Κάθε χρόνο τα άτομα που υποφέρουν από κάποιας μορφής νεφρική νόσο αυξάνονται παρά τις εξελίξεις στον τομέα της ιατρικής. Έχει παρατηρηθεί ότι η νεφρική νόσος, στις περισσότερες περιπτώσεις μπορεί να εξελιχθεί σε νεφρική ανεπάρκεια, είτε με πιο αργούς είτε με πιο γρήγορους ρυθμούς. Η νεφρική ανεπάρκεια αντιμετωπίζεται είτε με αιμοκάθαρση είτε με μεταμόσχευση. Το ανοσοποιητικό σύστημα διαδραματίζει σημαντικό ρόλο τόσο στην αντιμετώπιση όσο και στη πρόοδο των διάφορων μορφών νεφρικής νόσου. Ωστόσο, οι διάφοροι λεμφοκυτταρικοί τύποι, και ιδιαίτερα αυτοί της φυσικής ανοσίας, δεν έχουν μελετηθεί ιδιαίτερα και πολλές φορές από τις λίγες μελέτες που υπάρχουν προκύπτουν αντικρουόμενα αποτελέσματα. Η μελέτη μας επικεντρώθηκε στα Τ, στα ΝΚ και στα ΝΚ-Τ λεμφοκύτταρα. Επιλέξαμε να ερευνήσουμε αρχικά και τελικά στάδια νεφρικής νόσου. Έτσι οι ασθενείς κατηγοριοποιούνται σε τρεις ομάδες: ασθενείς με σπειραμαρονεφρίτιδα (αρχικό στάδιο), ασθενείς που υποβάλλονται σε αιμοκάθαρση και μεταμοσχευμένοι ασθενείς (τελικά στάδια). Σε κάθε πείραμα γίνεται σύγκριση με μια ομάδα ελέγχου (control, που αποτελείται από υγιή άτομα). Στην περίπτωση της σπειραματονεφρίδας παρατηρήθηκαν φυσιολογικά ποσοστά των λεμφοκυτταρικών τύπων και της έκφρασης του NKG2D υποδοχέα. Παρατηρήθηκε, όμως, αυξημένη έκφραση του TNF-α. Στην περίπτωση της αιμοκάθαρσης αν και παρατηρήθηκε μειωμένο ποσοστό ΝΚ και Τ κυττάρων, βρέθηκε ότι παρουσιάζουν αυξημένη κυτταροτοξικότητα και λειτουργικότητα. Αυτό φαίνεται και από την αύξηση στην έκφραση του CD107α και από την αύξηση στη συγκέντρωση του TNF-α. Όσον αφορά τα ΝΚ-Τ κύτταρα, αν και το ποσοστό τους είναι φυσιολογικό, εμφανίζουν αυξημένη έκφραση του υποδοχέα NKG2D. Τέλος, όσον αφορά την ομάδα των μεταμοσχευμένων ασθενών, δεν παρατηρήθηκε μεταβολή στο ποσοστό των Τ και των ΝΚ-Τ κυττάρων. Όταν όμως οι ασθενείς κατηγοριοποιούνται με βάση την φαρμακευτική αγωγή, το ποσοστό των ΝΚ-Τ κυττάρων είναι αυξημένο σε ασθενείς που τους χορηγείται κυκλοσπορίνη. Επίσης, η έκφραση του υποδοχέα NKG2D είναι φυσιολογική και στις δύο κατηγορίες λεμφοκυττάρων. Από την άλλη, αν και το ποσοστό των ΝΚ κυττάρων μειώνεται, αυτά εμφανίζουν φυσιολογική λειτουργικότητα και κυτταροτοξικότητα, όπως φαίνεται από τη φυσιολογική έκφραση του NKG2D υποδοχέα, της πρωτεΐνης CD107α και του TNF-α. / Every year, people who suffer from some form of kidney disease are increasing despite advances in medicine. It has been observed that the kidney disease, in most cases might progress to renal failure, either slower or faster. The renal failure is treated either by hemodialysis or renal transplantation. The immune system plays an important role in the treatment and in the progression of various forms of renal disease. However, the various types of lymphocytes, and especially those of innate immunity have not been widely studied and often the few studies that exist result in conflicting results. Our study focused on T, NK and NK-T lymphocytes. We chose to investigate initial and final stages of renal disease. Patients were categorized into three groups: patients with glomerulonephritis (initial stage), patients on dialysis and transplanted patients (final stages). In each experiment patients were compared with a control group consisting of healthy individuals. In the case of glomerulonephritis, we observed normal percentages of the lymphocyte population examined and normal expression of the NKG2D receptor, as well as increased expression of TNF-α. In the case of hemodialysis, NK and T cell percentages were reduced, while NK cells exhibited enhanced cytotoxicity and functionality. This was shown by the increase in the expression of CD107α and by the increase in the concentration of TNF-α. Regarding the NK-T cells, although their percentage was normal, they showed increased expression of NKG2D receptor. Finally, concerning the group of transplanted patients, no change was observed in the percentage of T and NK-T cells. However, when patients were categorized based on the medication, the percentage of NK-T cells was increased in patients receiving cyclosporine. In addition, the expression of NKG2D receptor was normal on T and NK-T cells. On the other hand, although the percentage of NK cells was reduced, those cells exhibited normal functionality and cytotoxicity, as shown by the normal expression of NKG2D receptor, CD107α protein and TNF-α.