La réponse immune lors d'une infection par le VHB est essentielle pour éliminer le virus. Le virus module le système immunitaire pour échapper à son contrôle. Le mécanisme par lequel le virus module l'immunité est encore mal connu. Les cellules dendritiques plasmocytoides (pDC) jouent un rôle crucial dans l'immunité antivirale de part leur capacité à capturer et apprêter les antigènes viraux afin d'induire une réponse immune adaptative. Les pDCs représentent un bon potentiel pour restaurer une immunité anti-VHB fonctionnelle, mais le VHB pourrait moduler les pDCs pour échapper au contrôle immunitaire. Dans une première partie, nous avons évalué le potentiel des pDCs à restaurer l'immunité anti-VHB en contexte d'hépatite B chronique. Nous avons utilisé une lignée de pDCs HLA-A*0201+ chargée avec des peptides HLA-A*0201 restreints dérivés des antigènes HBc et HBs du virus afin d'amplifier des lymphocytes T spécifiques ex vivo à partir de cellules de patients atteints d'hépatite B chronique. Nous avons ensuite établi un modèle de souris humanisées (Hepato-HuPBL) afin d'évaluer le potentiel thérapeutique de la stratégie in vivo. La stimulation de PBMC ou de lymphocytes infiltrant le foie (LIL) issus de patients HLA-A*0201+ par la lignée de pDC chargée avec le peptide HBc a permis d'amplifier des lymphocytes T spécifiques de HBc et fonctionnels dans 45,8% des cas. Le groupe de non répondeurs était caractérisé par la présence d'Ag HBe ou un plus fort niveau de lymphocytes T régulateurs. L'efficacité thérapeutique du vaccin de pDC a été évaluée dans un modèle de souris NOD-SCID β2m-/- reconstituées avec des PBMC issus de patient VHB et xenotransplantées avec des hépatocytes humains transfectés avec le VHB. La vaccination de ces souris avec la lignée de pDC chargée avec les peptides HBc et HBs a permis l'amplification de lymphocytes T CD8 spécifiques du VHB capables de lyser spécifiquement les hépatocytes infectés in vivo. Ainsi, la lignée de pDC chargée avec des peptides dérivés du VHB est capable d'amplifier des lymphocytes T CD8 spécifiques du VHB et fonctionnels in vitro et in vivo. Cette nouvelle stratégie d'immunothérapie pourrait donc restaurer une immunité antivirale et permettre l'élimination du virus chez les patients atteints d'hépatite B chronique. Dans une deuxième partie, nous avons évalué le rôle physiopathologique des pDCs au cours de l'infection chronique par le VHB et les conséquences fonctionnelles sur le cross-talk pDC/NK. Des défauts fonctionnels des pDCs et des cellules NK ont été observé chez les patients atteints d'hépatite B chronique. Cependant le cross-talk pDC/NK ainsi que les mécanismes en jeu n'ont pas encore été élucidé. Nous avons étudié le phénotype et la capacité de répondre à une stimulation TLR-L des pDCs de patients comparé aux pDCs de donneurs sains puis nous avons étudié les conséquences sur le cross-talk entre pDCs de patients (virémiques ou avirémiques) et cellules NK hétérologues. Les pDCs de patients montrent un phénotype plus activé que les pDCs de donneurs sains mais ne sont pas capables de répondre à une stimulation TLR9-L. De plus, les pDCs de patients virémiques ne sont pas capables d'activer les fonctions cytotoxiques des cellules NK. Cette perturbation du cross-talk pDC/NK semble liée à un défaut de production d'IFNα et d'expression d'OX40L par les pDCs de patients virémiques, ainsi qu'à la présence d'une quantité importante d'IP-10 dans le plasma des patients. Ainsi, le VHB pourrait échapper à l'immunité en altérant la fonction des pDCs et perturbant le cross-talk pDC/NK par un mécanisme dépendant de IP-10, OX40L et IFNα. / The immune control of HBV infection is essential for viral clearance. The virus is able to modulate the immune system to escape this control. The mechanisms involved remain largely unknown. Plasmacytoid dendritic cells (pDC) play a crucial role in anti‐viral immunity due to their ability to capture and process viral antigens and subsequently induce adaptive immune responses. PDCs are therefore promising to restore functional anti‐HBV immunity, but HBV could modulate the pDCs to escape the immune control. First, we investigated the potential of pDCs in triggering anti‐viral immunity against HBV during chronic infection. We used a HLA‐A*0201+ pDC line loaded with HLA‐A*0201-restricted peptides derived from HBc/HBs antigens to amplify specific T cells ex vivo from chronic HBV patients. Then we established an Hepato-HuPBL humanized mouse model to address the therapeutic potential of the strategy in vivo. Stimulation of PBMC or liver-infiltrated lymphocytes from HLA‐A*0201+ chronic HBV patients by the HBc peptide-loaded pDC line elicited functional HBV-specific CD8 T cells in 45.8% of cases. The “non-responder” group of patients was characterised by the presence of HBe Ag or higher level of regulatory T cells. The therapeutic efficacy