Adenosine is an important neuromodulator. In the present study, we used mice with expression of human ENT1 (hENT1) and deficiency of CD73, respectively, to address the relative importance of intracellular and extracellular pathways in adenosine regulation. [3H]Nitrobenzylthioinosine binding assays were performed and found increased expression of hENT1 with increased gene dose. We performed a series of behavioural experiments with caffeine and ethanol and compared hENT1 heterozygous and homozygous transgenic mice to their wild type littermates. We found that the expression of hENT1 leads to a change in behavioural responses relative to wild type mice, but no sign of a gene dose dependent increase was observed. With CD73 knockout mice, we performed a series of behavioural experiments with caffeine and ethanol that showed a change in adenosine related behaviours. We also performed experiments that tested anxiety-like behaviours and found reduced anxiety-like behaviours with CD73 knockout mice relative to wild type mice. These studies show that mice with enhanced expression of ENT1 or knockout of CD73 have altered extracellular level of adenosine.

Adenosine is an important neuromodulator. In the present study, we used mice with expression of human ENT1 (hENT1) and deficiency of CD73, respectively, to address the relative importance of intracellular and extracellular pathways in adenosine regulation. [3H]Nitrobenzylthioinosine binding assays were performed and found increased expression of hENT1 with increased gene dose. We performed a series of behavioural experiments with caffeine and ethanol and compared hENT1 heterozygous and homozygous transgenic mice to their wild type littermates. We found that the expression of hENT1 leads to a change in behavioural responses relative to wild type mice, but no sign of a gene dose dependent increase was observed. With CD73 knockout mice, we performed a series of behavioural experiments with caffeine and ethanol that showed a change in adenosine related behaviours. We also performed experiments that tested anxiety-like behaviours and found reduced anxiety-like behaviours with CD73 knockout mice relative to wild type mice. These studies show that mice with enhanced expression of ENT1 or knockout of CD73 have altered extracellular level of adenosine.

L'étude des populations de lymphocytes T CD4+ effecteurs (Teff) chez l'Homme présente un intérêt croissant dans les enjeux actuels que constitue l'élaboration de nouvelles immunothérapies. Ces travaux détaillent la caractérisation et la régulation d'une population de Teff exprimant l'ecto-nucléotidase CD73 ayant pour fonction de dégrader l'AMP extracellulaire en adénosine (Ado) immunosuppresseur. Cette population, enrichie en lymphocytes T helper de type Th1.17, est très polyfonctionelle et pro-inflammatoire. Les Teff CD73+ expriment peu de points de contrôles immunitaires inhibiteurs mais sont régulés par leur production autocrine d'Ado qui limite leurs fonctions effectrices et leurs capacités prolifératives. Les Teff CD73+ expriment par ailleurs fortement le transporteur ABC multidrug-resistance 1 (MDR1), responsable de l'exclusion de nombreuses drogues du cytoplasme des cellules. L'étude de cette population dans différents contextes pathologiques a permis de détailler le fonctionnement de cette population. J'ai pu mettre en évidence que l'expression de CD73 est en effet dynamique. Elle est notamment diminuée dans des pathologies arthritiques auto-immunitaires (la polyarthrite rhumatoïde et le rhumatisme psoriasique) dans lesquelles les Th17 et les Th1.17 sont fortement activés. La diminution d'expression de CD73 sur ces cellules constitue la levée d'un frein qui leur permet de contribuer pleinement à l'inflammation chronique caractérisant ces pathologies. Les Teff CD73+, présents dans les tumeurs solides humaines de sein et d'ovaire, pourraient en revanche présenter un avantage sélectif en contexte tumoral de par leur expression de MDR1 leur permettant de résister aux traitements de chimiothérapie. Ces traitements substrats de MDR1, combinés à des thérapies inhibant la fonction enzymatique de CD73, pourrait permettre la restauration d'une réponse immunitaire anti-tumorale efficace médiée par cette population pro-inflammatoire / Requirement for CD4+ effector T lymphocytes (Teff) comprehensive study in human is increasing since it can contribute to the emergence of new immunotherapy strategies. This work brings up important information concerning the characterization and regulation of a Teff population expressing the CD73 ecto-nucleotidase, which is able to degrade extracellular AMP into immunosuppressive adenosine (Ado). This population, highly polyfunctional and pro-inflammatory, is enriched in Th1.17 cells. CD73+ Teff express low levels of inhibitory immune checkpoints but are negatively regulated by the autocrine Ado production that limits their pro-inflammatory function and