Thesis (doctoral)--University of Lund, 1996. / Added t.p. with thesis statement added.

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains vi, 183 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Les patients admis en réanimation semblent pouvoir présenter des infections sévères à CMV, en dehors de tout traitement immunosuppresseur, mais l’incidence exacte de ces infections est difficile à évaluer. Nous avons réalisé un dépistage systématique du CMV chez 242 patients consécutifs, considérés non immunodéprimés avant leur admission en réanimation. Nous avons ainsi identifié que 16% des patients développaient au cours de leur séjour en réanimation une infection à CMV. La mortalité des patients ayant présenté une infection à CMV était supérieure à ceux n’ayant pas présenté l’infection. La physiopathologie des réactivations à CMV chez ces patients est incomprise. Nous avons sélectionné les patients de réanimation non-immunodéprimés et séropositif pour le CMV à l’admission, et nous avons comparé le statut des NK circulants de 15 patients ayant présenté une réactivation CMV dépistée par antigénémie circulante (les cas) à celui de 15 patients contrôles appariés aux premiers pour l’âge, le sexe, et la gravité à l’admission. Dans la période précédant la réactivation CMV, alors que les capacités de cytotoxicité sont comparables, nous mettons en évidence une déficience NK en termes de capacité de production d’interféron gamma chez les cas en comparaison des contrôles, et également de témoins sains. Le sepsis, cad le tableau clinique résultant de la réponse à toute infection, est dans sa forme la plus sévère, l’une des principales causes d’admission en service de réanimation. Nous avons réalisé un « immunomonitoring » complet des NK circulantes à la phase initiale d’états pro-inflammatoires chez 42 patients admis en réanimation pour des motifs infectieux (sepsis sévère ou choc septique) ou des tableaux de SIRS non infectieux. Les patients septiques présentaient une capacité réduite de dégranulation en comparaison des patients non-septique (SIRS). Ils présentaient également une réduction de la sécrétion d’interféron-, toujours en comparaison des patients SIRS. / Patients admitted to the intensive care unit (ICU) seem to have severe infections with CMV, without any immunosuppressive therapy, but the exact incidence of these infections is difficult to assess. We conducted a systematic screening of CMV in 242 consecutive patients non-immunosuppressed before ICU admission. We identified that 16% of patients developed CMV infection during their stay in ICU. The mortality of patients with CMV infection was higher than those without such infection. The pathophysiology of CMV reactivation occurring in critically ill patients with no previous immunosuppression is misunderstood. We selected non-immunocompromised ICU patients, CMV seropositive at admission, and we compared the status of circulating NK of 15 patients with CMV reactivation detected by antigenemia (cases) to 15 controls matched for age, gender, and severity on admission. In the period preceding the CMV reactivation, whereas capabilities cytotoxicity (degranulation) are comparable, we show impaired NK in terms of production of gamma interferon in cases compared to controls, and also healthy. Levels of IL-10 were significantly higher in cases with a strong correlation between the levels of this cytokine and severity of CMV reactivation as measured by the number of cells positive antigenemia. Sepsis, the clinical picture resulting from the response to any infection, in its severest form, is one of the main causes of admission in ICU. We conducted a complete immunomonitoring of circulating NK in the initial phase of pro-inflammatory state in 42 patients admitted to intensive care for reasons of infection (severe sepsis or septic shock) or of non-infectious SIRS (systemic inflammatory response syndrome). Septic patients showed a reduced ability of degranulation compared with non-septic patients (SIRS). They also showed a reduction in the secretion of gamma interferon, compared with patients with SIRS.