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Natural killer cells, or NK cells are an integral component of innate immunity and fullfills the function of recognizing and killing tumor and virus-infected cells. Their function is regulated by signals produced by the interaction of inhibitory and stimulatory receptors on their surface with their specific ligands on the targer cell surface. NKp80 is an activating receptor of NK cells and forms specific complex with cell receptor AICL, both of which belong to the family of C-type lectin-like receptors. Overexpression of AICL receptor is preferably specific for tumor cells of myeloid character. This master's thesis describes the production of AICL mutated form by expression in Escherichia coli BL21 Gold (DE3) followed by isolation and in vitro renaturation of the target protein. In a previous study it was found that an odd number of cysteines in the extracelular lectin domain of AICL causes wrong folding of the protein. Substituting an odd cystein for serine at position 87 lead to stable soluble form of AICL with an even number of cysteines in conserved positions, typical for CTLD receptors. Correctness of the formation of disulfide bonds between cysteines was verified by mass spectrometry. Significant amount of the protein gained allowed for setting up a wide variety of crystallization conditions....

Proteomic characterization of human NK cell membrane microdomains. (in Czech) Bc. Alan Kádek (Department of Biochemistry, Faculty of Science, Charles University in Prague, Czech Republic) Natural killer (NK) cells are one of the important components of innate immune system. Their main function is to fight against tumors, virally infected or otherwise malformed cells. Plasma membranes of NK cells contain regions with specific lipid composition compared to the surrounding membrane (called membrane microdomains or rafts). Because of their lipid composition, microdomains preferentially accommodate some immunologically relevant proteins and play a role during cellular polarization and signalization. Characteristic feature of membrane microdomains is their partial resistance to solubilization by mild non-ionogenic detergents. In this thesis, microdomains were isolated in a detergent-resistant membrane fraction (DRM) from human NK-92MI cell line and from NK cells immunomagnetically enriched from peripheral blood of non-leukemic donors. For the isolation, Triton X-100 or Brij-98 detergent solubilization and ultracentrifugation in a sucrose density gradient was used. Protein composition of isolated DRMs was analyzed by mass spectrometry employing an LC-MALDI-TOF/TOF method. Protein lists generated in these...

Les cellules NK sont capables d’ADCC (Antibody Dependent Cytotoxicity) suite à l’engagement durécepteur Fc!RIIIa/CD16a, et de fonctions effectrices directes antivirales et anti-tumorales: c’est la«cytotoxicité naturelle ». Ainsi activées elles peuvent également répondre en produisant des cytokines, commel’IFN-!. La dégranulation et la synthèse d’IFN-! par les cellules NK observées après engagement du récepteurCD16a, dont l’expression est indépendante du polymorphisme V158F, ont été largement supérieures à cellesobtenues avec les autres récepteurs activateurs. Son engagement par les AcMor thérapeutiques a produit desréponses fonctionnelles variables selon l’AcMor, et selon les donneurs de cellules. La perte d’expression duCD16a membranaire s’est révélé être un marqueur sensible de l’activation des cellules NK, même quand cedernier n’était pas engagé. Enfin, l’emploi de d’inhibiteur d’ADAM17 (TMI-2 et TIMP3) a permis d’observerle maintien de l’expression du CD16a après activation cellulaire sans augmenter les réponses fonctionnelles.Ce travail souligne la place centrale de l’engagement du CD16a dans l’activation NK. / NK cell can trigger ADCC (Antibody Dependent Cytotoxicity) through the engagement of theFc!RIIIa/CD16a receptor, and « Natural Cytotoxicity » after integration of cellular signals coming from theiractivating and inhibitory receptors. Moreover, activated NK cells produce cytokines such as IFN-!.Engagement by monoclonal antibodies (mAb) of CD16a was strongly more efficient than that of any otheractivating receptor to induce degranulation and IFN-! synthesis. Functional responses depend on thetherapeutic mAb used to engage CD16a and on the donor of NK cells. CD16a down-modulation was a verysensitive marker of NK cell activation, whatever the mean of activation. It was inhibited in the presence ofTMI-2 and TIMP3 (ADAM17 inhibitors), whereas CD16-dependent functional responses were not increased.This work highlighted the major role of the CD16a receptor in the activation of NK cells.