proliferative capacities. In addition, CD73+ Teff express high levels of the ABC transporter multi-drug resistance 1 (MDR1), responsible for the exclusion of cells’ cytoplasm of many drugs. The study of this population in different pathological contexts enabled to decipher its functions. I could evidence that CD73 expression is dynamic. CD73 is notably decreased in autoimmune arthritic pathologies (rheumatoid arthritis (RA) and psoriatic arthritis (PsA)) in which Th1.17 and Th17 are highly activated. CD73 decreased expression by these cells is a mechanism that alleviates self-inhibition by autocrine Ado production and enables them to fully contribute to chronic inflammation characterizing these pathologies. In tumor context, CD73+ Teff present in breast and ovarian tumors could on the contrary bear a selective advantage due to their high MDR1 expression enabling them to resist MDR1 substrates-based chemotherapy treatments. These chemotherapy treatments combined to therapies blocking CD73 enzymatic function could allow the restauration of an efficient anti-tumor immune response

Les nucléotides jouent un rôle majeur dans une pléiade de processus biologiques comme la composition des acides nucléiques, la signalisation, ou la régulation de la balance énergétique. Les nucléotides extracellulaires exercent également des fonctions biologiques. Par conséquent, des dérégulations des pools de nucléotides impactent l’homéostasie de multiples façons, par exemple en promouvant l’instabilité génétique ou un environnement immunosuppresseur. Or, ces paramètres font partie des « Hallmarks du Cancer » décrits par Hanahan et Weinberg. Ces observations confirment l’éventualité d’un rôle clé des nucléotides dans le cadre du cancer.cN-II et CD73 sont des 5’-nucléotidases impliquées respectivement dans les métabolismes nucléotidiques intra- et extracellulaire. Elles sont de nouvelles cibles thérapeutiques en oncologie. Cependant, leurs rôles dans la biologie de la cellule cancéreuse, ou le possible impact de leur utilisation en tant que cible thérapeutique sur le comportement des cellules tumorales sont peu connus. Considérant l’implication de ces enzymes dans les métabolismes nucléotidiques, nous avons enquêté sur les modifications de l’agressivité de la cellule cancéreuse ou sur sa capacité à interagir avec son microenvironnement, dans le cas d’une invalidation ou une diminution d’expression de cN-II et/ou CD73. cN-II semble donc impliquée dans l’adaptabilité métabolique et la combinaison des invalidations de cN-II et CD73 est associée à une modification d’expression d’enzymes du métabolisme du glucose. CD73 peut aussi moduler l’expression de gènes de la migration cellulaire. cN-II est impliquée dans la migration cellulaire, via l’axe COX-2/PGE2, et dans la sensibilité à des agents modulant ce paramètre. Ces caractéristiques sont plus marquées en association avec une invalidation de CD73. Ici, cN-II et CD73 ne semblent pas jouer de rôle dans la prolifération ou le dialogue avec une sous-population de cellule de l’immunité innée / Nucleotides play a major role in nucleic acids constitution and are involved in various cell phenomena. Indeed, intracellular ATP, GTP, AMP, GMP and their cyclic forms are components of cell signaling and define the energetic balance. Extracellularly, they also play multiple roles. Thus, when nucleotide pools are deregulated various processes are impacted. For example, a low availability of nucleotides supports genetic instability and aberrant levels of extracellular adenosine can lead to an immunosuppressive microenvironment. Interestingly, the cited parameters are among the Cancer Hallmarks described by Hanahan and Weinberg. These observations confirm the possibility of a key role of these molecules in this pathology. cN-II and CD73 are 5’-nucleotidases, involved in intra- and extracellular nucleotide metabolism respectively and have been identified as possible targets for new anti-cancer therapies. Nevertheless, very little is known about their biological roles on cancer cells and what parameters of cell biology could be impacted by such strategies. Considering the involvement of these purines in cell metabolism, we wondered what changes a decrease in cN-II and/orCD73 expressions or their silencing could trigger in cancer cells as well as in the interplay with their microenvironment.We studied cancer cell aggressiveness and the interplay with innate immune cells under cN-II and CD73 modulations. We observed that cN-II is involved in metabolic adaptability. The association of cN-II and CD73 invalidations results in glucose-metabolism-related gene modifications. CD73 can regulate migration-related genes expression but does not affect the process. cN-II is also involved in cell migration, via the COX-2/PGE2 axis. Again, these characteristics are accentuated when associated with CD73 deficiency. Here, cN-II and CD73 do not seem to be involved in cancer cell proliferation or in their interplay with a subset of innate immune cells