of the pDC-based vaccine was evaluated in NOD-SCID β2m-/- mice reconstituted with HBV patients' PBMC and xenotransplanted with human HBV-transfected hepatocytes. Vaccination of these mice with the HBc/HBs peptide‐loaded pDC line elicited HBV-specific T cells in vivo able to specifically lyses infected hepatocytes. Thus pDCs loaded with HBV‐derived peptides can elicit functional virus-specific T cells in vitro and in vivo. This new cell-based immunotherapeutic strategy could restore functional anti‐viral immunity and clear the virus in chronic HBV patients. In the second part, we investigated the pathophysiological role of pDCs from chronic HBV patients and the functional consequences on pDC-NK cross-talk. Functional impairment have been observed in both pDC and NK cells in chronic HBV patients. However, the cross-talk pDC/NK and the mechanisms involved have not been elucidated. We studied the phenotype and the ability to respond to a TLR-L stimulation of the pDCs from HBV patients compared to healthy donors, and we investigated the consequences on the cross-talk between patients' (viremic or aviremic) pDCs and heterologous NK cells. PDCs show higher levels of activation in patients compared to healthy donors but were not able to respond to TLR9-L stimulation. In addition, pDCs from viremic chronic HBV patients failed to trigger a normal NK cytolytic function after TLR9-L stimulation. This pDC-dependant NK dysfunction was related to impaired IFNα secretion and OX40L expression by pDCs from viremic patients, and related to high plasma IP-10 levels found in patients. In conclusion HBV could escape the immune system by impairing pDC function and subsequent pDC/NK cross-talk by a mechanism involving IP10, OX40L and IFNα.

Orientador: Mary Luci de Souza Queiroz / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T08:29:05Z (GMT). No. of bitstreams: 1 Torello_CristianeOkuda.pdf: 8596128 bytes, checksum: e07457446c814c5f191e0c629491876d (MD5) Previous issue date: 2009 / Resumo: Neste trabalho, investigamos os efeitos do extrato padronizado de Rhodiola rosea L. (ERR) sobre a resposta imunohematopoética de camundongos infectados com Listeria monocytogenes. Os resultados demonstram que o ERR aumenta a resistência dos animais frente uma dose letal de Listeria quando administrado profilaticamente nas doses de 100 e 250 mg/kg por sete dias consecutivos anteriores à inoculação. Paralelamente, reversão da mielossupressão induzida pela infecção, concomitante ao aumento na atividade de células natural killers (NK) e na produção das citocinas TNF-a, IFN-? e fatores estimuladores de colônias foram observados. Os resultados obtidos demonstram que o ERR compartilha da habilidade de regular positivamente os desequilíbrios hematopoiéticos e imunológicos envolvidos nos estágios iniciais da infecção com L. monocytogenes. Estes efeitos podem ser atribuídos à recuperação no equilíbrio da resposta hematopoiética através da produção de IL-1a e IL-6 pelas células estromais no microambiente medular e também da produção de fatores estimuladores de colônias a partir das 24 horas de infecção, promovendo aumento no número de progenitores de macrófagos e granulócitos na medula óssea. Além disso, a eficácia do ERR também depende do aumento na produção das citocinas TNF-a e IFN-?, do aumento na atividade funcional das células NK e polarização da resposta celular para Th1. Juntos, estes efeitos contribuem para o aumento na resistência a L. monocytogenes. / Abstract: In this work, we have investigated the effects of Rhodiola rosea L. extract (ERR) in Listeria monocytogenes infected mice. Our results demonstrated that ERR protects mice from a lethal dose of L. monocytogenes, when administered prophylactically at 100 and 250 mg/kg, for seven consecutive days prior to the infection. In addition, prevention of myelosuppression induced by infection, concomitant to increasing natural killers (NK) cells activity and production of TNF-a, IFN-? and colony-stimulating factors were observed. The results showed that ERR share the ability of regulating positively the hematopoietic and immunological unbalance involved in the initial stages of infection with this pathogen. These effects could be attributed to recovering of the hematopoietic response through the production of IL-1a and IL-6 by stromal cells in the bone marrow microenvironment and the production of colony-stimulating factors at 24 hours of infection, promoting an increase in the number of granulocytes and macrophages progenitors in the bone marrow microenvironment. Moreover, the efficacy of ERR depends on high levels of TNF-a and IFN- ?, increased NK cells activity and polarization to Th1 response. Together, these effects contribute to increasing resistance to L. monocytogenes. / Universidade Estadual de Campi / Farmacologia / Doutor em Farmacologia

