Infusões de células T e células NK (Natural Killer) de sangue periférico estão sendo realizadas para tratamento de malignidades. Os linfócitos propagados ex vivo, em normóxia (20% O2), são intravenosamente infundidos e precisam sobreviver a hipóxia associada a circulação venosa, da medula óssea (5% O2) e do microambiente tumoral (1% O2). O objetivo principal deste estudo foi determinar a capacidade proliferativa das células humanas T e NK em normóxia (20% O2) versus hipóxia (1% O2), por 28 dias, utilizando uma célula apresentadora de antígeno artificial (aAPC) para propagação em grau clínico. As células T expostas a hipóxia cresceram 100 vezes menos que as células T cultivadas em normóxia, enquanto que houve uma diminuição de 1000 vezes na taxa proliferativa das células NK hipóxicas, que exibiram um aumento na apoptose bem como um prejuízo na citotoxicidade. Hipóxia também induziu uma diminuição na expressão dos receptores KIR, NCR e NKG2D das células NK. Nesta mesma condição, a produção de IL-2 e IFNγ nas células T estavam diminuídas, sendo que nas células NK esse efeito foi mais acentuado. Hipóxia aumentou a expressão de genes relacionados com apoptose, angiogênese e metabolismo glicolítico, os quais estavam moderadamente aumentados nas células T, mas profundamente super-regulados nas células NK. Os níveis de ATP nas células T foram muito similares em ambas as condições de oxigênio, mas intensamente diminuídos nas células NK cultivadas em hipóxia. Também se observou, que a expressão do miR-210 induzido por hipóxia, estava super regulada nas células NK hipóxicas correlacionando com a perda da expressão da molécula NCAM/CD56. Em conjunto, os resultados deste estudo demonstram um maior impacto da hipóxia sobre as atividades proliferativas e citotóxicas das células NK estimuladas por aAPCs. Estudos adicionais são necessários para o entendimento do impacto deste comportamento das células NK em condições hipóxicas sobre a imunoterapia celular adotiva. / Infusions of T cells and natural killer (NK) cells from peripheral blood (PB) are being undertaken for the treatment of malignancies. Lymphocytes are propagated ex vivo in normoxia (20% O2) and intravenously infused and must survive hypoxia associated with venous blood and bone marrow (5% O2), and the tumor environment (1% O2). The objective this study was to determine the ability of T and NK cells to proliferate under normoxia (20% O2) versus hypoxia (1% O2) over 28 days using an artificial antigen presenting cells (aAPC) to propagate clinical-grade lymphocytes. T cells continuously exposed to 4 weeks of hypoxia grew at a rate of 100-fold less than T cells cultured in normoxia while the proliferative rate of NK cells lagged by 1,000-fold, behind normoxic conditions. Hypoxic cultured NK cells exhibit an increase in apoptosis as well as a correspondent impairment in cytotoxicity. In low oxygen tension the expression of KIR, NCR, and NKG2D receptors were decreased in NK cells. In hypoxia, the production of IL-2 and IFNγ were decreased in T cell and more so in NK cell. Chronic hypoxia increased the expression of related apoptosis, glycolytic metabolism and angiogenesis genes which were moderately increased in T cells but profoundly upregulated in NK cells. ATP levels in T cell were very similar in both oxygen conditions, but profoundly diminished in NK cells under hypoxia. We also noted that hypoxia inducible miR-210 levels are up regulated in hypoxic NK cells correlating with loss of CD56 expression. Taken together, this data show that a greater impact of hypoxia on proliferative and cytotoxic activity of NK cells activated by artificial antigen-presenting cells. More studies are needed to understand the impact of this behavior on the cell adoptive immunotherapy.

