TFs, histone modifying enzymes, histone modifications and nucleosomes as well as deep-sequencing of different RNA transcript populations. In summary, we find that (i) PICs assemble at tissue-specific enhancers leading to local transcription, (ii) large transcription initiation platforms (TIPs) at tissue-specific promoters and enhancers exist, which correlate with high CG-content of the DNA and transcription factor binding sites (TFBS), (iii) GC-content regulates the nucleosomal structure and initiation, including directionality, at promoters, (iv) Pol II is phosphorylated at a new residue of it C-terminal domain (CTD) in the 3’ regions of genes and (v) splicing events can influence the chromatin structure. Altogether, these results show that PIC formation at and transcription of enhancers are important for the regulation of T-cell target genes, that CpG islands represent important if not the major regulatory promoter element in mammals guiding tissue-specific gene expression and nucleosome structure, as well as novel mechanisms of Pol II elongation and the effect on chromatin structure.

Triptofano (TRP) é metabolizado por duas vias, a via serotonérgica e a via das quinureninas. Na via serotonérgica, TRP é metabolizado a serotonina (5-HT) e, em algumas células, à melatonina (MLT) que pode ser oxidada à N1-acetil-N2-formil-5- metoxiquinuramina (AFMK) e N1-acetil-5-metoxiquinuramina (AMK) por ação de peroxidases. Na via das quinureninas o TRP é diretamente metabolizado à N formilquinurenina (NFK) e em seguida a quinurenina (QUIN). A enzima indolamina 2, 3 dioxigenase (IDO) é uma das responsáveis por esta reação. Dada a importância da IDO na tolerância imunológica e pelo fato desta enzima ser induzível nos propusemos a avaliar a existência de uma regulação cruzada entre esta enzima e a via serotonérgica. Avaliando a interferência de AMK sobre a ação de IDO e a interferência de QUIN sobre a formação de AFMK por peroxidases, observamos uma possível interação entre as vias. AMK é um inibidor competitivo clássico de IDO e o Ki encontrado foi de 0,98 mM. QUIN é um inibidor acompetitivo linear simples da formação de AFMK e o Ki encontrado foi de 0,1 mM. A inibição da formação de AFMK também ocorre para a peroxidase humana (mieloperoxidase, MPO). Além de representarem uma regulação cruzada utilizada in vivo, as inibições encontradas podem ser relevantes para a proposta de novos inibidores de IDO e MPO na terapia imunomodulatória. Dado o nosso interesse pelas enzimas IDO e MPO, avaliamos ainda a localização intracelular destas enzimas em células de peritônio de camundongo, tanto residente como ativada com concanavalina A (Con A). O estímulo com Con A representa uma ativação de linfócitos T mediado por interferon gama (IFN-γ) e foi usado como modelo experimental para avaliar condições de localização em células ativadas. Por imunocitoquímica verificamos que IDO e MPO localizam-se próxima à membrana plasmática sendo que uma leve dispersão apenas de MPO foi observada em células ativadas com Con A. A localização intracelular das duas enzimas é no citoplasma, vesículas e núcleo. Curiosamente, verificamos MPO em células isoladas e também em agrupamentos celulares de duas ou mais células. Por citometria de fluxo identificamos macrófagos, linfócitos B1 e agrupamentos celulares como células que contém MPO. A mobilização de MPO durante a ativação celular, a presença de MPO em linfócitos e a presença de MPO e IDO em núcleos são informações novas que sugerem novas atividades para estas enzimas. / Tryptophan (TRP) is metabolized by two mains pathways, the serotoninergic pathway and the kynurenine pathway. In the serotoninergic pathway, TRP is metabolized to serotonin (5-HT) and, in some cells, to melatonin (MLT). The later can even be oxidized to acetyl-N1-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5 -methoxykynuramine (AMK) by peroxidases. In