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No. of bitstreams: 1 julianamelo.pdf: 1321154 bytes, checksum: 6613c9596412757aeb47ee110faa5b87 (MD5) Previous issue date: 2017-02-22 / A brucelose é uma doença infectocontagiosa causada por bactérias do gênero Brucella que acometem o homem e uma grande variedade de animais domésticos, resultando em prejuízos econômicos significativos aos sistemas de produção. Em humanos essa infecção pode causar febre ondulante, endocardite, artrite, osteomielite e complicações neurológicas enquanto em animais leva ao aborto e infertilidade. Sabe-se que a resposta imunológica a bactérias intracelulares como a Brucella ocorre essencialmente através da imunidade mediada por células, sendo macrófagos especialmente importantes no combate à infecção. Entretanto, apesar da efetividade da resposta, a B. abortus conta com diversos mecanismos de evasão, o que garante a sua sobrevivência no organismo hospedeiro. Dentre estes mecanismos, a modulação de células apresentadoras de antígenos tem sido apontada como um dos mais relevantes. Recentemente, diversos trabalhos têm evidenciado a importância das NTPDases e da molécula PD-1 na modulação da resposta imune. As NTPDases estão envolidas com a produção de adenosina que apresenta relevante caráter imunomodulador. Já a molécula PD-1 está associada a indução de um perfil anti-inflamatório com diminuição de IL-12 e aumento de IL-10. Neste contexto, o objetivo deste estudo foi determinar se a infecção com B. abortus é capaz de alterar a expressão destas moléculas e assim limitar a ação do sistema imune, favorecendo a sobrevivência do patógeno. Para isso, células RAW 264.7 foram infectadas com B.abortus e a modulação das moléculas CD80, CD86, CD40, MHCII, foi avaliada por citometria de fluxo. Em seguida, a expressão de CD39 também foi determinada por citometria de fluxo e por Western Blot. Por fim analisamos possíveis alterações na expressão da molécula PD-1 induzidas pela bactéria. Como resultados, foi confirmado que a infecção é capaz de inibir a expressão de CD80, CD86 e CD40, embora o mesmo não tenha sido observado com o MHCII. Além disso, a expressão de CD40 se mostrou diminuída mesmo após o estímulo com LPS. De forma surpreendente, foi observada uma diminuição na expressão das moléculas CD39 e PD-1, o que pode ser explicado pela menor ativação celular induzida pela infecção. Assim, os dados obtidos até o momento demonstram que as moléculas CD39 e PD-1 não são utilizadas pela Brucella para modular as APCs, mas são influenciados pela menor ativação celular induzida pelo patógeno. / Brucellosis is an infectious disease caused by bacteria of the genus Brucella that affect humans and a wide variety of domestic animals, resulting in significant economic losses to production systems. In humans the infection can cause undulant fever, endocarditis, arthritis, osteomyelitis and neurological complications while in animals leads to abortion and infertility. It is known that the immune response to intracellular bacteria such as Brucella occurs primarily by cell-mediated immunity, macrophage being especially important in fighting infection. However, despite the effectiveness of the response, B. abortus has several avoidance schemes, which ensures their survival in the host organism. Among these mechanisms, modulation of antigen-presenting cells has been identified as one of the most relevant. Recently, several studies have shown the importance NTPDase and PD-1 molecule to modulate the immune response. The NTPDase are envolidas with the production of adenosine which presents immunomodulatory relevant character. Since PD-1 molecule is associated with induction of an anti-inflammatory profile with decreased IL-12 and IL-10 increase. In this context, the aim of this study was to determine whether infection with B. abortus is able to alter the expression of these molecules and thus limit the action of the immune system, favoring the survival of the pathogen. To this end, RAW 264.7 cells were infected with B.abortus and modulation of CD80 molecules, CD86, CD40, MHCII, was evaluated by flow cytometry. Then the CD39 expression was also determined by flow cytometry and Western blot. Finally, we analyze possible changes in PD-1 molecule expression induced by bacteria. As a result, it was confirmed that the infection is capable of inhibiting expression of CD80, CD86 and CD40, although this has not been observed with MHCII. Additionally, CD40 expression showed decreased even after the stimulation with LPS. Surprisingly, it was observed a decrease in the expression of CD39 and PD-1 molecules, which can be explained by the lower cellular activation induced by the infection. Thus, the data obtained so far show that the PD-1 and CD39 molecules are not used by Brucella Modular APCs but are less influenced by cellular activation induced by the pathogen.