De nombreuses données expérimentales et cliniques ont montré l'importance des cellules Natural Killer (NK) dans l'immunosurveillance antitumorale. Les stratégies thérapeutiques basées sur les cellules NK pourraient donc être une alternative de choix dans le traitement de certains types de cancers. Nous avons focalisé notre étude sur deux types de cancer incurables malgré les récents progrès thérapeutiques : la leucémie lymphoïde chronique B (LLC-B) et le cancer de la prostate. Le but de notre étude est une meilleure compréhension des mécanismes mis en place par les cellules B leucémiques et les cellules tumorales prostatiques pour échapper à la réponse antitumorale des cellules NK. La connaissance de ces mécanismes d'échappement est un pré-requis indispensable à l'utilisation des cellules NK dans les thérapies antitumorales. Concernant la LLC-B, nos résultats suggèrent que les cellules NK de patients, fonctionnellement compétentes, ne peuvent pas initier une réaction immunitaire appropriée envers les cellules B leucémiques due au manque de reconnaissance de ces dernières. Concernant le cancer de la prostate, nos données préliminaires montrent que les cellules NK circulantes de patients sont également fonctionnellement compétentes, quelque soit le stade de la maladie, malgré la diminution significative de l'expression du récepteur NKp30. Ainsi, le degré d’immunogénicité des cellules B leucémiques et celui des cellules tumorales prostatiques devra être autant pris en compte que la fonctionnalité des cellules NK dans les stratégies visant à optimiser l'activité antitumorale de ces dernières. / Many experimental and clinical data have enlightened the importance of Natural Killer (NK) cells in tumor immunosurveillance. Therapeutic strategies based on NK cells could be an alternative in the treatment of certain cancers. We focused our study on two types of incurable cancers despite recent advances in treatment: B chronic lymphocytic leukemia (B-CLL) and prostate cancer. The aim of our study is a better understanding of the mechanisms set up by leukemic B cells and prostate cancer cells to escape from NK antitumor response. The knowledge of these escape mechanisms is an essential prerequisite to the use of NK cells in antitumor therapies. Regarding B-CLL, our results suggest that NK cells, although functionally competent, can not initiate an appropriate immune response against leukemic B cells due to a lack of recognition of the latter. Concerning the prostate cancer, our preliminary data show that circulating NK cells are functionally competent, whatever the stage of disease, despite the significant decrease in expression of the receptor NKp30. Thus, the degree of immunogenicity of leukemic B cells and of the prostate cancer cells must be taken into account as well as the functionality of NK cells in strategies aiming at improving NK antitumor activity.

