Made available in DSpace on 2018-08-01T21:35:47Z (GMT). No. of bitstreams: 1 tese_12379_Tese - COVRE L.P. 2018.pdf: 30345373 bytes, checksum: ec3dc8e879b3ba71ae62497eaf04c63e (MD5) Previous issue date: 2018-05-28 / As células natural killer (NK) são fundamentais na resposta imune inata. As funções dessas células são reguladas pela sinalização de receptores de ativação ou inibição. Durante seu desenvolvimento, as células NK podem sofrer uma diferenciação terminal progressiva, que está associada a modificações fenotípicas e deficiências funcionais. Neste trabalho, descrevemos aspectos relacionados a estas características decorrentes do envelhecimento ou induzidas durante a leishmaniose cutânea localizada causada por Leishmania braziliensis. Demonstramos que indivíduos idosos apresentaram um aumento na população de células NK expressando alta frequência do receptor inibitório KLRG1. Essas células possuem perfil de diferenciação terminal e ativam espontaneamente o sensor metabólico denominado proteína quinase ativada por AMP (AMPK). O estímulo através do receptor KLRG1 foi capaz de aumentar a fosforilação de AMPK, impedindo a desfosforilação dessa quinase mediada por PP2C. Além disso, a ativação de AMPK suprimiu a citotoxicidade, a produção de granzima B e de IFN-γ nas células NK. Células com a via KLRG1-AMPK ativa apresentaram reduções da capacidade proliferativa, da expressão da subunidade catalítica da telomerase (TERT) e no comprimento dos telômeros, bem como aumento do dano ao DNA. Todos esses fatores são associados a redução no crescimento celular e ao fenótipo de imunosenescência adquirido durante o envelhecimento. Da mesma forma, pacientes com leishmaniose cutânea localizada demonstraram um aumento do fenótipo maduro de células NK, com expansão da subpopulação CD56dim. Além disso, foram observados a diminuição dos marcadores imaturos NKG2A, CD161 e CD27, seguidos pelo aumento na expressão dos receptores NKG2C, KIR (CD158a) e acúmulo de células terminalmente diferenciadas caracterizadas como KLRG1bright e CD57bright. Células NK de pacientes com LCL apresentaram aumento da citotoxicidade, produção de granzima B e de citocinas inflamatórias quando comparadas com controles saudáveis. No entanto, também apresentaram uma baixa capacidade proliferativa, associada à presença de células KLRG1bright e CD57bright e encurtamento significativo dos telômeros quando comparado aos controles saudáveis. Juntos, nossos dados demonstram que o envelhecimento e a infecção causada por Leishmania podem afetar o fenótipo e a função de células NK, levando ao acúmulo de células terminalmente diferenciadas. Assim, a elucidação dos mecanismos que levam a esse comprometimento, como a via KLRG1 / AMPK, podem ser futuramente explorados como forma de retardar ou mesmo reverter as deficiências observadas nestas populações. Além disso, a identificação desses fatores pode ter implicações importantes para o desenvolvimento de estratégias terapêuticas, que irão auxiliar no processo de maturação funcional de células NK e aprimorar a resposta imunológica durante infecções e câncer.