Les cellules Natural Killer (NK) sont des lymphocytes du système immunitaire inné capables de tuer des cellules cibles et de produire des cytokines telles que l'interféron-γ. Au cours de mon travail de thèse, j'ai utilisé des approches de génétique directe et inverse dans le but d'étudier les mécanismes impliqués dans la régulation des capacités effectrices des cellules NK. La tolérance des cellules NK au soi est en partie assurée par les récepteurs inhibiteurs de surface qui sont spécifiques des molécules du complexe majeur d'histocompatibilité de classe I (CMH-I) exprimées par les cellules du soi. Cependant, des cellules NK qui ne sont pas capables de détecter l'expression du CMH-I ne sont pas autoréactives. Dans la première partie de ce travail de thèse, nous avons cherché à déterminer, chez la souris, les mécanismes de la tolérance NK, indépendante de la reconnaissance du CMH-I, qui est associée à une hyporeactivité des cellules NK. En utilisant des techniques de spectrométrie de fluorescence par corrélation à spot variable (svFCS), nous avons montré que dans les cellules NK hyporéactives les récepteurs activateurs et inhibiteurs sont confinés à la membrane plasmique par des réseaux structurés d'actine. A l'inverse, la reconnaissance par les cellules NK du CMH-I, qui « éduque » les cellules NK pour qu'elles acquièrent leurs capacités effectrices maximales, est associée une relocalisation des récepteurs activateurs au sein de nanodomaines. Ces résultats suggèrent que ce serait le confinement particulier des récepteurs activateurs à la membrane des cellules NK qui assure la tolérance au soi. / Natural killer (NK) cells are bone marrow-derived innate immune lymphocytes able to kill cellular targets and secrete cytokines such as interferon-γ. During my PhD work, I used reverse and forward genetic approaches to dissect the mechanisms involved in the regulation of NK cell effector capabilities at steady state. NK cell tolerance to self is partly ensured by major histocompatibility complex class I (MHC- I)-specific inhibitory receptors on NK cells, which detect MHC-I expression on self-cells and prevent NK cell activation. However, NK cells that do not detect self MHC-I are not autoreactive. In the first part of this PhD work, we sought to determine the mechanism at the basis of this MHC-I independent NK cell tolerance. Using spot variation fluorescence correlation spectroscopy (svFCS), we showed that MHC-I-independent NK cell tolerance in mice was associated with the presence of hyporesponsive NK cells in which both activating and inhibitory receptors were confined in an actin meshwork at the plasma membrane. In contrast, the recognition of self MHC-I by inhibitory receptors "educated" NK cells to become fully reactive, and activating NK cell receptors became dynamically compartmentalized in membrane nanodomains. We thus propose that the confinement of activating receptors at the plasma membrane is essential to ensuring self-tolerance of NK cells.

Ly49 receptors are the canonical natural killer cell class-I major histocompatibility complex receptors expressed in mice. They have a well-defined role in natural killer cell self/non-self discrimination and in the developmental licensing of functional natural killer cells. In this thesis, I report two novel aspects of Ly49 receptor biology. First, I show that their expression may be regulated by specific nucleosome occupancy on AML-1 binding sites within the distal Ly49 promoter. This finding sheds light on a potential regulatory pathway that has thus far been unexplored in studies of the Ly49 receptor family, and highlights the Ly49 family as an ideal model system in which to study the impact of nucleosome occupancy in general. Second, I show that Ly49 receptors have a central and indispensable role in the emerging phenomenon known as adaptive natural killer cell memory. Natural killer cells have recently been observed displaying adaptive, long-lived, antigen specific memory responses comparable to T cell memory responses, but no explanatory mechanism has been discovered to describe how adaptive memory is possible in these ‘innate’ immune cells. Using Ly49-deficient mice, I show that the inhibitory, self-specific Ly49 receptors Ly49C and Ly49I are required for adaptive memory responses to chemical haptens or protein antigens. Moreover, I show that Ly49C/I binding capabilities are required during all stages of the memory response, as is antigen presentation in the context of class I major histocompatibility complex, again analogous to T cell memory responses. I present initial findings implicating these Ly49 receptors as key components of the antigen recognition process itself, and propose a mechanism based in evolutionarily ancient immunology to explain how this specificity could arise. Finally, I demonstrate that Ly49-dependent natural killer cell memory is capable of mediating powerful anti-cancer vaccination effects using an aggressive model of melanoma. Together, these findings in Ly49 family expression regulation and its functional role in adaptive NK cell responses open several new avenues of study in Ly49 receptor biology and natural killer cell immunology.