Cancer immunotherapy is an encompassing term referring to therapeutic strategies that aim to boost the immune system to fight cancer. These strategies include administering immune cells that have been altered to have greater anti-tumor activity or using biologics and small molecules that target immune components to also promote tumor clearance. Natural Killer (NK) cells are cells of the innate immune system that recognize and kill abnormal cells such as cancer cells and play an important role in the anti-tumor response. Because of their crucial role in tumor immunity, NK cells are prime targets for immunotherapies. Repurposing small molecule drugs is an attractive strategy to identify new immunotherapies from already approved drugs. Here, we screened 1,200 approved drugs from the Prestwick Chemical Library to identify drugs that increase NK cell cytotoxicity. We used a high-throughput luciferase-release cytotoxicity assay to measure the killing of the myeloid leukemia cell line, K562 cells expressing nano luciferase (NL) by NK92 cells, a human NK cell line. From the drug candidates identified from the screening assay, the antibiotic colistin sulfate increased cytotoxicity of the NK92 cell line and unstimulated human NK cells towards K562-NL cells. This increase in NK cytotoxicity was short-lived as pre-treating NK92 cells with colistin for 1 hour or 24 hours did not increase cytotoxicity. Also, we show pre-treating K562-NL target cells with colistin does not sensitize them to NK-mediated killing. Further studies are needed to uncover the mechanism of action of colistin, thus contributing to knowledge of fundamental NK cell biology regarding NK cell cytotoxicity which will aid in identifying additional small molecule drugs that enhance NK cell activity.

Depuis peu, l’immunothérapie a émergé comme une nouvelle stratégie chez les patients atteint de Cancer Bronchique Non à Petites Cellules (CBNPC), confirmant ainsi le rôle du système immunitaire dans cette maladie. Malgré ces nouveaux traitements (thérapies ciblées, immunothérapie), les taux de réponses restent faibles avec un impact modeste sur la survie globale. Des biomarqueurs sont donc nécessaire pour définir les populations cibles de ces traitements. Une des pistes explorées est le statut immunitaire des patients, en effet celui-ci a un impact pronostic et pourrait influencer la réponse aux traitements standards tels que la chimiothérapie, les thérapies ciblées et même l’immunothérapie. Parmi les cellules du système immunitaire, les cellules Natural Killer (NK) auraient un rôle effecteur dans le CBNPC. Il est maintenant clairement établit que les cellules NK favorisent la mise en place d’une immunité adaptative fonctionnelle et efficace. Ainsi une altération des fonctions NK pourrait être un mécanisme associé à l’échappement à l’immunité adaptative de la tumeur. Dans notre première étude, nous avons mis en évidence que les exosomes de cellules dendritiques stimulaient les cellules NK via NKp30, cette activité étant associée à un gain de survie sans progression chez des malades inopérables porteurs d’un CBNPC avancé. Notre second projet a révélé, pour la première fois, un rôle pronostic des transcrits de NCR3 (gène de NKp30) chez des patients naïfs de tout traitement. L’activation des cellules NK via NKp30 pourrait être une stratégie efficace d’immunomodulation chez les patients atteints de CBNPC avancé. Ces travaux confirment le rôle important des cellules NK dans le CBNPC avancé. / Recently, immunotherapy has emerged as a new strategy in Non-Small Cell Lung Cancer (NSCLC) patients, confirming the key role of the immune system in this disease. Despite these new treatments (targeted therapies, immunotherapy), response rates remain low with a modest impact on overall survival. Biomarkers are needed to define the target population of these treatments. One of the options explored is the immune status; indeed the immune status of cancer patients has a prognosis impact and may influence the response to standard treatments such as chemotherapy, targeted therapies and even immunotherapy. Among the immune cells, Natural Killer cells (NK) have an effector role in NSCLC. It is now established that NK cells can promote a functional and efficient adaptive immunity. Therefore, an impaired NK functions could be a mechanism associated with the escape from adaptive immunity of the tumor. In our first study, we demonstrated that exosomes from dendritic cells stimulated NK cells through NKp30, this activity is being associated with improved survival in advanced NSCLC. Our second project has revealed, for the first time, a independent prognostic role of NCR3 transcript (NKp30 gene) for naïve advanced NSCLC. Activation of NK cells via NKp30 could be an effective strategy for immunomodulation in advanced NSCLC patients. These studies confirm a major role of NK cells in advanced NSCLC.