Les lymphocytes T régulateurs (Treg) jouent un rôle prépondérant dans la tolérance du système immunitaire. En physiopathologie, un défaut quantitatif ou fonctionnel en Treg favorise le développement de maladies auto-immunes tandis que leur présence participe au développement tumoral. En particulier, la présence de Treg dans le stroma immunitaire de la tumeur (TiTreg) est de mauvais pronostic pour la survie des patientes atteintes de cancers du sein et de l'ovaire. Les Treg sont recrutés dans la tumeur via l'axe CCL22/CCR4 et sont activés et amplifiés via leur interaction avec les pDC et l'axe de co-stimulation ICOS-ICOSL qui favorise leurs capacités suppressives. Ce projet contribue aux efforts réalisés ces dernières années visant à la compréhension des mécanismes d'immunosuppression des Treg opérant dans les tumeurs humaines. En effet, ce projet met en évidence que les Ti-Treg humains expriment fortement l'ectonucléotidase membranaire CD39. Cette enzyme extracellulaire catabolise l'Adénosine tri-phosphate (ATP) en Adénosine Monophosphate (AMP) pouvant être ensuite dégradé, via l'ectonucléotidase CD73, en Adénosine (Ado). Alors que l'ATP représente une Alarmine (signal de danger extracellulaire) qui en particulier contribue à l'activation de l'inflammasome, l'Ado possède un fort pouvoir immunosuppresseur qui est illustré chez les patients atteints d'une déficience de l'enzyme Adénosine Déaminase (ADA), ne pouvant dégrader l'Ado en Inosine (Ino), développent un Syndrome d'Immunodéficience Sévère. Contrairement au Treg murins, les Treg humains n'expriment pas CD73. Cependant nous avons pu identifier une population de lymphocytes T CD4+ mémoires non régulateurs (Tconv) exprimant CD73 et coopérant ainsi avec les Treg CD39+ pour la génération d'Ado. Cette population présente une capacité accrue de sécrétion de cytokines inflammatoires (IFNgamma, IL-17A, IL-22, GM-CSF) et l'expression de molécules (CXCR3, CCR6, MDR1) caractéristiques du profil Th1/17. De plus ces cellules semblent être moins sensibles à une régulation médiée par les points de contrôles dits immune-checkpoints (ICPs) tel que PD-1, CTLA-4, TIM-3, TIGIT. Par contre, les Tconv CD73+ sont sensibles à l'Ado généré lors de la coopération avec les Treg CD39+ qui engendre l'inhibition de leur prolifération et de leur sécrétion d'IFNgamma et de GM-CSF mais pas de l'IL-17A. L'Ado qui agit localement, peut également entrainer la suppression des Tconv CD73neg dans l'environnement proche. L'ensemble de ces résultats montre que l'expression de CD73 caractérise une population de T CD4 effecteurs polyfonctionnelle Th1/17 qui est une cible privilégiée et coopérative de l'immunosuppression des Treg dans l'environnement tumoral. En outre l'action d'Ado transforme ce puissant effecteur anti-tumoral en cellule potentiellement pro-tumorale via l'unique sécrétion privilégiée d'IL17 / Regulatory T cells (Tregs) play a key role in the immune system tolerance. In pathophysiology, a quantitative or functional defect in Treg promotes development of autoimmune diseases while their presence involved in tumor development. In particular, the presence of Treg in the immune stromal tumor environment (Ti-Treg) is associated with a poor prognosis for survival of patients suffering from breast cancer and ovarian cancer. Treg are recruited in the tumor through the CCL22 / CCR4 axis and are activated and amplified through their interaction with pDC expressing costimulatory axis ICOS-ICOSL and promoting their suppressive capacity. This project contributes to the efforts made in recent years to understand suppressive mechanisms of Treg operating in human tumors. Indeed, this project demonstrates that humans Ti-Treg strongly express the membrane ectonucleotidase CD39. This extracellular enzyme catabolizes Adenosine-triphosphate (ATP) to adenosine-monophosphate (AMP) which can then be degraded through the ectonucleotidase CD73 into Adenosine (Ado). While ATP is an Alarmine (extracellular danger signal) that particularly contributes to the inflammasome activation, Ado has strong