Submitted by VANESSA SANTOS MOURA null (vanessa_smoura@hotmail.com) on 2017-08-29T04:50:50Z No. of bitstreams: 1 Dissertação_Vanessa_Santos_Moura.pdf: 2606729 bytes, checksum: 3c16d37b47ce11933b2d1f88e4aa9b1f (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-08-29T18:18:44Z (GMT) No. of bitstreams: 1 moura_vs_me_jabo.pdf: 2606729 bytes, checksum: 3c16d37b47ce11933b2d1f88e4aa9b1f (MD5) / Made available in DSpace on 2017-08-29T18:18:44Z (GMT). No. of bitstreams: 1 moura_vs_me_jabo.pdf: 2606729 bytes, checksum: 3c16d37b47ce11933b2d1f88e4aa9b1f (MD5) Previous issue date: 2017-07-31 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O bolor verde e a podridão azeda destacam-se entre as doenças de pós-colheita em frutos cítricos, causados por Penicillium digitatum e Geotrichum citri-aurantii, diminuindo a qualidade e a quantidade dos frutos e, consequentemente, resultando em significativas perdas econômicas. Uma alternativa para controle destes fungos é através da toxinas killer produzidas por algumas espécies de levedura, capazes de matar fungos filamentosos. Saccharomyces cerevisiae produz toxinas killer proteicas que são letais para células sensíveis de levedura. Estas toxinas foram agrupadas em quatro tipos, K1, K2, K28 e Klus, codificado por elementos extra cromossomais associados a partículas virais na forma de dsRNA. Este trabalho tem como objetivo caracterizar a toxina killer de S. cerevisiae ACB-K1 e testar sua atividade antagônica em patógenos pós-colheita de citros. O isolado ACB-K1 apresentou atividade killer, sobre levedura sensível (S. cerevisae NCYC 1006) além do fitopatógeno P. digitatum, não apresentando porém inibição contra o patógeno G. citri-aurantii. A toxina apresentou máxima atividade em pH 4,1 a 22 °C, tanto para a levedura sensível quanto para o fitopatógeno P. digitatum. A toxina apresentou estabilidade em diferentes pH de 4,1 a 6,0, após a incubação de 24h a 22 °C sobre o fungo. O isolado ACB-K1 apresentou dsRNA, sendo detectadas duas formas (LA e M-dsRNA), sugerindo que a base genética para a produção da toxina é extra cromossomal, dado confirmado pela cura do fenótipo killer a 40 °C. As frações obtidas por cromatografia de exclusão molecular em gel de Sephadex G75 demonstraram características de biocontrole contra o fitopatógeno P. digitatum. / Green mold and sour rot are among post-harvest diseases in citrus fruits, caused by Penicillium digitatum and Geotrichum citri-aurantii, reducing a quality and quantity of fruits and, consequently, resulting in significant economic losses. An alternative for the control of fungi is using killer toxins produced by some species of yeasts, capable of killing filamentous fungi. Saccharomyces cerevisiae produces protein killer toxins that are lethal to yeast sensitive cells. These toxins were grouped into four types, K1, K2, K28 and Klus, encoded by extrachromosomal elements associated with viral particles in the form of dsRNA. This work aims to characterize a killer toxin of S. cerevisiae ACB-K1 and to test its antagonistic activity in post-harvest citrus pathogens. The isolate ACB-K1 showed activity killer on sensitive yeast (S. cerevisae NCYC 1006) besides the phytopathogenic P. digitatum, but did not present inhibition against the pathogen G. citri-aurantii. The killer toxin showed maximum activity at pH 4.1 at 22 ° C for both a sensitive yeast and the phytopathogenic P. digitatum. The toxin presented stability at pH range from 4.1 to 6.0, after a 24h incubation at 22 ° C on the fungus. The ACB-K1 isolate showed dsRNA and two forms were detected (LA and M-dsRNA), suggesting that a genetic basis for a toxin production is extrachromosomal, confirmed by curing the killer phenotype at 40 ° C. The fractions obtained by exclusion chromatography Sephadex G75 gel, demonstrated biocontrol characteristics against the phytopathogen P. digitatum.

As células Natural Killer (NK), quando estimuladas por meio de seus receptores, rapidamente produzem citocinas e quimiocinas, incluindo IFN?, TNF?, TGF?, GMCSF, MIP1?, MIP1?, IL-10 e outras, as quais podem afetar a função de outras células hematopoéticas. Considerando as evidências recentes de que as célulastronco hematopoéticas (CTH) respondem diretamente à sinalização de várias citocinas, acreditamos que a produção de citocinas mediada pelas células NK possa regular a função da CTH e que a sua desregulação possa favorecer a transformação maligna. As neoplasias mieloproliferativas (NMP) negativas para o rearranjo t(9;22)/BCR-ABL1, incluindo as entidades Policitemia Vera (PV), Trombocitemia Essencial (TE) e Mielofibrose Primária (MFP), são doenças hematopoéticas originadas de alteração clonal da CTH, e podem servir de modelo para o estudo dessa regulação NK-CTH. Além de mutações que ativam vias de proliferação e sobrevivência celular, como a mutação JAK2V617F, presente em mais da metade das NMP, outros mecanismos contribuem para a patogênese e manutenção da doença, tais como mutações adicionais e regulação da hematopoese neoplásica pelo microambiente da medula óssea. Este último inclui não apenas células do estroma, mas também células do sistema imune. Dessa forma, com objetivo de investigar a potencial contribuição das células NK para a patogênese das NMP, caracterizamos células do sangue periférico de pacientes com NMP do Ambulatório de Hematologia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto, bem como células esplênicas obtidas de animais de um modelo murino condicional knockin de expressão heterozigótica da Jak2V617F (Jak2VF) quanto à frequência, expressão de receptores e função das células NK. Observamos menor porcentagem de células NK e maior expressão do receptor inibitório NKG2A nos