Orientadores: Carlos Alberto Petta, Fernando Guimarães / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T09:37:25Z (GMT). No. of bitstreams: 1 Alves_PauloCesarMartins_M.pdf: 1689043 bytes, checksum: d194c0a2789183c6c8600428851acdbc (MD5) Previous issue date: 2010 / Resumo:A possibilidade de se gerar preparações de células efetoras enriquecidas com células natural killer (NK) de pacientes com câncer tem sido foco de estudos que buscam fontes de células efetoras autólogas para o tratamento de tumores humanos. Objetivos: O objetivo neste estudo foi avaliar a possibilidade de se expandir células NK a partir do sangue de pacientes com neoplasia de ovário, assim como o potencial citotóxico destas células. Sujeitos e métodos: Células mononucleares do sangue periférico (CMSP) de 13 pacientes voluntárias com neoplasia de ovário, Grupo Benigno (n=7) e Grupo Maligno (n=6), foram cultivados em meio CellGro suplementado com anti-CD3 (20ng/ml, adicionado aos primeiros 10 dias), IL-2 ( 1000 UI / ml) e SFB (10%) por 21 dias. A ação citotóxica das preparações de células efetoras foi avaliada por citometria de fluxo utilizando como células alvo linhagens tumorais K562 e OVCAR3.Resultados: No Grupo Benigno, as células cultivadas por 21 dias apresentaram variação percentual positiva dos subtipos de linfócitos NK (17,1 ± 5,2%) e NK-T (10,4 ± 3,1%) associados à variação percentual negativa das células T (-6,2 ± 6,2 %). No Grupo Maligno, as células cultivadas por 21 dias apresentaram variação percentual positiva para todos os subtipos (NK 12,5 ± 6,0%, NK-T 4,2 ± 0,7% e T 15,3 ± 5,6%). As preparações de células enriquecidas com células NK, de ambos os grupos, foram citotóxicas contra as linhagens de células tumorais. A citotoxicidade foi significativamente (p <0,05) maior quando utilizadas células efetoras cultivadas por 21 dias, em comparação com células efetoras obtidas no dia 14 da cultura. Também foi observado maior ação citotóxica (p<0,05) nas frações, separadas magneticamente, contendo células CD56+ em comparação com as frações CD56-. Conclusões: Preparações de células efetoras ricas em células NK podem ser expandidas in vitro a partir de CMSP de pacientes com neoplasia de ovário. Estas células são providas de função citolítica, indicando o sistema de cultivo empregado como uma fonte viável de células efetoras autólogas / Abstract: The possibility of generating natural killer (NK) enriched cell preparations from cancer patients has been focused on studies seeking for autologous effector cell source for treatment of human malignancies. The objective of the present work was to evaluate the possibility of expanding NK cells from peripheral blood of patients with ovarian neoplasia, and to evaluate their cytotoxic function. Peripheral blood mononuclear cells (PBMC) from 13 volunteer patients with ovarian neoplasia, 7 benign and 6 malignant tumors, were cultured in CellGro medium supplemented with anti-CD3 (20ng/ml, for the 9-10 initial days), IL-2 (1000 UI/ml) and FBS (10%) for 21 days. The cytotoxic capacity of NK cells was evaluated using a flow cytometry-based cytotoxicity assay. The K562 and the OVCAR3 cell lines were used as target cells. Day-21 cultured cells of the benign neoplasia patients resulted in positive percentual variation of NK (+17.1±5.2%) and NK-T (+10.4±3.1%) lymphocyte subtypes associated with negative percentual variation of T cells (-6.2±6.2%). Differently, day-21 cultured cells of the malignant neoplasia patients presented positive percentual variation for all of the lymphocyte subtypes (NK +12.5±6.0%, NK-T +4.2±0.7% and T +15.3±5.6%). NK enriched effector cell preparations from both, benign and malignant ovarian neoplasia patients, were cytotoxic against the K562 and OVCAR3 cell lines. Cytotoxicity was significantly (p<0.05) higher using effector cells from day-21 cultures, compared with effector cells from day-14. Cytotoxicity was significantly (p<0.05) higher using magnetically separated effector cell fractions containing CD56+ cells compared with effector cell fractions deprived of these cells. The present study demonstrated that NK enriched effector cell preparations can be expanded in vitro from PBMC of ovarian neoplasia patients. These cells are provided with cytolytic function, indicating the culture system employed as a feasible source for autologous effector cells that should be evaluated for the NK-based adoptive therapy of cancer / Mestrado / Ciencias Biomedicas / Mestre em Tocoginecologia

Bei den konventionellen Krebstherapien kommt es häufig zu einer Wiederkehr des Tumors, da meist einzelne Tumorzellen und abgesiedelte Metastasen im Körper verbleiben. Vor diesem Hintergrund hat die Entwicklung neuer Behandlungsmethoden, die spezifisch die Tumorzellen erkennen und eliminieren und zudem gesunde Körperzellen schonen, eine große Bedeutung in der heutigen Krebsforschung. Eine erfolgsversprechende Strategie ist die Generierung von tumorspezifischen, zytotoxischen Immuneffektorzellen, zum Beispiel T-Lymphozyten und Natürlichen Killerzellen, durch die genetische Modifikation mit einem chimären Antigenrezeptor (CAR). Dabei gibt es bereits weitreichende Studien mit T-Lymphozyten, so dass sich nun das Forschungsinteresse immer mehr auf die NK-Zellen richtet. Im Gegensatz zu CAR-armierten T-Lymphozyten sind sie in der Lage ihr antitumorales Potenzial nicht nur gegen Antigen-positive sondern auch MHC-Klasse I-negative Tumorzellen zu richten. Mögliche Zielstrukturen der CAR sind tumorassoziierte