Cytotoxiques en lysant différents types de cellules et régulent la réponse immunitaire. Leur rôle dans l’asthme et ses exacerbations reste encore à identifier même si des modifications phénotypiques ont été observées chez des patients asthmatiques et qu’il a été récemment montré dans un modèle murin qu’elles n’intervenaient pas dans le développement de l’asthme allergique. L’objectif de la thèse était de mieux comprendre la place des cellules NK dans la pathologie asthmatique en se focalisant sur deux aspects : l’exacerbation viro-induite et l’inhibition par des composants microbiens.L’hypothèse pour la 1ère partie de la thèse était que les cellules NK de patients asthmatiques pouvaient présenter une dysfonction dans leur réponse à des agents microbiens qui pourrait favoriser l’exacerbation de la réaction asthmatique. Pour cela, nous avons analysé l’activation, la cytotoxicité et la production de cytokines de cellules NK provenant de patients asthmatiques sévères stimulées avec des molécules mimant des microorganismes ou un rhinovirus vivant (HRV), en comparaison avec des donneurs sains. Nous avons montré que les cellules NK de patients sévères étaient moins cytotoxiques que les cellules NK de donneurs sains en réponse à la stimulation avec un agoniste de Toll-Like Receptor 3 ou du TLR7/8 et avec HRV. En outre, lorsqu’elles sont stimulées avec de l’IL-12 et de l’IL-15, des cytokines produites pendant l’infection virale, les cellules NK de patients asthmatiques sévères expriment moins d’IFN-γ que les cellules NK de donneurs sains. Nos résultats suggèrent que l’activation des cellules NK de patients asthmatiques pourrait être insuffisante pendant les infections respiratoires et pourraient participer à l’aggravation de l’asthme.L’hypothèse pour la 2ème partie de la thèse était que les cellules NK pourraient participer à l’inhibition de la réaction asthmatique allergique dans un modèle murin. Dans des souris C57BL/6 sensibilisées à l’ovalbumine, l’instillation de FSL1, un agoniste de TLR2/6 inhibe la réaction asthmatique allergique. Cette inhibition étant associée à des modifications de la population des cellules NK, nous avons analysé leur rôle grâce à des souris déficientes en cellules NK. En l’absence de cellules NK, les souris développent un asthme allergique, et l’inhibition par FSL1 est maintenue. Par conséquent, les cellules NK ne jouent pas de rôle dans le développement de l’asthme allergique expérimental, ni dans son inhibition induite par un agent microbien. Cependant, elles pourraient être modifiées par l’environnement allergique, et avoir ainsi un rôle dans les exacerbation viro-induites. Cette question cruciale rejoint le travail réalisé dans la première partie de la thèse.En conclusion, nos résultats suggèrent que les fonctions des cellules NK seraient modifiées dans la pathologie asthmatique, qu’elle soit allergique ou non. Notre hypothèse est que le défaut d’activation des cellules NK participerait aux exacerbations viro-induites de l’asthme. Les perspectives de ces travaux sont de poursuivre la caractérisation des cellules NK chez les patients asthmatiques sévères et d’évaluer le rôle des cellules NK dans un modèle murin d’exacerbation de la réaction asthmatique.L’hypothèse pour la première partie de la thèse était que les cellules NK de patients asthmatiques pouvaient présenter une dysfonction dans leur réponse à des agents microbiens qui pourrait favoriser l’exacerbation de la réaction asthmatique. Pour cela, nous avons analysé l’activation, la cytotoxicité et la production de cytokines de cellules NK provenant de patients asthmatiques sévères stimulées avec des molécules mimant des microorganismes ou un rhinovirus vivant (HRV), en comparaison avec des donneurs sains. Nous avons montré que les cellules NK de patients sévères étaient moins cytotoxiques que les cellules NK de donneurs sains en réponse à la stimulation avec un agoniste de Toll-Like Receptor 3 ou du TLR7/8 et avec HRV [...] / Asthma is a chronic inflammatory disease of the airways affecting 334 million of people worldwide. Among asthma patients, 5% suffers from severe asthma. Severe asthma represents a major unmet need because, despite heavy treatments, patients still suffer from uncontrolled asthma symptoms, frequent exacerbations and a dramatic decrease in their respiratory capacity. The role of microorganisms in asthma is complex. On one hand, a group of epidemiologic and experimental studies have shown that chronic exposure with bacteria or microbial compounds, particularly during early childhood, would provide protection against allergic asthma. On the other hand, respiratory viruses are responsible for 80% of exacerbations and are associated with an increasing risk of developing asthma in children, whether allergic or not. Natural Killer cells (NK) are lymphocytes involved in innate antiviral immunity. They have cytotoxic functions by lysing different types of cells but also regulatory functions by producing cytokines and activating other immune cells. Their role in asthma and its exacerbations has yet to be identified, although phenotypic changes have been observed in asthmatic patients and it has recently been shown in a mouse model that they are not involved in the development of allergic asthma. The objective of the thesis was to better understand the role of NK cells in asthmatic pathology by focusing on two aspects : virus-induced exacerbation and inhibition by microbial compounds.The hypothesis for the first part was that NK cells from asthma patients may present a dysfunction in their response to microbial agents that could promote the exacerbation of the asthmatic reaction. To do this, we analysed NK cell activation, cytotoxicity and production of cytokines from severe asthmatic patients stimulated with molecules mimicking microbes or a human rhinovirus (HRV), compared to healthy donors. We showed that NK cells from severe asthma patients were less cytotoxic than NK cells from healthy donors in response to stimulation with a Toll-like Receptor 3 or TLR7/8 agonist and HRV. Moreover, when stimulated with IL-12 and IL-15, cytokines produced during viral infections, NK cells from severe asthma patient express less IFN-γ than NK cells from healthy donors. Our results suggest that the activation of NK cells in asthma patients may be insufficient during respiratory infections and may contribute to the worsening of asthma.The hypothesis for the second part was that NK cells may participate to the inhibition of a mouse model of allergic asthma. In C57BL/6 mice sensitized with ovalbumin, instillation of FSL1, agonist of TLR2/6, inhibits the features of experimental asthma. Since this inhibition is associated with changes in the population of NK cells, we analysed their role using mice deficient in NK cells. In the absence of NK cells, mice develop allergic asthma, and inhibition by FSL1 is maintained. Therefore, NK cells do not play a role in the development of experimental allergic asthma or in its inhibition induced by a microbial agent. However, they may be modified by the allergic environment, and thus have a role in viro-induced exacerbations. This crucial question is in line with the work done in the first part of the thesis.In conclusion, our results suggest that the functions of NK cells may be modified in asthmatic pathology, whether allergic or not. Our hypothesis is that the defect in NK cell activation may participate to virus-induced asthma exacerbations. Perspectives of this work are to further characterize NK cells in severe asthma patients and to evaluate the role of NK cells in a mouse model of asthma exacerbation.