animais Jak2 wt/VF. Pacientes portadores de NMP apresentaram número absoluto de células NK-CD16+ reduzido em relação aos controles saudáveis. O número de células NK-CD16+ foi menor na MFP em relação aos controles e à TE, particularmente naqueles portadores da mutação JAK2V617F. Encontramos menor expressão do receptor de ativação NKG2D nos portadores de PV positivos para a mutação JAK2V617F. Houve menor expressão do receptor de ativação NKp46 nos portadores de NMP, particularmente nos portadores da mutação JAK2V617F, e nos pacientes com MFP. Observamos também menor expressão do receptor NKG2A nos portadores de TE negativos para a mutação JAK2V617F. Em concordância com a redução de células NK, a porcentagem de linfócitos totais mostrou-se reduzida nos pacientes com NMP, particularmente nos portadores de MFP, independentemente da mutação JAK2V617F. Encontramos redução percentual e absoluta do subtipo de células NK CD56brightCD16- (cuja principal função é secretória) nos pacientes com NMP, especialmente na presença da mutação JAK2V617F, nos pacientes com PV e MFP, e menor frequência absoluta do subtipo CD56-CD16bright (com função primariamente citotóxica) nos portadores de MFP. Em contraste, não verificamos deficiência citotóxica das células NK dos animais Jak2 wt/VF em relação aos controles Jak2 wt/wt. Adicionalmente, as células NK dos animais Jak2-mutados demonstraram menor capacidade de secreção da citocina MIP-1?, reconhecida por regular a função de CTH, em relação aos animais controle. Finalmente, houve expressão significativamente aumentada do gene MyD88 nos animais mutados em relação aos controles, sugerindo que a via de sinalização dos receptores do tipo Toll (TLR) pode estar envolvida na regulação NKCTH nas NMP. Em resumo, detectamos deficiência numérica e funcional de células NK em células primárias murinas e humanas de NMP. Nossos achados sugerem potencial regulação da hematopoese maligna pelas células NK nestas neoplasias e podem contribuir para a identificação de novas estratégias terapêuticas que possam interferir nesta complexa interação. / Natural Killer (NK) cells, when stimulated by their receptors, rapidly produce cytokines and chemokines, including IFN?, TNF?, TGF?, GM-CSF, MIP1?, MIP1?, IL-10 and others, which may affect the function of other hematopoietic cells. Considering the recent evidence that hematopoietic stem cells (HSC) directly respond to cytokine signaling, we hypothesized that NK cells mediated cytokine production can regulate HSC function and that their dysregulation may favor malignant transformation. BCR-ABL1-negative myeloproliferative neoplasms (MPN), including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF), are hematopoietic diseases originated from HSC clonal transformation, and thus can serve as a model for studying this NK-HSC regulation. In addition to mutations that activate cell proliferation and survival pathways, such as the JAK2V617F mutation, present in more than half of MPN cases, other mechanisms contribute to the pathogenesis and maintenance of the disease, such as additional mutations and regulation of neoplastic hematopoiesis by the bone marrow microenvironment. This latter includes not only stromal cells but also cells of the immune system. Therefore, in order to investigate the potential contribution of NK cells to the pathogenesis of MPN, we characterized the frequency, receptor expression and function of NK cells from patients with MPN from the Clinical Hospital of the Medical School of Ribeirão Preto, University of São Paulo, as well as from splenic cells obtained from animals of a conditional knockin murine model of Jak2V617F heterozigous expression. Lower percentage of NK cells and higher NKG2A inhibitory receptor expression was observed in Jak2 wt/VF animals as compared to Jak2 wt/wt controls. In agreement, patients with MPN presented reduced absolute numbers of NK-CD16+ cells when compared to healthy controls. The number of NKCD16+ cells was lower in the PMF than in controls or ET patients, particularly in those bearing the JAK2V617F mutation. We found lower expression of the NKG2D activatory receptor in PV patients with the JAK2V617F mutation. There was lower expression of the NKp46 activatory receptor in MPN patients, particularly in those with the JAK2V617F mutation, and in PMF patients. We also observed reduced expression of the NKG2A receptor in non-JAK2 mutated ET patients. In agreement with the NK cell reduction, the percentage of total lymphocytes was reduced in patients with MPN, particularly in PMF, regardless of the JAK2V617F mutation. We found an absolute decrease of the CD56brightCD16- NK subtype (whose main function is secretory) in patients with MPN, especially when the JAK2V617F mutation was present, in patients with PV and PMF. Also, lower absolute frequency of CD56-CD16bright NK subtype (primarily cytotoxic) was found in PMF patients. In contrast, we did not find cytotoxic deficiency in the Jak2 wt/VF NK cells as compared to the Jak2 wt/wt controls. In addition, Jak2-mutated NK cells presented reduced ability of secreting the cytokine MIP-1?, known to regulate HSC function. Finally, there was significantly increased expression of the MyD88 gene in the Jak2-mutated animals as compared to controls, suggesting that the Toll-like receptors (TLR) signaling pathway may be involved in NK-HSC regulation in MPN. In summary, we detected numerical and functional deficiency of NK cells in murine and human primary cells of MPN. Our findings suggest a potential regulation of malignant hematopoiesis by NK cells in these neoplasms and may contribute to the identification of new therapeutic strategies that may target this complex interaction.