Antigene, wie das Prostata-spezifische Stammzellantigen (PSCA). Es wird auf über 94 % der humanen primären Prostatakarzinome und deren Knochenmetastasen verstärkt exprimiert, jedoch kaum auf Normalgewebe. PSCA ist somit ideal für eine Immuntherapie geeignet. Die bisher in Studien verwendeten CAR-armierten NK-Zellen wiesen eine feststehende Spezifität gegenüber einem bestimmten Tumorantigen auf. Allerdings ist die Expression von Tumorantigenen innerhalb des Tumors sehr heterogen oder wird durch Tumorevasionsmechanismen herunterreguliert. Dies begrenzt die Reaktivität CAR-armierter NK-Zellen. Durch die Generierung eines CAR, dessen Spezifität gegenüber einem Tumorantigen ausgetauscht werden kann, wäre der universelle Einsatz CAR-armierter NK-Zellen in der adjuvanten Immuntherapie von Tumorerkrankungen möglich. Im Hauptteil dieser Arbeit wurden die permanente NK-Zelllinie YTS und primäre humane NK-Zellen mittels lentiviralen Gentransfers mit einem PSCA-spezifischen CAR, bestehend aus dem gegen PSCA gerichteten Einzelkettenantikörper αPSCA und dem aktivierenden NK-Zellrezeptor NKp46, armiert. Die generierten NK-Zellen wiesen eine über längere Zeiträume stabile Oberflächenexpression des CAR αPSCA-NKp46 auf. Die Kreuzvernetzung des CAR mit seinem Antigen führte zunächst zu keiner selektiven Immunantwort der CAR-armierten YTS und primären NK-Zellen gegenüber histogenetisch verschiedenen, PSCA-exprimierenden Tumorzelllinien. Erst nach gleichzeitiger Überexpression des mit NKp46 assoziierten Signaladaptermoleküls CD3-ζ wurde eine Aktivierung der Effektorfunktionen der YTS NK-Zellen induziert. Dies zeigte sich zum einen in der Expression von CD107a als Degranulationsmarker sowie der Freisetzung des inflammatorischen Zytokins IFN-γ. Zum anderen wiesen die CAR-armierten und CD3-ζ-exprimierenden YTS NK-Zellen eine spezifische Zytotoxizität gegenüber MHC-Klasse I- und PSCA-exprimierenden Tumorzellen auf. Im anschließenden Teil der Arbeit wurde ein modular aufgebauter CAR generiert, bei dem der Einzelkettenantikörper und folglich die Spezifität gegenüber Tumorantigenen austauschbar ist. Dazu wurden YTS NK-Zellen durch lentiviralen Gentransfer mit dem biotinylierbaren NKp46-NK-Zellrezeptor NKp46-KiBAP modifiziert, der über mehrere Monate stabil auf der Oberfläche exprimiert wurde. Die exogene als auch endogene Biotinylierung des Rezeptors wurde mittels einer Biotinproteinligase demonstriert. Unter Ausnutzung der sehr starken Biotin-Avidin-Bindung wurde die Assoziation mit einem Einzelkettenantikörper nachgewiesen. Dafür wurde exemplarisch der gegen PSCA gerichtete, biotinylierbare Einzelkettenantikörper αPSCA-BAP verwendet. Diese Arbeit zeigt, dass eine spezifische Erkennung und effiziente Lyse von PSCA-exprimierenden Tumorzellen durch die generierten CAR-armierten NK-Zellen erfolgte, wobei zum ersten Mal NKp46 als Bestandteil eines CAR verwendet wurde. Zudem wurde ein modular aufgebauter CAR generiert, dessen Spezifität gegenüber Tumorantigenen austauschbar ist. Diese neuartige Strategie ermöglicht erstmalig eine flexible Armierung von NK-Zellen und stellt damit einen wesentlichen Vorteil bei der Behandlung verschiedener Krebserkrankungen dar.

Natural killer (NK) cells represent a crucial component of the innate immune system and are primarily regulated by the interactions of their activation and inhibitory receptors with ligands available on target cells. The genetically linked Ly49 and NKR-P1 family of receptors constitute two of the major regulatory receptor systems used by NK cells and have been shown to bind different ligands. Whereas the Ly49 receptors survey MHC-I ligands on target cells, the NKR-Pl receptor family members bind to various members of the C-type lectin-related (Clr) family. Interestingly, NKR-P1 and Clr haplotypes possess a stable genomic polymorphism across multiple mouse strains, suggesting that this inhibitory receptor:ligand relationship has an important role in the maintenance of host cellular cognate specificities. The NKR-P1 and Clr receptor-ligand pairs identified in mice include the NKR-P1B:Clr-b and the NKR-P1G:Clr-f interacting pairs. Previous RT-PCR and in situ RNA hybridization data generated by our laboratory determined that kidney tubular epithelium as well as the small and large intestinal epithelial cells specifically and highly expresses the Clr-f transcripts. Contrarily, the Clr-b transcripts were only detected on hematopoietic cells of various lymphoid organs and kidneys. Moreover, foregoing studies revealed that the loss of Clr-b following viral or chemical induced stress mediates NK cell killing of the target cell, suggesting a tissue-specific immune-surveillance mechanism in parallel with the global MHC-I-dependent missing-self model. However, the role of the NKR-P1B:Clr-b recognition-system have never been examined in the intestine. Additionally, the role of Clr-f in the kidney and intestines, where they are highly expressed, has not been investigated. For these reasons, I aimed in my thesis to provide a better understanding of the functional aspect of the NKR-P1B:Clr-b and NKR-P1G:Clr-f recognition systems in mediating gut mucosal and renal homeostasis, respectively. First, in order to determine the role of NKR-P1B and Clrb receptor:ligand pair as a “missing-self” immunosurveillance system in the gut, I started by identifying the expression pattern of both the receptor and ligand on various intestinal cells. My results demonstrate that NK cells do not represent the major NKR-P1B-expressing