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Preparation of fusion ligands and evaluation of their binding to NK cell receptors
Nepokojová, Tereza ; Vaněk, Ondřej (advisor) ; Grantz Šašková, Klára (referee)
Natural killer cells (NK cells) are an important part of innate immunity. On their surface they express a complex group of receptors that use different signalling motifs to activate or inhibit NK cell cytotoxic activity. NK cells are capable to kill aberrant cells (namely, viral, infected, and tumour cells) by using special cytotoxic mechanisms to trigger apoptosis. The activating receptors recognize tumour or stress-induced ligands, e.g., NKG2D receptor recognizes the MICA ligand and NKp30 recognizes the B7-H6 ligand. Therefore for human immune system it is only natural that cancer cells are destroyed by NK cells. The current therapeutic goals in the treatment of cancer are primarily focused on strengthening the body's own natural ability to fight with cancer and one possible way is stimulation of NK cells to win this deadly fight. In addition to NK cells, antibodies are also widely used for the treatment of cancer, as well as other immune-related disorders. Most of them are monoclonal antibodies, but antibody fragments are getting attention and are being tested more and more in recent years. This work describes the preparation of three bifunctional fusion proteins: B7-H6-L-aHER2, MICA-L-aHER2, and aHER2-L-MICA, which contain immunoligands for the activating receptors of NK cell and VHH fragment...
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The influence of S159A mutation on the oligomeric state of human NK cell receptor NKR-P1A
Hausleitner, Filip ; Vaněk, Ondřej (advisor) ; Kožíšek, Milan (referee)
Natural killer cells (NK cells) are lymphocytes that possess cytotoxic activity against tumour or virally infected cells independent of preceding antigen sensitisation. To kill such cells, they utilise their activating and inhibitory surface receptors that interact with target cell surface molecules. The immune response carried by NK cells depends on the balance of both activating and inhibitory signals. Human NK cell surface receptor NKR-P1A belongs to the structural family of C-type lectin-like receptors. This receptor interacts with its ligand LLT1, which belongs to the same protein family, with low affinity and high specificity. The NKR-P1A:LLT1 complex formed between NK cell and its target cell inhibits NK cell cytotoxicity, and hence is a part of the regulation of immune response. This thesis studied the effect of S159A mutation on the stoichiometric state of soluble human NKR-P1A ectodomain in solution. Therefore, a mutant form of NKR-P1A G90-S225 S159A ectodomain was successfully produced in stably transfected human embryonic kidney cells 293 (HEK293S GnTI" ). This construct was purified by affinity and size-exclusion chromatography, and analysed by SDS-PAGE and analytical ultracentrifugation. Our results show that the preclusion of N-linked glycosylation in the position 157 promotes the...
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Preparation of tumour ligand B7-H6 with coiled-coil tag and verifying of its binding to receptor NKp30
Krejčová, Kateřina ; Vaněk, Ondřej (advisor) ; Moserová, Michaela (referee)
Natural killer cells (NK cells) are part of innate antitumor immunity. These cells have the capacity to prevent viral infection or malignant transformation without prior antigen sensitization. Activation of NK cells consist of different recognition strategies. Mechanism of activation is based on down-regulated expression of MHC gp I molecules on the cell surface. NK cells possess both activation and inhibitory receptors that transmit activation or inhibitory signals which determine if NK cells are activated or not, and thus whether the target cell will be lysed or spared. NKp30 is a type I transmembrane receptor which recognize the stress-induced cell surface ligand B7-H6. Interaction of these two proteins leads to the initiation of immune response. The main aim of this thesis is the preparation of cell ligand B7-H6 with coiled-coil peptide tag in human embryonic kidney cell lines HEK293 GnTI- and HEK293T. Successful preparation of B7-H6 with coiled-coil tag on its C-terminus was verified by mass spectrometry. Its interaction with natural cytotoxicity receptor NKp30 was also proven by sedimentation analysis. Key words: NK cells, recombinant expression, B7-H6, HEK293, coiled-coil (This thesis is written in Czech)
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The impact of obesity on the immune system with a focus on NK cells
Císařová, Radka ; Krulová, Magdaléna (advisor) ; Černý, Jan (referee)
Obesity is expanding rapidly even in developing countries and has negative effect on human health. The emerging low-grade chronic inflammation of visceral adipose tissue is generally accepted as the cause of this negative effect. The most important factor in the inflammation development is the induction of inflammatory phenotype of macrophages and their subsequent accumulation in visceral adipose tissue. The first impulse for these changes is ambiguous but can be associated with changes in phenotype and activation status of NK cells. NK cell mediated cytotoxicity against viral infected or cancer cells is impaired by obesity. Leptin level is chronically elevated in obesity and has the counter effects on NK cells. Short-term leptin exposure causes lower cytotoxicity of NK cells but long-term leptin exposure has opposite effect. The few studies about phenotype of NK cells in visceral adipose tissue during obesity point on higher production of INF- and expression of NKG2D by NK cells. Further studies of NK cell phenotype in the context of obesity and in particularly in the context of visceral adipose tissue of obese subjects are important for understanding the negative effect of obesity.