Natural killer (NK) cells were so named for their uniqueness in killing certain tumor and virus-infected cells without prior sensitization unlike T lymphocytes. NK cells possess a myriad of activation receptors and cytokine receptors that allow them to recognize stress ligands on infected/tumor cells and respond to the cytokines produced during the inflammatory process. Upon activation, NK cells produce pro-inflammatory cytokines, cytotoxic granules and chemokines to recruit other cells which ultimately result in killing of target cells. These functions of NK cells are modulated in vivo by several immune mediators; IL-15 being the most potent in enabling NK cell homeostasis, maturation and activation. Indeed, IL-15 knockout mice have no detectable NK cells. During microbial infections, NK cells stimulated with IL-15 display enhanced cytokine responses. This priming effect has previously been shown with respect to increased IFN-γ production in NK cells upon IL-12 and IL-15/IL-2 co-stimulation. In this study, I explored if this effect of IL-15 priming can be extended to other cytokines and observed enhanced NK cell responses to stimulation with IFN-, IL-21, IL-2 and IL-4 in addition to IL-12. Notably, we also observed elevated IFN-γ production in primed NK cells upon stimulation through the Ly49H activation receptor. IL-15 treatments induced NK cell proliferation, enhanced NK cell responses to activating stimuli and equipped them with cytotoxic granules thereby “readying” them for battle against infections and tumors. Here, we try to understand the signaling mechanisms underlying IL-15 treatments that activate NK cells. Currently, the fundamental processes required for priming and whether these signaling pathways work collaboratively or independently for NK cell functions are poorly understood. To identify the key signaling events, we examined IL-15 priming on NK cells in which the pathways emanating from IL-15 receptor activation were blocked with specific inhibitors. Our results demonstrate that the PI3K-AKT-mTOR pathway is indispensable for cytokine responses in IL-15 primed NK cells. Furthermore, this pathway is also implicated in a broad range of IL-15 induced NK cell effector functions such as proliferation and cytotoxicity. Given that NK cells are critical for control of viral infections like murine cytomegalovirus (MCMV), we decided to analyze the consequences of blocking the PI3K-AKT-mTOR pathway in NK cells on its anti-viral responses. Likewise, NK cells from mice treated with rapamycin to block the mTOR pathway displayed defects in proliferation, IFN-γ and granzyme B production resulting in elevated viral burdens upon MCMV infection. Taken together, our data demonstrates the requirement of PI3K-mTOR pathway for enhanced NK cell functions by IL-15. It also shows that IL-15 primes NK cell responses to several cytokines and to Ly49H activation receptor stimulation. To our knowledge this is first report to demonstrate the requirement of mTOR activity in NK cells for efficient control of acute MCMV infections; thereby coupling the metabolic sensor mTOR to NK cell anti-viral responses.

Children treated for acute lymphoblastic leukemia (ALL) are immunodeficient and therefore at an increased risk of infection and cancer recurrence. Natural killer (NK) cells are a subset of lymphocytes that are very efficient at combatting infections and cancer; however, children treated for ALL have impaired NK cell number and function. Exercise has the potential to bolster NK cell number and function, at least in healthy children and adults. Limited evidence suggests exercise may also have beneficial effects on NK cells in children treated for cancer. However, these previous exercise immunology studies in children with cancer have yielded low sample sizes. Therefore, the aim of this study was to assess the: 1a) feasibility, 1b) acceptability and 1c) safety of performing an exercise intervention in children with ALL. The secondary objectives were to assess the 2a) effects of acute exercise on NK cell number, function and receptor expression in children receiving maintenance therapy for ALL compared to healthy children, as well as to 2b) assess how the NK response changes over 4 months of therapy, and to 2c) assess the link between physical activity and NK cell number and function at rest in children receiving maintenance therapy for ALL. Children undergoing maintenance therapy for ALL (n=4) were recruited from McMaster Children’s Hospital, and healthy sex and pubertal-status matched children (n=4) were recruited from the Hamilton community. ALL patients completed a total of 3 exercise visits, occurring monthly after their regularly scheduled chemotherapy session. At each exercise visit, children were asked to complete 30 minutes of continuous biking, followed by 1 hour of rest. Blood samples were drawn at rest prior to exercise (PRE), immediately after exercise (POST) and 1 hour into recovery (REC). Healthy children only completed one exercise visit. During recovery, participants were asked to complete a physical activity enjoyment scale (PACES) questionnaire and a structured interview in order to assess exercise acceptability and to gauge participant feedback on study components, respectively. Participants were outfitted with an accelerometer to track physical activity levels between visits. Feasibility was assessed by tracking recruitment statistics, study completion rates and exercise completion rates. Acceptability of accelerometer wear was assessed by tracking accelerometer wear and log rates. Safety was assessed by tracking adverse events. All parameters were reported using descriptive statistics. We approached 22 patients to participate, and 4 children completed the study (100% completion rate) out of a goal of 15. Primary deterrents to participation were that patients and families did not want to extend time spent at the hospital or had time restrictions and that patients were uncomfortable with blood collection methods. Exercise was feasible (94% exercise completion rate), acceptable (4.2 ± 0.38 out of 5 PACES score), and safe. Accelerometer wear rates (61.9% (range 3.7-100.0%)) and log completion rates (69.0% (25.9-100.0)) were moderate. Exercise transiently increased NK cell number and function in healthy children and some children with ALL. There were no patterns in the change of the NK cell response to acute exercise over time. We were unable to assess the link between physical activity and NK cells due to a paucity of data. This study cautiously suggests that exercise is a feasible, acceptable and safe intervention that may increase NK cell number and function in children treated for ALL. / Thesis / Master of Science in Medical Sciences (MSMS) / Children treated for leukemia have weak immune systems, making them more susceptible to developing infections and cancer recurrence. Natural killer cells are a special immune cell that is very effective at combatting cancer and infections; however, children treated for leukemia have very low amounts of natural killer cells and they do not function well. Exercise is a simple way to boost the immune system in healthy adults and children, by increasing the number and function of natural killer cells. We don’t know what effect exercise has on natural killer cells in children with leukemia. Previous studies looking at the effects of exercise on the immune system of children with cancer have not been able to recruit enough children to participate. Therefore, it is also important to investigate why children with cancer may not want to participate in exercise studies looking at immune function. The main goals of this thesis were to assess how likely we are to recruit enough children being treated for leukemia to participate in a study looking at how exercise changes natural killer cells, if our participants enjoyed being part of this study, and how safe exercise is for children being treated for leukemia. We also wanted to learn about how natural killer cells respond to exercise in children being treated for leukemia. We found that most of the children and families that decided not to participate in our study felt they did not have time, and the second most common reason for not participating was because the children experienced anxiety surrounding blood draws for the study. The children that decided to participate in the study enjoyed the exercise and being in the study. We also found that the exercise was safe. Finally, we saw that exercise was able to increase natural killer cell numbers and function in some, but not all, children treated for leukemia. The results of this study suggest that exercise may be a realistic and safe way to improve immune function in some children with leukemia.