cells in the gut lamina propria. Instead, ILC3 subsets constituted the predominant cell population expressing the receptor, whereas γδT cells composed a small fraction of NKR-P1B+ lymphocytes. In addition, the NKR-P1B expression on myeloid cells was exclusive to colon macrophages and DC subsets. Interestingly, the highest percentage of NKR-P1B+ immune cells was found in the gut, which suggests the dominant role of NKR-P1B in regulating immune functions at the level of intestinal mucosa. As expected, the expression of the NKR-P1B ligand, Clr-b, appeared on all innate immune cell types in the gut. Next, using oral infection models of Salmonela typhimurium and Citrobacter rodentium, I showed that NKR-P1B-deficient NK cells, ILC3 and γδ T cells are hyporesponsive compared to their WT counterparts. In particular, gut NKR-P1B-deficient NK cells and γδT cells secreted low levels of IFNγ cytokine while infected with S.typhimurium. Importantly, the decreased IFNγ secretion by NK and γδT cells was associated with an increased dissemination of the bacterium into the knockout spleens at day 5 post-infection. Likewise, I detected a significant decrease in IL-22 cytokine production by NKR-P1B-deficient ILC3 compared to their WT counterparts at both steady state and following C.rodentium infection. Next, I address the potential role of Clr-f in the kidney. Renal tubular epithelial cells have been shown to express high levels of Clr-f transcripts. Epithelial cells constitute the major cellular component of kidney tubules and are well known to mediate metabolic waste excretion, reabsorption of essential molecules as well as other physiological functions, such as ions exchange and water retention. To determine the role of Clr-f in renal epithelial cells, I generated a Clr-f-deficient mouse with the help of two of my previous lab colleagues. Importantly, chemical analysis on urine and serum samples from knockout and WT littermates indicated that Clr-f-deficient kidneys display a decreased filtration capacity. In particular, higher creatinine levels were detected in the Clr-f deficient serum. In addition, Clr-f-deficient mice appeared to have a lower fractional excretion of sodium (FENa) in their urine filtrates in comparison to WT excreted urine. Blood pressure measurements on the same mice at 12 and 24 weeks of age revealed a hypotensive phenotype in the Clr-f-deficient mice. Furthermore, pathological assessment of Clr-f-deficient kidneys exhibited moderate and aggravated lesions of the tubular epithelium along with marked glomerular mesangiolysis. Lastly, flow cytometry analysis on isolated lymphocytes from Clr-f-deficient and WT mice demonstrated comparable immune infiltrates between the two mouse genotypes. Altogether, our data shows that the absence of Clr-f results in the development of glomerular and tubular lesions in an immune-independent manner leading to an abnormal kidney function. Additionally, the disruption of NKR-P1B:Clr-b recognition system results in abnormal innate immune cell number and function in the mouse intestine. These novel findings sheds light on the important role of Clr-f in maintaining healthy kidney morphology and function, as well as the crucial role for NKR-P1B:Clr-b interactions in mediating intestinal homeostasis at steady and infected states.

Les cellules Natural Killer (NK) sont des lymphocytes capables de tuer les cellules tumorales avec une expression altérée des molécules du Complexe Majeur d’Histocompatibilité (CMH) de classe I . Chez l’homme, cette reconnaissance de “l’absence du soi” est relayée par l’absence d’engagement des antigènes des leucocytes humains (HLA) par les récepteurs inhibiteurs des cellules NK. Chez l’homme, ces récepteurs inhibiteurs incluent les récepteurs KIR qui ont comme analogues fonctionnels chez les rongeurs les lectines Ly49. Certaines tumeurs échappent à la surveillance des NK en augmentant leur expression des molécules HLA. Donc, bloquer les interactions entre les molécules KIR et HLA est une stratégie anti-tumorale intéressante qu’INNATE PHARMA a décidé d’explorer en développant l’anticorps thérapeutique anti-KIR 1-7F9. Mais, ces interactions sont nécessaires pour l’acquisition des fonctions des NK, i.e leur éducation. De plus, elles sont impliquées dans la tolérance aux cellules du soi par les cellules NK. Anticiper la toxicité de 1-7F9, évaluer ses propriétés anti-tumorales et son impact sur l’éducation des NK étaient les objectifs de notre travail. Deux modèles murins ont été développées à ces fins. Le premier, est basé sur les souris B6 : les récepteurs inhibiteurs Ly49 C/I ont pour ligand les molécules H-2b. Le second modèle est basé sur des souris transgéniques pour un unique récepteur KIR et son ligand, en l’absence des molécules endogènes de CMH de classe I murines. Ces deux modèles ont montré que le blocage des récepteurs inhibiteurs n’altère pas la tolérance au soi des NK et est une stratégie anti-tumorale efficace qui n’altère pas la fonctionnalité des NK. / Natural Killer cells (NK cells) are lymphocytes able to kill tumors with aletered expression of Major Histocompatibility Complex (MHC) class I molecules. This “missing self” recognition is mediated by the lack of engagement of Human Leukocytes Antigens (HLA) with NK inhibitory receptors that include Killer Immunoglobulin like Receptors (or KIR) in humans. In rodents, the functional analogues of KIR are Ly49 lectins. Some tumors escape NK cell immune surveillance by increasing the