Natural killer (NK) cells play a significant role in the detection and destruction of virally infected and tumor cells. The NKR-P1 receptors regulate NK cell function by an alternative missing-self recognition system. Although the NKR-P1 receptors were among the first surface NK receptors identified on rodent NK cells more than 20 years ago, there is still very little known about their biological function and their physiological ligands. Furthermore, no three-dimensional structure of any of the NKR-P1 family receptors has been published so far. To understand the functional architecture of mouse NKR-P1 receptors, we developed a simple and efficient protocol providing large amounts of pure soluble NKR-P1 proteins. The crystal structure of mouse NKR-P1A, determined at 1.7 A resolution, is the first structure of a representative of the NKR-P1 family. Crystal structure is formed by a compact C-type lectin-like domain and an extended loop that participates in domain swapping. A potential role of the swapped loop has been suggested in natural ligand binding by in silico studies. However, chemical cross-linking and H/D exchange in combination with high resolution mass spectrometry revealed this loop in close proximity to the compact core in solution. The discrepancy between the crystal and solution structure...

Rheumatoid arthritis (RA) is a worldwide problem representing one of the most prevalent autoimmune diseases in the world. Despite the commonness of the disea- se, its pathogenesis has not been fully described. Immune cells ranging from antigen- presenting cells to T, B and NK cells playing various roles participate in the rheumatic process. In this work we concentrated on NK cells expressing a repertoire of activating and inhibitory receptors which influence their function in health and disease. We focused on the analysis of NK cell function and described its possible modulation by rheumatic autoantigens and multivalent glycodendrimers bearing 4 (GN4C) or 8 (GN8P) N-acetyl glucosamine moieties. The effect on NK cells and the glycosylation pathways was further studied in vitro. Finally, an in vivo study was performed on an animal model of RA - col- lagen-induced arthritis (CIA) to assess the effect of the compounds on clinical develop- ment of the disease and selected immune parameters. Comparison of NK cell cytotoxicity in patients suffering from RA, other inflam- matory diseases and healthy donors showed its impairment particularly in RA patients. Peripheral blood NK cells reacted to GN8P glycoconjugate by inhibition of their effector function in CD161 high-expressing samples. The MGAT5...

Natural killer (NK) cells are an intensively studied part of immune system possessing unique ability to recognize and induce death of tumor and virus-infected cells without prior antigen sensitization. Their function is regulated by a fine balance of signals induced by multiple activating and inhibitory cell surface receptors and their interaction with the ligands present on the target cell. This can be illustrated on the homodimeric rat inhibitory receptor NKR-P1B and its ligand Clrb which play, besides other things, crucial role in the immunological response of NK cells to the infection with rat cytomegalovirus (RCMV), one of the most studied NK cell function model in rat model organism. During RCMV infection the target cell downregulates cell surface expression of Clrb, thus decreasing inhibitory signal transmitted through the NKR-P1B receptor to the NK cell, which would ideally lead to NK cell activation and lysis of the infected cell. However, RCMV carries a gene for "decoy" surface receptor - RCTL that mimics Clrb and thus helps to escape the immunological response of NK cells. Moreover, while this escape strategy was demonstrated in the WAG rat strain, it has been shown that the NKR-P1B homologue from SD rat strain binds only Clrb and does not recognize RCTL. Thus the SD rat strain is less...