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Natural killer cells play a significant role in the immune response against tumor and infected cells. NK cells express a wide variety of surface receptors, including NKRP1, a C-type lectin-like family of both activating and inhibitory receptors. Recently, ligands have been found for some of these previously orphan molecules, some of them lying within the same family. This is also the case of rat Clr-b as a cognitive ligand for rat NKRP1B. It has been shown that in rat, this inhibitory NKRP1B-Clr-b mutual receptor system is subverted by rat cytomegalovirus protein RCTL, a viral version of Clr-b, which serves as a decoy ligand for NK cells. The aim of my diploma thesis was cloning and production of the above mentioned C-type lectin-like proteins based on transient transfection of HEK293T cell line in a suspension culture. This expression system allows us not only to obtain proteins of our interest with a satisfactory yield but also in their native conformation, removing the need for time consuming and often fruitless refolding procedures required in case of using the E. coli expression system. Success was achieved in case of Clr-b and NKRP1B receptors from both WAG and SD strains. Proteins were purified using IMAC followed by gel filtration, identified by mass spectrometry and characterized by disulfide...

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....

The Natural Killer (NK) cells have an important role in the nonspecific immunity of the or- ganism. They have the ability to identify and to kill tumor cells and cells infected by a virus without preceding sensitization by antigen. Their function is directed by the amount of sti- mulation and inhibition receptors interacting with ligands on the tumor or infected cell. This thesis focuses on the preparation and the study of the complex of rat NK cellular inhi- bition receptor NKR-P1B ("natural killer cell receptor - protein 1B") and its ligand Clrb ("C-type lectin-related ligand b"). The Clrb initiates the inhibition of NKR-P1B, meaning that if the cell express Clrb, it won't be destroyed. If the cell gets infected by the rat cytome- galovirus, it loses Clrb from its surface and its destruction is therefore no longer prevented. Cells infected with this virus defend themselves from destruction by expression of the viral gene of C-type lectin RCTL, which is a homolog of Clrb. Transient transfection of human embryonic kidney 293 cell line with simple glycosylation (HEK293S GnTI− ) was used for the recombinant preparation of the soluble form of these two receptors of the rat NK cells. The native forms of the receptors - disulfidic homo- dimers - were prepared as the fusion construct with IgG Fc (using...

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...

NK cells, which are part of the innate immune system, are increasingly gaining attention, especially due to their cytotoxic ability to kill tumor cells of certain lines and certain viral, bacterial or parasitic infestation of the body. They lay a role in organ transplantation, the fight against HIV and other autoimmune diseases. NK cells have been studied since the 70th of the 20th century, but the structures and physiological ligands of their receptors remain only partially understood, as does the exact role of these cells in the organism. They communicate with others through their receptors, that recognize the lack of expression of MHC class I glycoproteins on the surface of target cells, thereby preventing cell recognition by cytotoxic lymphocytes. This diploma thesis deals with the research of receptors from cattle (Bos taurus), which is not a traditional laboratory animal and my task was to contribute to research of the structure of this group of proteins. I dealt with the recombinant production of some of the most important representatives of NK receptors CD69, NKRP1 and NKG2D in bacterial cells. The findings published in this thesis are a continuation of my bachelor thesis and together can be beneficial for further research into structural proteins and thus may help as in veterinary medicine...