expression of HLA molecules on their surface. So, blocking interactions between KIR and HLA molecules constitutes an interesting therapeutic strategy that INNATE PHARMA decided to explore, developing the anti-KIR monoclonal antibody 1-7F9. Nevertheless, these interactions are necessary for the acquisition of NK functional properties, i.e for their “education”. They are also involved in self-tolerance on educated NK cells. For the pre-clinical development of 1-7F9, it was necessary to anticipate anti-KIR mAb safety and toxicity, evaluate its anti-cancer potential and its impact on NK education. A first part of our work was performed in a surrogate B6 mouse model:Ly49 C/I inhibitory receptors have H-2b molécules as endogenous ligand. Their interactions were blocked with anti-Ly49 C/I monoclonal antibody. In a second part, we report the generation of transgenic mice expressing a single inhibitory KIR in the context of its HLA ligand and in absence of endogenous mouse MHC class I molecules. Both models showed that the blockade of NK inhibitory receptors interactions with their endogenous ligands did not break self tolerance, had a strong anti-tumor effect and did not abrogate NK functionality.

Les cellules NK sont classiquement décrites comme des lymphocytes effecteurs du système immunitaire inné, dotées d’un jeu limité de récepteurs permettant la reconnaissance de cellules tumorales ou infectées par des pathogènes, et dépourvues de capacités de mémoire immunitaire. Des travaux récents montrent cependant que les cellules NK semblent douées de diverses propriétés « adaptatives » proches de celles des lymphocytes T (LT). L’étude princeps de ce nouvel aspect de la biologie des cellules NK a été réalisée dans un modèle murin d’hypersensibilité retardée (HSR) aux haptènes, et démontre qu’en l’absence des effecteurs classiques de ces réactions (LT), les cellules NK sont capablesd’induire des réactions d’HSR. La première partie de ce travail de thèse a consisté à étudier la contribution des cellules NK dans l’initiation et le développement des réactions d’HSR en présence des effecteurs classiques (LT). Nous montrons ainsi que bien que des cellules NK « mémoires » spécifiques de l’haptène soient retrouvées dans le foie des souris de type sauvage sensibilisées, leur contribution à la réaction d’HSR est mineure. Par contre, si ces cellules NK mémoires sont transférées à une souris receveuse dépourvues de LT nonsensibilisée, elles sont capables d’induire des réactions d’HSR lors d’un nouveau contact avec l’haptène. Dans la deuxième partie, nous avons comparé les réactions d’HSR induites par les cellules NK et les lymphocytes T mémoires dans ce système de transfert. Nous mettons en évidence que les réactions développées par les cellules NK sont d’une durée limitée et qu’elles impliquent un oedème avec peud’infiltration par les cellules immunitaires, au contraire des réactions induites par les cellules T mémoires. Enfin, dans la troisième partie de ce travail, nous avons analysé le compartiment cellulaire NK circulant chez des patients souffrant de pathologies inflammatoires cutanées dans lesquelles les LT ont un rôle clairement identifié à ce jour. Nous rapportons des modifications qualitatives et quantitatives de ces cellules, suggérant leur implication potentielle dans la physiopathologie de ces maladies. L’ensemble de ces données confirme donc l’existence de cellules NK « mémoires », dont le rôle physiologique en présence des effecteurs adaptatifs classiques reste encore aujourd’hui à démontrer. / NK cells are classically defined as lymphocytes of the innate immune system, equipped with a limited set of receptors involved in the recognition of tumoral or infected cells, and devoid of immune memory. However, recent studies showed that NK cells seem endowed with various "adaptive" properties similar to those of T lymphocytes (TL). The original description of this new aspect of NK cell biology was made in a murine model of delayed-type hypersensitivity (DTH) to haptens. In this model, NK cells were found to be able to induce DTH reactions in the absence of classical DTH effectors (TL). The aim of the first part of this PhD thesis was to study the contribution of NK cells in the initiation and development of HSR reactions in the presence of classical effectors (TL). We show that although hapten-specific "memory" NK cells are generated in the liver of hapten-sensitized wild type mice, their contribution to HSR reactions is minor. By contrast, if "memory" NK cells are transferred to unsensitized recipient mice lacking T cells, they can induce DTH reactions upon a new contact with the hapten. In the second part, we compared the DTH reactions induced by NK cells and memory T lymphocytes in thistransfer system. We showed that hapten-induced skin reactions mediated by NK cells are of limited duration and associated with a weak cellular infiltrate, in contrast to memory T cell-mediated reactions. Finally, in the third part of this work, we analyzed the circulating NK cell compartment in patients suffering from inflammatory skin diseases thought to be induced by T cells. We report qualitative and quantitative changes of NK cells in patients in comparison with healthy controls, suggesting the potential involvement of NK cells in the pathophysiology of these diseases. Altogether, our data confirm the existence of "memory" NK cells, whose physiological role in the presence of conventional adaptive effectors still remains to be assessed.

