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Preparation and study of human NK cell receptor AICL
Nový, Jiří ; Vaněk, Ondřej (advisor) ; Novák, Petr (referee)
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....
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Lectin receptor-ligand interaction important in experimental tumor therapy
Grobárová, Valéria ; Černý, Jan (advisor) ; Filipp, Dominik (referee) ; Krulová, Magdaléna (referee)
Lectin-saccharide interactions are involved in many biological processes essential for the survival and proper function of multicellular organisms. C-type lectin-like receptors, predominantly expressed by cells of the innate immune system, recognize saccharide structures on microbes and also aberrant glycosylation pattern of cancer cells. The NKR-P1 receptor family was among the first natural killer (NK) receptor families that were identified, however ligands for some of members remain still elusive. Recently, publications describing N-acetylglucosamine-terminated oligosaccharide structures as possible ligands for NKR-P1 receptor have been subjects for correction/retractions after investigation of the Ethical Committee of the Institute of Microbiology, ASCR, v. v. i. and Charles University in Prague. Re-evaluation of glycodendrimer effect, particularly effect of N-acetyl-D-glucosamine octabranched dendrimer on polyamidoamine scaffold (GN8P), revealed mostly indirect role of NK cells on modulation of immune responses. Properly folded soluble recombinant rat NKR-P1A and mouse NKR-P1C lack binding activity to neoglycoproteins modified with GlcNAc-terminated structures. Moreover, new possible target cell populations (NKT cells and macrophages) for saccharide binding were identified.
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Studies on structure and biological functions of NKR-P1 receptors
Rozbeský, Daniel ; Novák, Petr (advisor) ; Konvalinka, Jan (referee) ; Drbal, Karel (referee)
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...
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NK cells and their receptors in immune regulation - possible targets for immunomodulation
Svoboda, Jan ; Fišerová, Anna (advisor) ; Pěknicová, Jana (referee) ; Kročová, Zuzana (referee)
(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...
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Úloha vrozené a získané imunity v imunoterapii melanomu a pankreatického adenokarcinomu
NEDBALOVÁ, Pavla
This study examines the role of innate and adaptive immunity in the immunotherapy based on the combination of the ligands stimulating phagocytosis anchored in the tumour cells membrane and the mixture of TLR agonists. This immunotherapy is primarily focused on the innate immunity activation and induces strong inflammatory infiltration, which neutrophils and NK cells are part of. Therefore, the next aim of this study was to evaluate the anti-tumour activity of neutrophils and NK cells. For examination malignant melanoma and pancreatic adenocarcinoma mouse tumour models were used.
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Preparation of recombinant forms of the extracellular part of mouse leukocyte receptors from NKR-P1 family.
Adámek, David
Mouse NK cell receptors belonging to NKR-P1 family plays role in activation, inhibition and cytokine secretion by these cells. Aim of this thesis is preparation of extracellular parts of C57BL/6 mouse strain activating receptors mNKR-P1A and mNKR-P1C. Production vectors with coding sequences of both proteins were prepared. Next, optimization of production in E. coli was done and appropriate in vitro refolding and purification protocol were developed. Purified proteins were characterized by mass spectrometry and labeled by a fluorescent dye. Primary screening for potential ligand was performed. Further work will involve structural characterization of the receptors and identification of their ligands. These data may help to clarify the function of NK cells.
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Preparation of recombinant forms of the extracellular part of mouse leukocyte receptors from NKR-P1 family.
Adámek, David ; Man, Petr (advisor) ; Brdička, Tomáš (referee)
Mouse NK cell receptors belonging to NKR-P1 family plays role in activation, inhibition and cytokine secretion by these cells. Aim of this thesis is preparation of extracellular parts of C57BL/6 mouse strain activating receptors mNKR-P1A and mNKR-P1C. Production vectors with coding sequences of both proteins were prepared. Next, optimization of production in E. coli was done and appropriate in vitro refolding and purification protocol were developed. Purified proteins were characterized by mass spectrometry and labeled by a fluorescent dye. Primary screening for potential ligand was performed. Further work will involve structural characterization of the receptors and identification of their ligands. These data may help to clarify the function of NK cells.
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Potentiation of the biological activity of IL-2 and IL-15 in vivo
Votavová, Petra ; Kovář, Marek (advisor) ; Reiniš, Milan (referee)
5 Abstract Interleukin-2 possesses strong stimulatory activity for activated T and NK cells and thus it is an attractive molecule for immunotherapy. However, its unfavourable pharmacological properties, extremely short half-life and severe toxicities associated with high-dose IL-2 are the most serious and limiting drawbacks. Moreover, IL-2 has been also implicated in the homeostasis of T regulatory cells where it plays a decisive role as an essential growth factor of these cells. Several different approaches to improve the therapeutic potential of IL-2 have been studied. Recently described IL-2/anti-IL-2 mAb immunocomplexes which show much higher and selective biological activity in contrast with free IL-2 in vivo are probably the most promising of them. In this study, we compared the biological activity of free IL-2 with IL-2/anti-IL-2 mAb immunocomplexes in order to demonstrate their benefits over free IL-2. We also demonstrated that IL-2/anti-IL-2 mAb immunocomplexes possess noticeable antitumor activity in two syngeneic mouse tumor models, namely EL4 T lymphoma and B16F10 melanoma, if administered early in tumor progression. Therefore, we justified potential use of IL-2/anti-IL-2 mAb immunocomplexes in tumor immunotherapy. We covalently conjugated IL-2 to synthetic semitelechelic polymeric carrier based...
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Expression of the recombinant extracellular parts of leukocyte receptors AICL a NKR-P1CBALB
Čonka, Martin ; Novák, Petr (advisor) ; Cebecauer, Marek (referee)
v anglickém jazyce NK cells represent a population of lymphocytes which are able to kill certain tumor cells or virally infected cells. The subject of the diploma thesis is a mouse NK cell receptor mNKR- P1CBALB and a human leukocyte receptor hAICL. The mNKR-P1CBALB belongs to the activating receptors and is able to activate the cytotoxic functions of NK cells. The hAICL receptor is a ligand to the NKp80 which is an activating receptor of NK cells. Interaction between these two proteins leads to the activation of effector functions of NK cells as well. The aim of this work was the preparation of the recombinant extracellular parts of receptors mNKR-P1CBALB and hAICL, the optimalization of their in vitro refolding and the characterization of proteins using mass spectrometry. The proteins samples will be used for further structural study of the extracellular parts of these leukocytes receptors.
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Proteomic characterization of human NK cell membrane microdomains
Kádek, Alan ; Man, Petr (advisor) ; Martínková, Markéta (referee)
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...
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