immunosuppressive effect which is illustrated in patients with deficiency of the enzyme Adenosine Deaminase (ADA), which cannot degrade Ado into Inosine (Ino) and develop an Immunodeficiency Syndrome Severe. Unlike murine Treg, human Treg do not express CD73. However we could identify a non-regulatory population of CD4+ T cells (Tconv) expressing CD73, and thus cooperating with CD39+ Treg for Ado generation. This population has an increased capacity of secretion of inflammatory cytokines (IFNgamma, IL-17A, IL-22, GM-CSF) and the expression of molecules (CXCR3, CCR6, MDR1) characteristics of Th1/17 profile. Moreover, these cells appear to be less sensitive to regulation mediated by the immunocheckpoints (ICPs), such as PD-1, CTLA-4, TIM-3, TIGIT. Nonetheless CD73+ Tconv are sensitive to Ado generated in cooperation with CD39+ Treg which induce the inhibition of their proliferation and their secretion of IFNgamma and GM-CSF but not IL-17A . Ado acting locally, can also inhibit the Tconv CD73neg in the surrounding environment. All these results show that the expression of CD73 characterizes a population of multifunctional effector T CD4 Th1/17, which is a specific and cooperative target of Treg immunosuppression in the tumor environment. In addition the action of Ado transforms this potent anti-tumor effector to potentially pro-tumor cells which only secret IL17

Millions of people worldwide die of cancer every year. In the last decade, im- munotherapy offered new treatment options achieving long-lasting remissions in a number of patients. Several new immunotherapy-based drugs have been ap- proved by Food and Drug Administration. However, majority of patients either do not respond or soon relapse. Combination of therapies as well as exploring new immune checkpoints seems promising. This thesis focuses on the new immunotherapeutic target CD73. CD73 is membrane ectonucleotidase, widely expressed on the regulatory leukocytes and on cancer cells. The enzymatically active CD73 contributes to the tumour mi- croenvironment by production of immunosuppressive adenosine. This novel im- mune checkpoint is being intensively studied. This thesis aims on development of new approaches for targeting and inhibition of CD73. Soluble recombinant CD73 (rhCD73) was prepared in mammalian expression system and transfectants stably expressing membrane-bound CD73 were prepared as well. Inhibitors necessary for both of my goals have been designed based on published inhibitor of CD73. Development and evaluation of novel antibody mimetic for CD73 characteri- sation was done. The so-called iBody, HPMA polymer conjugate decorated with CD73 inhibitor for targeting, fluorophore for...

Millions of people worldwide die of cancer every year. In the last decade, im- munotherapy offered new treatment options achieving long-lasting remissions in a number of patients. Several new immunotherapy-based drugs have been ap- proved by Food and Drug Administration. However, majority of patients either do not respond or soon relapse. Combination of therapies as well as exploring new immune checkpoints seems promising. This thesis focuses on the new immunotherapeutic target CD73. CD73 is membrane ectonucleotidase, widely expressed on the regulatory leukocytes and on cancer cells. The enzymatically active CD73 contributes to the tumour mi- croenvironment by production of immunosuppressive adenosine. This novel im- mune checkpoint is being intensively studied. This thesis aims on development of new approaches for targeting and inhibition of CD73. Soluble recombinant CD73 (rhCD73) was prepared in mammalian expression system and transfectants stably expressing membrane-bound CD73 were prepared as well. Inhibitors necessary for both of my goals have been designed based on published inhibitor of CD73. Development and evaluation of novel antibody mimetic for CD73 characteri- sation was done. The so-called iBody, HPMA polymer conjugate decorated with CD73 inhibitor for targeting, fluorophore for...

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