Les cellules NK sont des lymphocytes de l’immunité innée qui ont un rôle majeur dans le contrôle précoce des infections virales et dans l’immunosurveillance des tumeurs. Cependant, en cas de stimulation chronique, un état d’anergie, où les cellules NK n’exercent plus leur fonction a été mis en évidence. Les mécanismes conduisant à cette perte de fonction demeurent mal caractérisés et s’il s’agit d’une cause ou d’une conséquence de la chronicité n’est pas non plus déterminé. Cibler les cellules NK constitue une perspective thérapeutique de choix mais nécessite une meilleure compréhension de ces aspects. L’objectif de ce travail de thèse s’articule autour de trois points. Premièrement, nous avons généré un modèle tumoral murin sensible aux cellules NK permettant l’étude de la réponse anti-tumorale et l’établissement d’une stimulation chronique. Deuxièmement, l’utilisation de ce modèle a permis d’investiguer les mécanismes conduisant à la perte de fonctionnalité des cellules NK et de tester des stratégies de restauration. Enfin, nous avons mené une étude parallèle, chez l’homme, par analyse d’une cohorte de patients chroniquement infectés par le virus de l’Hépatite B dans le but de déterminer le phénotype des cellules NK et d’identifier les causes conduisant à leur perte de fonction dans ce contexte / NK cells are innate lymphocytes which play a crucial role in the early control of viral infection and in tumor immunosurveillance. However, a state of tolerance, where NK cells are poorly functional, occurs in the context of chronic stimulation. The mechanisms leading to this process remain poorly understood and whether this is a cause, or a consequence of chronicity is unknown. Targeting NK cells appears to be a potent therapeutic strategy but requires further investigation. With the lack of clarity in the field this work had three main objectives. First, we engineered a tumoral mouse model that was strongly immunogenic for NK cells and thus allowed us to study the anti-tumoral response of NK cells and to trigger chronic stimulation. Then, we used this model to investigate the mechanisms driving NK cell loss of function and to test potential therapeutic strategies to reverse this state. Finally, in the context of human chronic infection we analyzed samples from HBV infected patients in order to determine the phenotype, function and signaling capacity of NK cells to identify the drivers of NK cell dysfunction

Despite the existence of a treatment, there is still not a cure for HIV-1 infection and there arearound 700 000 deaths per year from AIDS-related diseases. A major barrier for a cure is theestablishment of latent reservoirs that are impossible to distinguish from healthy cells and thuscan escape the immune system. One potential solution is called shock-and-kill strategy, whichaims to induce HIV-1 reactivation, exposing latently infected cells to the immune system andmaking them susceptible to cell death. In our lab, it was seen that when NK cells are stimulatedwith a pan-caspase inhibitor, they acquire the “shock” ability, but it is still unknown how. Inthis project, we observed that the supernatant from pan-caspase inhibitor-stimulated NK cellscan increase HIV-1 reactivation in two different latency models. Furthermore, the protein levelsof three HIV-1 suppressors were found to be increased in the same supernatant. For this reason,their effect in HIV-1 reactivation in latently infected cells was analysed. Although we did notobserve an increase in HIV-1 reactivation, the upregulation of these three proteins can be usefulin the clinical context. Since they are HIV-1 suppressors, their presence can prevent theinfection from spreading after latent cells are reactivated. Altogether, our results show that NKcells stimulated with a pan-caspase inhibitor are secreting a biological product that inducesHIV-1 reactivation. This indicates that there is a pathway in NK cells that can potentially beexploited in order for them to be able to induce HIV-1 reactivation.