the kynurenine pathway, TRP is metabolized to N-formylkynurenine (NFK) and to kynurenine (KYN). Indoleamine 2, 3 dioxygenase (IDO) is one of those responsible for this reaction. Since IDO is importat in immune tolerance and the fact that this enzyme is inducible by cytokines we proposed whether there is a cross regulation between this enzyme and the serotoninergic pathway. A possible interaction between MLT and TRP oxidation pathways was shown by the AMK influence on IDO activity and QUIN interference on AFMK formation by peroxidases. AMK was shown to be an IDO classical competitive inhibitor with a Ki of 0.98 mM. QUIN was a peroxidase (horseradish peroxidase, HRP) classical uncompetitive inhibitor and Ki was found to be 0,1 mM. AFMK formation inhibition was also found in human peroxidase (myeloperoxidase, MPO). Beyond the in vivo crosstalk, new IDO and MPO inhibitors in immunomodulatory therapy would be proposed by the compounds shown in this study. Given our interest in IDO and MPO, we also evaluated their intracellular localization in both resident and concanavalin A (Con A) activated mice peritoneum cells. Con A stimulation is a IFN-γ mediated T lymphocytes activation and was our experimental model to evaluate activated cells. In light microscopy we observed IDO and MPO localization near the membrane and MPO only had a dispersed localization in Con A activated cells. Cytoplasm, nucleus and vesicles were the intracellular localization of both enzymes. Interestingly, we found MPO in isolated cells and in cell clusters of two or more cells. MPO was founded on macrophages, B1 cells and cell clusters by flow cytometry. The MPO mobilization during cell activation, the presence of MPO in lymphocytes and the presence of MPO and IDO in nuclei are new informations to suggest new activities for these enzymes.

A abordagem imunoterapêutica do câncer tem sido cada vez mais explorada. Entre os fatores que a tornam atraente, mas também a limitam, está o uso de material antigênico do próprio paciente. Assim, pretendeu-se estabelecer condições de extração e amplificação de mRNA tumoral, como fonte renovável de antígenos. Pretendeu-se avaliar também a eficácia da vacina, com o uso de monócitos transfectados com RNA tumoral total para mimetismo das células tumorais. Diferentes concentrações (0,1 mg a 10 mg) de RNA total de SK-BR-3 e diferentes tempos (12, 24 e 48 h) foram usados para transfecção. Na avaliação do potencial linfo-estimulador dos monócitos foi usado o ensaio de proliferação linfocitária e a secreção de citocinas durante a co-cultura. Como resultado viu-se que monócitos transfectados se tornaram mais ativados e foram capazes de induzir linfoproliferação. Esses resultados indicaram ser possível o desenvolvimento de um método para avaliação das respostas celulares induzidas contra células tumorais em pacientes com câncer que foram vacinados. / The cancer immunotherapeutic approach has been increasingly exploited. Among the factors that make it attractive, but also limited, is the use of patient antigenic material. Thus, we propose to establish conditions for extraction and amplification of mRNA tumor, as renewable source of antigens. It is also intended to assess the vaccine effectiveness, using monocytes transfection with total tumor RNA for mimicry the tumor cells. Different concentrations (0.1 to 10 mg) of SK-BR-3 total RNA and different times (12, 24 and 48 h) were used in transfection. To assess the lympho-stimulator potential of transfected monocytes was used the test of lymphocyte proliferation, and cytokines secretion during co-culture. The result was that transfected monocytes became more activated and were able to induce lymphoproliferation. These results indicated that the development of a method for evaluating cellular responses induced against tumor cells in cancer patients who were vaccinated is possible.