Study of receptor-ligand pair NKR-P1F and Clrg Mouse NKR-P1F:Clr-g receptor:ligand pair is important component of the receptor "zipper" that occurs at the contact between natural killer cell and its target cell, and represents a recently discovered example of lectin-lectin interactions important for recognition among immune cell subsets. In order to study structure of these proteins and interactions between them, we have prepared pET-30a(+) bacterial expression vectors coding parts of extracellular domains of the two receptors. After induction of protein production with IPTG, the proteins precipitated into inclusion bodies, from which they could be refolded in vitro. Refolded proteins were purified using combination of ion exchange and size exclusion chromatography. NKR-P1F construct yielded only small amounts of soluble protein using standard refolding protocols. Furthermore we have experienced difficulties with reproducibility of the refolding results. In the case of Clrg the standard protocols for protein refolding were not sufficient. In order for the Clrg to fold properly, the odd cysteine which does not fit into the pattern usual for this family of receptors was substituted for serine and resulting C148S construct was shown to be more useful. Further, using (benzyldimethylammonio)propanesulfonate in...

(english) Natural Killers - NK cells play an important role in immune surveilance and regulation either by direct cytotoxicity towards infected, transformed or otherwise damaged cells, or by production of cytokines and chemokines. The resulting response of NK cells is given by the sum of stimulating and inhibiting signals, tranduced by a wide array of receptors. Killer Ig-like receptors KIR2DL4 and LILRB1, which recognize self HLA-G molecules in pregnancy, as well as NKR-P1 receptors, which differ in the number of isotypes, are species-dependent and reduced during phylogenesis. NKG2D, reacting to stress-inducible proteins, and adenosine receptors (AR), which supress the inflamatory reaction, remain evolutionary conserved. The aim of this work was to study the involvement of NK cells and their receptors in several immune disorders and in various species, to provide new insights into their function and posisible immune modulation. We have shown here, that the choice of species in the study of NK cell effector functions may be crucial in some cases. The reaction to glycans, using synthetic GlcNAc-terminated glycomimetics GN8P, exerted opposing effects on NK cell function in humans and C57Bl/6 mice. In humans, the glycomimetic decreased cytotoxic activity of high NKR-P1A expressing NK cells, while in...

The kinetics of tumor growth and progression are governed by the interaction between tumor cells, the non-malignant stroma and both innate and adaptive immune cell lineages. Innate immunity has a critical role in the control of tumor cell growth and metastasis. The microenvironment of many tumors is populated with innate immune cells, including regulatory natural killer (NK) cells and dendritic cells (DCs), tumor associated macrophages, and myeloid derived suppressor cells, that suppress normal immune function. Much of our understanding of interactions between tumors and the innate immune system is based on experimental studies performed in mouse “syngenic” models. However, there is clear need for a mechanistic understanding of the human innate immune system within the tumor microenvironment. The goal of my thesis is to characterize the interactions between human innate immune cells and tumors and to define specific pathways and cell lineages that are targets for immune modulation. A central focus of my thesis is the use of cutting-edge humanized mouse models based on the immunodeficient NOD-scid IL2Rgnull (NSG) mouse strain to study human immuno-oncology. In the first section of my thesis I describe studies that evaluate the influence of inflammatory stimuli on innate immune control of tumors. Agents that induce inflammation have been used since the 18th century for the treatment of cancer. The inflammation induced by agents such as toll-like receptor (TLR) agonists is thought to stimulate tumor-specific immunity in patients and augment control of tumor burden. While NSG mice lack murine adaptive immunity (T and B cells), these mice maintain a residual murine innate immune system that responds to TLR agonists. Here I describe a novel NSG mouse strain lacking TLR4 that fails to respond to lipopolysaccharide (LPS). NSG-Tlr4null mice support human immune system engraftment and enables the study of human specific responses to TLR4 agonists. My data demonstrate that specific stimulation of TLR4 activates human innate immune system and promotes regression of human patient derived xenograft (PDX) tumors. In the second section of my thesis I describe the development of an NSG mouse strain that constitutively expresses human Interleukin 15 (IL15) and supports the development of functional human NK cells. Using humanized NSG-IL15 transgenic mice (NSG-Tg(Hu-IL15), my data clearly demonstrate a critical role for human NK cells in limiting growth of a PDX melanoma. In the third section of my thesis I describe, the use of the bone marrow/liver/thymus (BLT) humanized mouse model to study the interactions between the human immune system and PDX melanoma and to evaluate the response of the melanoma to immunotherapy modalities. My results collectively suggest that mice engrafted with human immune systems and bearing human tumors can be harnessed as translational models, which are critically needed as tools to study tumor immunotherapy. These humanized mouse models are an ideal translational tool to advance our understanding of human immuno-oncology and for development and testing of novel immune therapies for the treatment of malignancies.