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NMR study of the extracellular part of the mouse Nkr-p1b receptor from natural killer cells
Skála, Kristián ; Chmelík, Josef (advisor) ; Martínek, Václav (referee)
Protein Nkr-p1b is a surface receptor of cytotoxic NK cells, that mediates inhibitory signal toward the body's own cells. In this study, the ligand binding domain of the mouse protein receptor Nkr-p1b (mNkr-p1b LBD) was prepared by recombinant expression in E. coli cells. Isolated protein was subsequently used for NMR structural analysis. Prediction of protein secondary structures ratio was carried out using three different methods (CD, PSIPRED and TALOS). Results correlate well with the structure of CTLD domain, that plays a key role in ligand binding and thus to function of Nkr-p1b receptor. We managed to prepare this protein in a form suitable for NMR experiments. Based on the data obtained by NMR spectra analysis, a preliminary model of the mNkr-p1b LBD protein structure was created. However, for more precise learning of the 3D structure accurate positions of individual atoms need to be determined by other NMR spectra evaluation in the next phase. Explaining the structure of the ligand binding domain of mNkr-p1b protein could help to better understand the complex mechanism of activation of NK cell cytotoxic activity, thereby contributing to its controlled use as a therapeutic against some viral and tumor diseases.
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Structural studies of rat NK cell receptor NKR-P1B and its ligand Clrb
Skořepa, Ondřej ; Vaněk, Ondřej (advisor) ; Kavan, Daniel (referee)
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...
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Progesterone influence on the maternal immune system in pregnancy
Škvorová, Anna ; Koucký, Michal (advisor) ; Černý, Jan (referee)
Pregnancy represents a major challenge to the maternal immune system. From an immunological point of view, a fetus is a semi-allograft. The mechanisms providing immunological paradox of fetal tolerance are still not well known and require further research. A complex network of immuno-endocrine interactions ensures fetal growth and development within the maternal uterus. The hormone playing an indispensable role in pregnancy is progesterone. The aim of this thesis is to summarize current knowledge of the effects of progesterone on the immune system in pregnancy and its mechanisms. Progesterone can affect target cells via the classical nuclear progesterone receptors, which act as transcription factors, or it can act using a variety of other ways, including non-genomic rapid signaling. Progesterone optimizes conditions for successful establishment and maintenance of pregnancy, changes the amount, localization and characteristics of immune cells and production of cytokines. It reduces the antigen-presenting capacity of dendritic cells, monocytes, and macrophages, suppresses NK cell cytotoxicity, supports the proliferation of uterine NK and dendritic cells, affects B cells and induces the formation of T regulatory cells and their recruitment into the fetal-maternal interface. The wide range of...
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Preparation of the soluble form of a mouse NKR-P1A protein for the NMR study
Skála, Kristián ; Chmelík, Josef (advisor) ; Adámková, Lyubina (referee)
Natural killer cells (NK cells) are a type of lymphocyte. According to their function they are defined as cytotoxic cells which cause cell death without prior sensitization. NKR-P1 is one of the families of NK surface receptors. This family belong to C-type lectin like with inhibitory or activatory function. In this work we concern of soluble form of mouse protein Nkr-p1a, that is isoform of activatory receptor Nkr-p1a. This receptor is expected to be intracellular due to lack of major part of its transmembrane domain. We focus on the optimization of Nkr-p1a production parameters. As production system we used bacterial strain E. coli BL21(DE3) Gold, in which the target protein is produced and subsequently isolated in the form of inclusion bodies. Obtained recombinant protein was refolded and purified. As purification step we used high-performance liquid chromatography. We optimized concentration of inductor of expression, production time and temperature. The objective is to set up protocol for preparation of isotopically labeled protein for nuclear magnetic resonance structure characterization. (in czech)
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Production and purification of recombinant receptor Clrb
Prokopová, Tereza ; Vaněk, Ondřej (advisor) ; Černá, Věra (referee)
The Natural Killer (NK) cells play a vital role in the nonspecific immunity. They are capable of efficient immunity reaction without any antigen specific receptors on their surface. NK cells recognize non-self molecules and they also recognize their molecules serving as health markers, and absence of these molecules such as MHC glycoproteins on the target cell surface. The NK cells are able to recognize viral infection or tumor transformation in the organism. If natural killer cell is in contact with a cell carrying an abnormally low MHC class I glycoproteins, it will create a signal which informs the cell is infected with a virus. NK cells trigger apoptosis of the target cell without prior stimulation, proliferation and differentiation. They also promote inflammatory responses by the production of chemokines and cytokines. The response is always the interplay of activating and inhibitory signals that the cell receives from its surroundings. The latest research shows that the targeted modulation of NK cells leads to less complications in bone marrow transplantation. They can be potentially used in immunotherapy, e.g. in the treatment of autoimmune diseases. Therefore, NK cells are a highly-studied group of cells. This thesis is focused on a production of Clrb ("C- type-lectin-related protein b")....
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