Les anticorps monoclonaux représentent aujourd’hui un outil de choix en thérapeutique et en diagnostic. Les anticorps thérapeutiques sont des biomédicaments en plein essor depuis les années 1970 et représentent 10% du marché des produits pharmaceutiques. Les anticorps monoclonaux sont utilisés dans divers domaines : en cancérologie, pour lutter contre les maladies auto-immunes ou en infectiologie. Le nombre des anticorps monoclonaux en développement ne cesse d’augmenter. Les premiers anticorps monoclonaux utilisés en thérapie étaient d’origine murine et leur administration à l’Homme est susceptible de déclencher des effets secondaires. De nouveaux anticorps visant à limiter voir faire disparaitre ces effets indésirables tels que d’abord les anticorps chimériques, puis les anticorps humanisés et enfin les anticorps totalement humains ont été développés. 9 anticorps totalement humains sont actuellement sur le marché et d’autres sont en cours de développement. Le phage display, les souris transgéniques et l’utilisation de lymphocytes B humains sont les trois stratégies mises en œuvre pour produire des anticorps totalement humains. L’utilisation des lymphocytes B humains, peu étudiée à cause d’un faible rendement et de problèmes de stabilité, a connu ces dernières années un regain d’intérêt grâce à l’immortalisation virale par le virus Epstein-Barr et à la découverte de myélomes humains. Dans ce contexte, l’objectif de mon projet de thèse a été la production d’anticorps monoclonaux humains à partir de lymphocytes B humains. Pour ce faire, deux approches basées sur l’immortalisation virale par le virus Epstein-Barr couplée ou non à une immortalisation cellulaire par des myélomes ont été mises en œuvre. La première approche utilise des lymphocytes B mémoires isolés de sang périphérique de donneurs infectés ou vaccinés. L’entérotoxine B de Staphylococcus aureus (SEB) a été utilisée comme modèle.La deuxième approche implique une immunisation in vitro de lymphocytes B naïfs extraits de sang périphérique. Cette stratégie pourrait permettre la production d’anticorps humains contre des antigènes pour lesquels il n’existe pas de donneurs infectés ou vaccinés. Deux modèles, le peptide N-terminal de la neurotoxine A de Clostridium Botulinium A (BoNT/A) et la protéine de fusion ZZTat101, comportant le domaine ZZ de Staphylococcus aureus lié covalemment à la protéine transactivatrice Tat du virus de l’immunodéficience humaine VIH-1, ont été employés. Nous avons réussi à obtenir des IgMs dirigés contre la neurotoxine Clostridium Botulinium A, ainsi que des IgMs (et peut-être des IgGs) dirigés contre la protéine Tat. L’immortalisation par Epstein-Barr, nous a permis d’isoler 7 lignées de lymphocytes immortalisés sécrétant des anticorps IgMs anti-TBA-Nter humains. L’immunisation in vitro produisant essentiellement des IgMs, la possible production d’IgGs après stimulation par la protéine ZZTat101 se révèle un résultat très intéressant. Nous avons montré que la production d’anticorps par ZZTat101 impliquait les 7 cystéines, la région 22-57 et la liaison aux héparanes sulfates de Tat. / The number of monoclonal antibodies used as drug or under clinical investigation increases rapidly. The first murine monoclonal antibodies (mAbs) used in therapy induces human anti-mouse antibodies (HAMAs) when administered to patients. Such HAMAs hamper the therapeutic efficacy of mAbs and induce side effects. To limit these effects, new antibodies were developed during the, last 30 years. Chimeric, humanized and fully human antibodies were engineered. The use of human monoclonal antibodies (hAbs) appears ideal to solve the problem of HAMAs. Nowadays 9 fully human antibodies are available and others are evaluated in clinical trials or currently investigated in research labs. Three methods exist to produce fully human antibodies: the phage display, the transgenic mice and the use of human B lymphocytes. The majority of fully human antibodies resulted from the phage display and the transgenic mice methods. The use of human B lymphocytes is less investigated due to a poor yield and stability problems. These last years, the immortalization process, thanks to the involvement of the Epstein-Barr virus and human myeloma, induced a rise of interest for human B lymphocytes. In this context we decided to develop fully human monoclonal antibodies using human B lymphocytes through immortalization using the Epstein-Barr virus followed or not by an immortalization with a human/mouse heteromyeloma HM. The first approach is based on hAbs production from peripheral blood memory B lymphocytes isolated from infected or vaccinated donors. The Staphylococcus aureus enterotoxine B (SEB) was used as a model. Memory B lymphocytes were purified and cultured in the presence of Epstein-Barr virus (EBV). The transformation of memory B lymphocytes by EBV allowed the generation of immortalized B lymphocytes lines producing IgGs antibodies directed against SEB. We succeeded in isolating 6 EBV-immortalized memory B lymphocytes lines secreting anti-SEB IgGs antibodies. After many attempts to immortalize EBV immortalized memory B lymphocytes lines secreting anti-SEB antibodies with myeloma, the fusion of a EBV immortalized memory B lymphocytes with the human/mouse heteromyeloma HM led to an hybridoma. Unfortunately this hybridoma has rapidly lost its capacity to secrete d’IgGs anti-SEB. In the second approach the hAbs production implies the in vitro immunization of peripheral blood naïve lymphocytes. This strategy could allow the hAbs production against antigens for which no infected or vaccinated donors may be available. The Clostridium Botulinum neurotoxin A (BoNT/A), the most powerful toxin, and its N-terminal peptide (TBA-Nter) or the fusion protein ZZTat101 were used as models. ZZTat101 is a fusion between the ZZ domain of Staphylococcus aureus and the Tat protein of the human immunodeficiency virus HIV-1. Monocytes, B lymphocytes and T lymphocytes were isolated from human PBMC depleted of Natural killer. These cells were tools to develop efficient in vitro immunization protocols. IgMs directed against TBA-Nter and also IgMs (and possibly IgGs) directed against Tat were obtained. The use of the Epstein-Barr virus induced 7 EBV immortalized lines secreting anti-TBA-Nter IgMs antibodies. Unfortunately, after fusion with the heteromyeloma HM no hybridoma was isolated against TBA-Nter and Tat. The ZZTat101 mechanism involved on humoral response was studied, showing that the 7 cysteines, the region 22-57 and the ability of Tat to bind heparane sulfate are necessary to trigger the humoral response.