Introduction : Les cellules souche mésenchymateuse (CSM) présentent une puissante activité immunomodulatrice sur les lymphocytes T et les Natural Killer (NK), impliquées dans les réactions allogéniques. Les propriétés immunomodulatrices des CSM dépendent de contacts cellulaires et des facteurs secrétés. Ainsi les exosomes produits par ces cellules pourraient constituer des nouveaux produits thérapeutiques.L’objectif de ce travail est d’étudier, in vitro, l’effet d’exosomes dérivés de CSM sur les lymphocytes B, T et les NK.Méthodologie : Les CSMs utilisées sont issues de foies fœtaux humains. Les exosomes ont été isolés à partir du milieu de culture des CSMs par une série d’ultracentrifugation à 100000g.Résultats : Contrairement aux CSMs qui inhibent la prolifération des lymphocytes T et B, leurs exosomes n'ont pas d'effet sur leur prolifération. Cependant ils inhibent la prolifération, l’activation et la cytotoxicité (expresion CD107a) des NK. Nous avons mit en évidence, par FACS, la présence de TGFbeta; à la surface des exosomes. De plus leur fonction inhibitrice est abrogé en présence d’un anticorps bloquants anti-TGFbeta;. Réciproquement l’exposition de cellules NK à du TGFbeta; inhibe la cytotoxicité et la prolifération de cellules. Enfin, en présence d'exososmes nous avons montré, par IF, une translocation de Smad 2/3 (messager du signal TGFbeta;) dans les noyaux des cellules NK, inhibé par l'ajout d'anticorps anti-TGFbeta;.Conclusion: Ces résultats suggère que les propriétés immunomodulatrices de CSMs sur NK pourraient dépendre de TGFβ présenté ou associé aux exosomes. / Introduction: Mesenchymal stem cells (MSCs) are powerful immunomodulators regulating the function of B and T lymphocytes and natural killers cells (NK) involved in allogeneic reactions. Their immunomodulatory properties depend on cell contact and secretion factors produced by MSCs. Thus exosomes produced by these cells could provide new therapeutic tools.Objective: The objective of this work is to study the effect of MSC derived exosomes in vitro on B and T lymphocytes and NK cells.Method: MSCs used for this study are isolated from human fetal liver. Exosomes were isolated from MSC culture medium by a serie of ultracentrifugation at 100000g.Results: MSCs inhibit the proliferation of T lymphocytes. Unlike MSCs, their exosomes do not abrogate the proliferation of T and B cells. However they inhibit the proliferation, activation and cytotoxicity (CD107a expression) of NK cells. By FACS analysis we showed a surface expression of TGFb; by exosomes. Inhibition of NK cells activation by exosomes is altered by a neutralizing anti-TGFb; antibody. Contrary when NK cells are cultured with TGFb; the same effect qs exosomes is demonstrated. By IF, we found a nuclear translocation of Smad 2/3 (TGFb; signal transducer) in NK cells cultured with exosomes, which is inhibited by the qddition of anti-TGFb; antibody.Conclusion: These results suggest that the immunomodulatory properties of MSCs on NK could depend on exosome presentation or association with TGFb;.

Depuis ces 10 dernières années, l’immunothérapie est à l’avant-garde de la thérapie anticancéreuse. Les cellules Natural Killer (NK) font partie du système immunitaire inné et possèdent la capacité unique de lyser les cellules tumorales sans activation préalable par un antigène spécifique. Elles jouent un rôle majeur dans le contrôle de plusieurs cancers hématologiques et solides dont les tumeurs stromales gastrointestinales (GIST). Leur activation dépend de l’équilibre entre leurs récepteurs activateurs et inhibiteurs. Les Natural Cytotoxicity Receptors (NCR) font partis des récepteurs activateurs les plus importants dans leur reconnaissance des cibles et comprennent le NKp30, NKp44 et NKp46. Le NKp30 possède 3 isoformes: NKp30a et NKp30b sont immunostimulantes induisant la sécrétion d’Interféron (IFN) γ et de Tumor necrosis factor (TNF) α alors que NKp30c est immunosuppressive favorisant la production d’interleukine 10 (IL-10). L’IFNγ est un puissant activateur des cellules immunitaires tandis que l’IL-10 est une cytokine anti-inflammatoire. Le TNFα a été décrit initialement comme un facteur sérique induisant la nécrose des tumeurs, cependant son rôle a depuis été élargi à des fonctions homéostatiques. De nombreuses études laissent à penser que les fonctions antitumorales des cellules NK ne se limitent pas à l’élimination des cellules tumorales. Malgré les progrès importants réalisés dans la compréhension des cellules NK, de nombreux travaux sont encore à mener pour exploiter pleinement leur potentiel antitumoral.Notre équipe a démontré l’importance capitale des cellules NK dans les GIST. Ainsi l’infiltrat NK prédit la survie sans progression des patients. De plus nous avons montré que l’expression préférentielle de l’isoforme immunosuppressive NKp30c impactait négativement le pronostic des patients GIST. Suite à ces résultats, nous avons cherché à mieux caractériser l’impact des isoformes du récepteur NKp30 chez les patients GIST en réponse à l’IM. Dans un premier temps, nous avons démontré qu’un haut ratio d’expression entre NKp30b et NKp30c prédisait une meilleure réponse à l’imatinib mesylate (IM, un inhibiteur de tyrosine kinase, traitement de référence des GIST) et que l’expression des isoformes de NKp30 impactait l’environnement