Les cellules NK sont des lymphocytes de l'immunité innée impliqués dans la reconnaissance et l'élimination de cellules tumorales ou infectées par des pathogènes intracellulaires. La biologie des cellules NK est régulée par des facteurs intrinsèques comme les facteurs de transcription ainsi que par des facteurs environnementaux comme les cytokines, produites en condition homéostatique ou inflammatoire. Certaines cytokines, comme l'IL-15, l'IL-12 ou l'IL-18 sont connues pour potentialiser les fonctions effectrices des cellules NK. L'IL-15, en activant la voie STAT5 permet, d'une part, d'assurer la survie des cellules NK et, d'autre part via la kinase mTOR, d'induire leur prolifération, d'augmenter leur métabolisme ainsi que leurs fonctions effectrices. D'autres cytokines comme le TGF-ß sont connues pour inhiber les fonctions des cellules NK. Le TGF-ß1 est une des cytokines les plus immunosuppressives du système immunitaire et, en étant secrété´ par différents types de cancers, il participe a` l'échappement tumoral. Depuis longtemps, les effets du TGF-ß in vitro sont connus pour contrer ceux de l'IL-15. L'objectif de ce travail a été´ d'étudier les effets du TGF-ß sur la biologie des cellules NK. Nous avons observé´ que l'ajout de TGF-ß, in vitro, induit un blocage rapide de l'activation de la voie mTOR par l'IL-15, que le TGF-ß a des effets très proches de ceux de la rapamycine, un inhibiteur spécifique de mTOR et que, in vivo chez la souris, l'activation constitutive des voies de signalisation activées par le TGF-ß induit un phénotype proche de celui de la délétion de mTOR dans les cellules NK / NK cells are innate lymphocytes involved in the recognition and elimination of tumor or infected cells. The biology of NK cells is regulated by intrinsic factors such as transcription factors but also by cytokines produced at steady state or under inflammatory conditions. Some of these cytokines like IL-15, IL-12 or IL-18 are known to increase NK cells functions. IL-15 allows NK cell survival via STAT5 and, via mTOR, increase NK cell proliferation, metabolism and acquisition of functions. In the other hand, cytokines like TGF-ß are known to inhibit NK cell function. TGF-ß1 is a major immunosuppressive cytokine, often secreted by tumor cells and participates to tumor escape. The inhibitory effects of TGF-ß in vitro on IL-2/15 mediated NK cell activation have long been shown, but the mechanism remains unknown. The objective of this work was to characterize the effects of TGF-ß at a molecular level. We have observed that TGF-ß induces a rapid blockade of IL-15 induced mTOR activation, in vitro. TGF-ß and the mTOR inhibitor rapamycin have similar effects. Finally, using genetic mouse models in vivo, constitutive TGF-ß signaling or mTOR deletion results in similar developmental arrests in NK cells

Dans les pays occidentaux, les lymphomes T périphériques et NK représentent environ 10% des lymphomes non-Hodgkiniens. Le lymphome NK/T de type nasal est l'une des entités de présentation extra-ganglionnaire les plus fréquentes, en Asie, et en Amérique Centrale et du Sud. Il survient classiquement dans la sphère nasopharyngée avec une prédilection pour les adultes jeunes. Morphologiquement, la tumeur est souvent angiocentrique avec une invasion de la paroi des vaisseaux par les cellules tumorales d'aspect variable. Ces lymphomes ont le plus souvent une origine NK avec un phénotype CD3+ (cytoplasmique), CD5-, CD56+, CD4-/CD8-, expression des molécules cytotoxiques et absence de réarrangement des gènes des récepteurs T. Le virus d'Epstein-Barr est présent dans la quasi-totalité des cellules tumorales dans sa forme clonage épisomale, avec une latence de type II, suggérant son rôle dans l'oncogenèse. A côté des mutations fréquentes des gènes FAS et TP53 (p53) et des méthylations de TP73 et CDKN2A (p16), des délétions du bras long du chromosome 6q sont fréquemment observées. Très récemment, des méthylations et des mutations des gènes suppresseurs de tumeur PRDM1, ATG5, et AIM1 localisés en 6q21 ont été retrouvées dans les lignées de lymphome NK/T de type nasal. Nous avons réalisé une analyse combinée du profil d'expression génique et du profil génomique par hybridation comparative sur puces, d'échantillons tumoraux de lymphome NK/T de type nasal (n=9) et de lignées, comparés à celle de lymphocytes NK normaux et de lymphomes T périphériques, sans autre spécificité (PTCL, NOS). Nous avons identifié la signature moléculaire particulière du lymphome NK/T de type nasal caractérisée par un haut niveau des trascrits de