Les lymphocytes T CD4+ auxiliaires ou T helper en anglais sont capables de soutenir une grande diversité de fonctions grâce à leur capacité à se différencier en différents sous-types effecteurs en fonction de l’antigène rencontré et de l’environnement cytokinique dans lequel ils se trouvent. Les connaissances actuelles sur la différenciation des cellules T helper mettent en avant l’existence de réseaux transcriptionnels particulièrement complexes et spécifiques à chaque sous-ensemble T helper. En 2008, les cellules T CD4 sécrétrices d’IL-9 (Th9) sont identifiées comme un nouveau sous-type de cellules T helper. Différenciées en présence d’IL-4 et TGF-β, les cellules Th9 sécrètent de l’IL-9 et de l’IL-21, et contribuent au développement de maladies auto-immunes et allergiques. Les lymphocytes Th9 présentent également des propriétés anti-tumorales particulièrement intéressantes.Le réseau transcriptionnel des cellules Th9 résulte d’un équilibre entre les voies de signalisation induites par les différentes cytokines nécessaires à sa polarisation. L’IL-4 permet l’activation de STAT6 et l’expression de GATA3 et IRF4, tandis que le TGF-β conduit à l’activation de la voie des Smad et l’expression du facteur PU.1. Le module transcriptionnel IRF4/BATF ainsi que le facteur PU.1 sont des messagers indispensables au développement des cellules Th9 et à la sécrétion d’IL-9.IRF8 est un facteur de transcription critique pour le développement des cellules myéloïdes et des lymphocytes B. Récemment, il est apparu qu’IRF8 était impliqué dans la polarisation de sous-ensembles T helper. En effet, IRF8 limite la sécrétion d’IL-17 par les cellules Th17, de même qu’il réprime l’expression de l’Il4 et l’Il17 dans les cellules Treg. Structurellement proche d’IRF4, IRF8 interagit avec des cofacteurs tels que PU.1 ou BATF afin de réguler l’activité transcriptionnelle.Ce travail présenté ici révèle que le facteur IRF8 participe à la polarisation des cellules Th9 in vitro et in vivo. Le TGF-β nécessaire à la différenciation des cellules Th9 régule directement l’expression d’Irf8 grâce à l’activation de Smad3. Comme dans d’autres types cellulaires, la fonction transcriptionnelle d’IRF8 est dépendante de ces partenaires d’interaction. Nous montrons qu’en présence des facteurs PU.1, IRF4 et BATF, IRF8 participe à un complexe multiprotéique nécessaire à l’induction des cytokines caractéristiques des cellules Th9, notamment l’Il9 et l’Il21. Nous démontrons également qu’en présence de la protéine ETV6, IRF8 est capable de former un complexe initiant la répression de l’activité transcriptionnelle de l’Il4. Nous soulignons ainsi le rôle bivalent joué par IRF8 dans le développement des cellules Th9 dépendamment de ses partenaires. Pour finir, l’expression d’Irf8 est nécessaire aux cellules Th9 pour exercer leurs fonctions anti-tumorales. / CD4 helper T cells support a wide range of functions due to their ability to differentiate into different effector subsets depending on the antigen encountered and the cytokine environment in which they are. Current knowledge on the differentiation of helper T cells highlights the existence of complex transcriptional networks specific to each T helper subset. In 2008, IL-9 secreting CD4 T cells (Th9) are identified as a new helper T cell subtype. Differentiated in the presence of IL-4 and TGF-β, Th9 cells secrete IL-9 and IL-21, and contribute to the development of autoimmune and allergic diseases. Th9 lymphocytes also exhibit strong anti-tumor properties.The transcriptional network of the Th9 cells results from a balance between the signaling pathways induced by the different cytokines required for its polarization. IL-4 allows activation of STAT6 and expression of GATA3 and IRF4, whereas TGF-β