cytokinique de la tumeur. De plus, nous avons établi pour la première fois le lien entre la présence de ligands solubles de NKp30, B7 Homolog 6 soluble (sB7-H6) et BCL2 Associated Athanogene 6 soluble (sBAG6), et la diminution de la survie sans évènement des patients GIST traités à l’IM.Malgré l’infiltration immunitaire de nombreuses tumeurs, les fonctions antitumorales des lymphocytes sont inhibées par le microenvironnement tumoral. Ainsi, nous avons étudié quelles voies de signalisation étaient associées à l’inhibition des cellules NK présentes dans cet environnement. Pour cela, nous avons réalisé un microarray à partir des cellules NK infiltrant les GIST et avons mis en évidence le rôle délétère de la voie TNFα/TNF Receptor 2/Baculoviral IAP Repeat Containing 3 (BIRC3)/TNF Receptor Associated Factor 1 (TRAF1) dans la fonctionnalité des cellules NK. En effet, l’activation de cette voie dans les cellules NK entraine la diminution de la transcription du gène du récepteur activateur NKp46 ainsi que son expression membranaire. Cette diminution était corrélée avec l’expression de l’isoforme NKp30c. Par ailleurs, nous avons pu mettre en évidence chez la souris que le TNFα facilitait la dissémination métastatique de la lignée tumorale sensible aux cellules NK B16F10.Nos résultats sur les cellules NK ont renforcé leur grand potentiel en tant que cible thérapeutique pour l’immunothérapie anticancéreuse. En effet, l’importance du récepteur NKp30 et de ses isoformes dans la prédiction de la réponse à l’IM dans les GIST et la mise en évidence d’un nouveau mécanisme inhibiteur des cellules NK par la voie TNFα/TNFR2/BIRC3/TRAF1 ouvrent la voie à de nouvelles stratégies dans le traitement des cancers. / Over the last 10 years, immunotherapy has been at the forefront of cancer therapy. Natural Killer (NK) cells are part of the innate immune system and have the unique ability to lyse tumor cells without any antigen specific priming. They have a key prognostic role in several hematological and solid cancers including gastrointestinal stromal tumors (GIST). A balance between activating and inhibitory receptors triggers NK cell activation. Natural cytotoxicity receptors (NCR) are among the most clinically relevant activating receptors and include NKp30, NKp44 and NKp46. NKp30 can be expressed in 3 different isoforms: NKp30a and NKp30b are both immunostimulatory, inducing interferon (IFN) γ and tumor necrosis factor (TNF) α secretion whereas NKp30c is immunosuppressive, producing interleukin 10 (IL-10). IFNγ is a potent activator of immune cells whereas IL-10 is an anti-inflammatory cytokine. TNFα was first described as a serum factor, inducing tumor necrosis but its role has since been broadened to homeostatic functions. Ample evidence suggests that anti-tumor functions of NK cells are tightly regulated and expand far beyond the simple killing of malignant cells. Despite the tremendous progress in understanding NK cell biology, further work is warranted to fully exploit the anticancer potential of these cells.Our group demonstrated the crucial role that NK cells have in GIST. Indeed, NK cell infiltrate positively correlates with progression-free survival. Moreover, we showed that the preferential expression of the immunosuppressive isoform NKp30c, negatively impacts the clinical outcome of GIST patients. To further extend these observations, we explored the influence of various NKp30 isoforms in GIST patients.Firstly, we revealed that a high ratio between the expression of NKp30b and NKp30c isoforms predicted a stronger imatinib mesylate (IM) response (a tyrosine kinase inhibitor, TKI – first line standard of care in GIST) and that tumor cytokine milieu is modified following NKp30 isoform expression. Furthermore, we demonstrated a link between the presence of soluble ligands of NKp30, soluble B7 Homolog 6 (sB7-H6) and soluble BCL2 Associated Athanogene 6 (sBAG6), and a decrease in event-free survival in IM-treated GIST patients.Despite the presence of immune infiltration in many tumors, antitumor functions of lymphocytes are inhibited by the tumor microenvironment. Thus, we explored which signaling pathways were associated with NK cell inhibition in the tumor microenvironment. To do so, we performed a microarray from GIST infiltrating NK cells which highlighted the deleterious effect of TNFα/TNF Receptor 2/Baculoviral IAP Repeat Containing 3 (BIRC3)/TNF Receptor Associated Factor 1 (TRAF1) pathway on the function of NK cells. Next, we demonstrated that activation of this pathway in NK cells decreased gene transcription and protein expression of the activating receptor NKp46 (also called Natural Cytotoxicity Triggering Receptor 1 NCR1). This decrease positively correlated with NKp30c isoform expression. Moreover we showed that in mice, TNFα increases the metastatic dissemination of the NK sensitive tumor cell line, B16F10.Results from our research on NK cells strengthen the potential of NK cells as a therapeutic target for anti-tumor immunotherapy. Taken together, this thesis demonstrates the key role of the NKp30 receptor and its isoforms in the IM therapy as predictive marker in GIST response and describes for the first time a new NK cell inhibitory mechanism via the TNFα/TNFR2/BIRC3/TRAF1 pathway, paving the way for novel therapeutic strategies in cancer treatment.