marqueurs de cellules NK et de molécules cytotoxiques, notamment de granzyme H dans les lymphomes NK/T de type nasal comparé aux PTCL, NOS. Par immunohistochimie, nous avons validé l'expression "spécifique" de granzyme H par les cellules tumorales du lymphome NK/T de type nasal, qui pourrait constituer un nouveau marqueur de ces lymphomes. Comparé aux cellules NK normales, le lymphome NK/T de type nasal a une signature plus proche des cellules NK activées que des NK au repos et sur-expriment des gènes associés à la biologie vasculaire, des gènes induits par l'EBV, et PDGFRA. Nous avons confirmé l'expression protéique de PDGFRAa et de sa forme phosphorylée, et montré in vitro la sensibilité de la lignée tumorale MEC04 à l'imatinib mesytale. La dérégulation des voies de signalisation AKT, JAK-STAT et NF-kB, suggérée par les analyses bioinformatiques, a été corroborée par la mise en évidence d'une expression nucléaire des formes phosphorylées d'AKT, de STAT3 et de RelA dans les lymphomes NK/T de type nasal. De plus, plusieurs gènes dérégulés dans ces voies moléculaires sont localisés dans des régions altérées de manière récurrente par des gains ou des pertes (AKT3 (1q44), IL6R (1q21.3), CCL2 (17q12), TNFRSF21 (6p12.3)). En plus de l'activation constitutive de STAT3 confirmée par l'expression nucléaire de phospho-STAT3, l'inhibition de croissance et l'augmentation de la mort cellulaire des cellules de la lignée MEC04 résultant de l'inhibition de STAT3 conforte le rôle de STAT3 dan la lymphomagenèse du lymphome NK/T nasal. L'analyse intégrée a également mis en évidence la dérégulation du gène suppresseur de tumeur HACE1 en 6q21, confirmée par RT-PCR quantitative. Bien que les mécanismes exacts conduisant à l'activation de plusieurs voies moléculaires, de même qu'à la dérégultaion de HACE1 ne soient pas déterminés, nos résultats identifient plusieurs voies oncogéniques impliquées dans le lymphome NK/T de type nasal ainsi que de nouveaux biomarqueurs diagnostiques - comme granzyme H - et des cicles thérapeutiques d'intérêt. L'étude en cours du profil d'expression des microARNs pourrait apporter un éclairage sur les mécanismes impliqués dans certaines voies identifiées / In Western countries, mature natural killer (NK)- and T-cell lymphomas account for 15% to 20% of aggressive lymphomas and around 10 % of all non-Hodgkin lymphomas. This number is higher in Asia, with 25% in Japan and 39% in Taiwan. Among those T- and NK-cell lymphomas with primary extranodal presentation, extranodal NK/T-cell lymphoma of nasal type (nasal NKTCL) is one of the most common entities in Asian, Central and South American populations. It classically arises in the nasal region showing a predilection for young adults with male predominance. This tumor morphologically exhibits an angiocentric and angio destructive growth pattern, admixed with polymorphous non-neoplastic infiltrates. Most tumor cells have a cytoplasmic CD3+, CD5-, CD56+, CD4-/CD8- phenotype with expression of cytotoxic granule-associated proteins and without rearrangement of T-cell receptors genes. Killer immunoglobulin-like receptors have been reproted to be expressed in a subset of this lymphoma and its expression might be associated with prognosis. Epstein-Barr virus is present in virtually all neoplastic cells in its clonal episomal form with type II latency program, implying a role in oncogenesis. Although the results were variable between different studies, methylations of TP73 (p73) and CDKN2A (p16) and mutations of FAS and TP53 (p53) were frequently found in nasal NKTCL. Genomic alterations have also been reported in nasal NKTCL with frequent deletion in chromosome 6q. A very recent study also identified both methylations and mutations of three putative tumor suppressor genes PRDM, ATG5, and AIM1 mapping to del6q21 in nasal NKTCL cell times. We performed integrative gene expression profiling and array-based comparative genomic hybridization analyses of nasal NKTCL tumors as well as tumour-derived cell lines, compared to that of normal NK cells and peripheral T-cell lymphomas, not otherwise specified (PTCL, NOS). We identified the distinctive molecular signature of nasal NKTCL with high transcript levels for NK-cell markers ans cytotoxic molecules, especially granzyme H in nasal NKTCL compared to PTCL, NOS. By immunohistochemistry, we validated expression of grnzyme H which appears a novel sensitive biomarker of nasal NKTCL. Compared to normal NK cells, nasal NKTCL tumors were closer to activated than resting cells and overexpressed several genes related