leads to activation of the Smad pathway and expression of the transcription factor PU.1. The IRF4 / BATF transcriptional module and the PU.1 factor are essential messengers for the development of Th9 cells and IL-9 secretion.IRF8 is a crucial transcription factor for the development of myeloid cells and B lymphocytes. Recently it appeared that IRF8 was involved in helper T subset polarization. Indeed, IRF8 limits the secretion of IL-17 by Th17 cells, as well as repressing the expression of Il4 and Il17 in Treg cells. Structurally close to IRF4, IRF8 interacts with cofactors such as PU.1 or BATF in order to regulate transcriptional activity.This work reveals that the IRF8 transcription factor contributes to the polarization of Th9 cells in vitro and in vivo. The TGF-β needed for Th9 cell differentiation activate Smad3 pathway which directly modulates the Irf8 expression. As in many cellular subtypes, the transcriptional function of IRF8 is dependent on these interaction partners. We show that in the presence of the transcription factors PU.1, IRF4 and BATF, IRF8 participates in a multiprotein complex essential for the induction of the Th9 cytokines, Il9 and Il21. We also demonstrate that in the presence of the ETV6 protein, IRF8 is able to form a complex responsible for the repression of Il4 expression. We underline the bivalent role played by IRF8 in the development of Th9 cells depending on its partners. Finally, expression of Irf8 is crucial for Th9 cells to exercise their antitumor functions.

Various recent studies have suggested that regular intake of some non-steroidal anti-inflammatory drugs (NSAIDs) have a preventative effect against several types of tumours including breast cancer. The term nanotechnology refers to technology in which one-billionth of a meter is used as a scale for chemical particle size. This work aims to study the effect of both ibuprofen and aspirin on DNA damage using peripheral blood lymphocytes from breast cancer patients and comparing the results with those from healthy females as a control using the Comet and micronucleus assays. Western blot analysis (WBA) was used to investigate the effect of these drugs on XRCC3 and p53 proteins, whereas QPCR was to evaluate this effect on p53, cox1 and cox2 genes. Two hundred fifty ng/ml of ibuprofen (NP and bulk) and 500 ng/ml of aspirin (NP and bulk) were used to treat the lymphocytes. Both aspirin and ibuprofen caused a reduction in DNA damage and micronucleus formation. Aspirin, both forms, showed a reduction in DNA damage in the Comet and micronucleus assays. Ibuprofen both forms, by contrast, showed a statistically significant reduction in micronucleus frequency in the micronucleus assay, while its preventative effect with the Comet assay was weak or insignificant. NPs of both agents were more effective than bulk sizes. Using the Comet repair assay, aspirin and ibuprofen nano form catalysed DNA repair to a greater extent than their bulk forms. Also, both sizes showed better repair with NSAIDs compared to samples repaired without NSAIDs. In WBA aspirin increased the expression of XRCC3 protein in healthy cells. However, both NSAIDs decreased that expression in cells from BC patients. Furthermore, aspirin increased p53 expression in BC patients lymphocytes. With the QPCR method, results of both aspirin forms increased the expression of the p53 gene in BC patient cells statistically significantly. Both drugs reduced cox1 expression in healthy volunteers and cancer patients lymphocytes. Moreover, cox2 reduction was only in lymphocytes from BC patients. The results of this work are consistent with the view that NSAIDs, particularly aspirin and ibuprofen, could have a promising role in cancer treatment including breast cancer.