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Diverses études ont souligné le rôle prépondérant du dialogue entre les cellules NK et les cellules dendritiques au cours des réponses immunes. Cependant, les récepteurs impliqués dans ce processus restent incertains. Au cours de ce travail, nous nous sommes attachés à identifier les récepteurs mis en jeu lors de la reconnaissance virale à l’aide de modèles humains et murins. Pour cela, nous avons mimé l’infection virale en utilisant deux ARN bicaténaires synthétiques – poly(AU) et poly(IC) – et montré qu’ils sont tous deux capables d’activer TLR3 mais que seul poly(IC) engage les hélicases RIG-I et MDA5. Les deux ARN induisent l’activation des cellules NK au sein des PBMC humaines, mais seul poly(IC) induit la production d’IFN-gamma. Les DC myéloïdes (mDC) sont requises pour cette activation sans nécessité d’un contact cellulaire entre les cellules NK et les mDC. En outre, les IFN de type I et l’IL-12 secrétés par les DC sont respectivement nécessaires à l’initiation du potentiel lytique et à la production d’IFN-gamma. Poly(IC), au contraire de poly(AU), a une action synergique avec l’IL-12 produite par les mDC pour induire la production d’IFN-gamma en agissant directement sur les cellules NK. Enfin, l’activation conjointe de TLR3 et des hélicases RLR sur les mDC et RIG-I sur les cellules NK, nécessaire à la production d’IFN-gama en réponse à l’ARN bicaténaire, a été confirmée à l’aide de souris déficientes pour TLR3 et Cardif et d’un ligand spécifique de RIG-I. En conclusion, nous rapportons pour la première fois la nécessité pour un composé microbien d’engager deux familles de récepteurs sur deux populations cellulaires distinctes pour induire une réponse innée éfficace. / Crosstalk between NK cells and DC is critical for the response to the microbial mimic poly(IC) but the dsRNA receptors involved in each cell types remained to be defined. We show herein that two dsRNA, poly(AU) and poly(IC), similarly engaged TLR3 while only poly(IC) triggered the RIG-I and MDA-5 helicases. Both dsRNA triggered NK cell activation within PBMC but only poly(IC) induced IFN-gamma. mDC were required for NK cell activation by the two dsRNA, suggesting that they triggered at least TLR3 on mDC. DsRNA induction of cytolytic potential and IFN-gamma production in NK cells did not require contact with mDC but was dependent on the secretion of type I IFN and IL-12, respectively. Poly(IC) but not poly(AU) synergized with mDC-derived IL-12 for high IFN-gamma production by acting directly on NK cells. Finally, the requirement of TLR3 and the RLR on mDC and the involvement of the RIG-I but not TLR3 on NK cells for the production of IFN-gamma induced by dsRNA was confirmed using TLR3 and Cardif deficient mice and RIG-I specific activator. This cooperation was further confirmed using inactivated FLU virus infected-target cells both in human and mouse system demonstrating that NK cells were able to sense viral material by a direct transfer from infected cells likely through lytic immunological synapse without prior infection of NK cells. Thus, we report for the first time the requirement of cotriggering