to vascular biology, EBV-induced genes and PDGFRA. Notably, we confirmed the expression of PDGFRa and its phosphorylated form at the protein level, and in vitro the MEC04, nasal NKTCL-cell line, was sensitive to imatinib mesylate. Deregulation of the AKT, JAK-STAT and NF-kB pathways suggested by bioinformatical analysis, was corroborated by nuclear expression of phosphorylated AKT, STAT3 and RelA in nasal NKTCL, and several deregulated genes in these pathways mapped to regions of recurrent copy number aberrations (AKT3 (1q44), IL6R (1q21.3), CCL2 (17q12), TNFRSF21 (6p12.3)). In addition to constitutive activation of STAT3 as confirmed by the demonstration of phosphorylated STAT3 in the nuclei of neoplastic nasal NKTCL cells, growth inhibition and cell death of nasal NKTCL cells induced by STAT3 inhibition implied the role of STAT3 in the nasal NK/T-cell lymphomagenesis. Integrative analysis and qRT-PCR analysis also evidenced deregulation of another tumor suppressor HACE1 in the frequently deleed 6q21 region. Although the exact mechanism of activation of several pathways as well as that of HACE1 deregulation remains to be determined, our studies highlight emerging oncogenic pathways in nasal NKTCL and identify novel diagnostic and therapeutic targets. The ongoing investigation of microRNA expression profiling might shed light in a better understanding of the pathogenesis of nasal NKTCL and especially of the activation of oncogenic pathways. Connectivity map analysis may also help to depict other targeted therapies useful to improve the prognosis of this agressive lymphoma

As células Natural Killer (NK), quando estimuladas por meio de seus receptores, rapidamente produzem citocinas e quimiocinas, incluindo IFN?, TNF?, TGF?, GMCSF, MIP1?, MIP1?, IL-10 e outras, as quais podem afetar a função de outras células hematopoéticas. Considerando as evidências recentes de que as célulastronco hematopoéticas (CTH) respondem diretamente à sinalização de várias citocinas, acreditamos que a produção de citocinas mediada pelas células NK possa regular a função da CTH e que a sua desregulação possa favorecer a transformação maligna. As neoplasias mieloproliferativas (NMP) negativas para o rearranjo t(9;22)/BCR-ABL1, incluindo as entidades Policitemia Vera (PV), Trombocitemia Essencial (TE) e Mielofibrose Primária (MFP), são doenças hematopoéticas originadas de alteração clonal da CTH, e podem servir de modelo para o estudo dessa regulação NK-CTH. Além de mutações que ativam vias de proliferação e sobrevivência celular, como a mutação JAK2V617F, presente em mais da metade das NMP, outros mecanismos contribuem para a patogênese e manutenção da doença, tais como mutações adicionais e regulação da hematopoese neoplásica pelo microambiente da medula óssea. Este último inclui não apenas células do estroma, mas também células do sistema imune. Dessa forma, com objetivo de investigar a potencial contribuição das células NK para a patogênese das NMP, caracterizamos células do sangue periférico de pacientes com NMP do Ambulatório de Hematologia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto, bem como células esplênicas obtidas de animais de um modelo murino condicional knockin de expressão heterozigótica da Jak2V617F (Jak2VF) quanto à frequência, expressão de receptores e função das células NK. Observamos menor porcentagem de células NK e maior expressão do receptor inibitório NKG2A nos animais Jak2 wt/VF. Pacientes portadores de NMP apresentaram número absoluto de células NK-CD16+ reduzido em relação aos controles saudáveis. O número de células NK-CD16+ foi menor na MFP em relação aos controles e à TE, particularmente naqueles portadores da mutação JAK2V617F. Encontramos menor expressão do receptor de ativação NKG2D nos portadores de PV positivos para a mutação JAK2V617F. Houve menor expressão do receptor de ativação NKp46 nos portadores de NMP, particularmente nos portadores da mutação JAK2V617F, e nos pacientes com MFP. Observamos também menor expressão do receptor NKG2A nos portadores de TE negativos para a mutação JAK2V617F. Em concordância com a redução de células NK, a porcentagem de linfócitos totais mostrou-se reduzida nos pacientes com NMP, particularmente nos portadores de MFP, independentemente da mutação JAK2V617F. Encontramos redução percentual e absoluta do subtipo de células NK CD56brightCD16- (cuja principal função é secretória) nos pacientes com NMP, especialmente na presença da mutação JAK2V617F, nos pacientes com PV e MFP, e menor frequência absoluta do subtipo CD56-CD16bright (com função primariamente citotóxica) nos portadores de MFP. Em contraste, não verificamos deficiência citotóxica das células NK dos animais Jak2 wt/VF em relação aos controles Jak2 wt/wt. Adicionalmente, as células NK dos animais Jak2-mutados demonstraram menor capacidade de secreção da citocina MIP-1?, reconhecida por regular a função de CTH, em relação aos animais controle. Finalmente, houve expressão significativamente aumentada do gene MyD88 nos animais mutados em relação aos controles, sugerindo que a via de sinalização dos receptores do tipo Toll (TLR) pode estar envolvida na regulação NKCTH nas NMP. Em resumo, detectamos deficiência numérica e funcional de células NK em células primárias murinas e humanas de NMP. Nossos achados sugerem potencial regulação da hematopoese maligna pelas células NK nestas neoplasias e podem contribuir para a identificação de novas estratégias terapêuticas que possam interferir nesta complexa interação. / Natural Killer (NK) cells, when stimulated by their receptors, rapidly produce cytokines and chemokines, including IFN?, TNF?, TGF?, GM-CSF, MIP1?, MIP1?, IL-10 and others, which may affect the function of other hematopoietic cells. Considering the recent evidence that hematopoietic stem cells (HSC) directly respond to cytokine signaling, we hypothesized that NK cells mediated cytokine production can regulate HSC function and that their dysregulation may favor malignant transformation. BCR-ABL1-negative myeloproliferative neoplasms (MPN), including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF), are hematopoietic diseases originated from HSC clonal transformation, and thus can serve as a model for studying this NK-HSC regulation. In addition to mutations that activate cell proliferation and survival pathways, such as the JAK2V617F mutation, present in more than half of MPN cases, other mechanisms contribute to the pathogenesis and maintenance of the disease, such as additional mutations and regulation of neoplastic hematopoiesis by the bone marrow microenvironment. This latter includes not only stromal cells but also cells of the immune system. Therefore, in order to investigate the potential contribution of NK cells to the pathogenesis of MPN, we characterized the frequency, receptor expression and function of NK cells from patients with MPN from the Clinical Hospital of the Medical School of Ribeirão Preto, University of São Paulo, as well as from splenic cells obtained from animals of a conditional knockin murine model of Jak2V617F heterozigous expression. Lower percentage of NK cells and higher NKG2A inhibitory receptor expression was observed in Jak2 wt/VF animals as compared to Jak2 wt/wt controls. In agreement, patients with MPN presented reduced absolute numbers of NK-CD16+ cells when compared to healthy controls. The number of NKCD16+ cells was lower in the PMF than in controls or ET patients, particularly in those bearing the JAK2V617F mutation. We found lower expression of the NKG2D activatory receptor in PV patients with the JAK2V617F mutation. There was lower expression of the NKp46 activatory receptor in MPN patients, particularly in those with the JAK2V617F mutation, and in PMF patients. We also observed reduced expression of the NKG2A receptor in non-JAK2 mutated ET patients. In agreement with the NK cell reduction, the percentage of total lymphocytes was reduced in patients with MPN, particularly in PMF, regardless of the JAK2V617F mutation. We found an absolute decrease of the CD56brightCD16- NK subtype (whose main function is secretory) in patients with MPN, especially when the JAK2V617F mutation was present, in patients with PV and PMF. Also, lower absolute frequency of CD56-CD16bright NK subtype (primarily cytotoxic) was found in PMF patients. In contrast, we did not find cytotoxic deficiency in the Jak2 wt/VF NK cells as compared to the Jak2 wt/wt controls. In addition, Jak2-mutated NK cells presented reduced ability of secreting the cytokine MIP-1?, known to regulate HSC function. Finally, there was significantly increased expression of the MyD88 gene in the Jak2-mutated animals as compared to controls, suggesting that the Toll-like receptors (TLR) signaling pathway may be involved in NK-HSC regulation in MPN. In summary, we detected numerical and functional deficiency of NK cells in murine and human primary cells of MPN. Our findings suggest a potential regulation of malignant hematopoiesis by NK cells in these neoplasms and may contribute to the identification of new therapeutic strategies that may target this complex interaction.