Nous avons analysé, chez les patients atteints d'hépatite virale C chronique, les lymphocytes exprimant des récepteurs de molécules HLA de classe I (NKG2A et les membres CD158a,b et CD158b,j du groupe KIRs) qui sont important pour réguler les cellules NK et qui peuvent aussi être exprimés par les cellules T (LT)CD8+ activés/mémoires. Nous n'avons pas détecté de phénomènes d'accumulation généralisée de ces cellules dans le foie ou le sang des patients. Nonobstant, l'infection VHC semble influencer le phénotype des LT CD8+. Par rapport aux paramètres cliniques, nous avons observé une corrélation positive entre la fréquence intra-hépatiques de cellules T CD8+ NKG2A+ et le degré de sévérité des lésions. Ces résultats montrent de multiples altérations de la représentation de sous populations lymphocytaires T au cours de l'hépatite virale C chronique et révèle des associations positives de certaines de ces sous populations avec des paramètres cliniques majeurs de la maladie.

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...

Natural killer (NK) cells are an essential part of immune system, providing self-surveillance of virally infected, stress transformed or cancerous cells. NKR-P1 receptors and their ligands from clec2 gene family represent an alternate missing-self recognition system of NK cells based on interaction of highly related C-type lectin-like receptors. Human NKR-P1 has been described more than twenty years ago but still remains the sole human orthologue of this receptor family, particularly numerous in rodents. On binding to its cognate ligand LLT1, NKR-P1 can relay inhibitory or co-stimulatory signals. Although being interesting targets for their potential role in tumor immune evasion and autoimmunity, nature of their interaction is still unclear. To elucidate the architecture of their interaction, we developed a generally applicable method for recombinant expression of human NKR-P1 and LLT1 and their homologues based on transfection of HEK293S GnTI- cells. Further, we described a stabilizing mutation His176Cys, that enables for expression of highly stable and soluble LLT1. Finally, we have crystallized LLT1 and human NKR-P1 in different glycosylation states both as individuals and in complex. While both structures of LLT1 and NKR-P1 follow the classical C-type lectin-like superfamily fold, contrary to...

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.

Natural killer or NK cells are immunocytes that mediate innate immunity against pathogens and tumors without pre-exposition to the antigen. They are holding rapid antiviral defense during the initial phase of immune response, before starting the production of antibodies and the development of specific cytotoxic T -lymphocytes. On the surface of NK cells is expressed wide range of inhibition and activation receptors. Important family of those receptors are C - type lectin like from which the family of NKR - P1 ("natural killer cell receptor - protein 1") was discovered first. Diploma thesis deals with the preparation/study of mice NK cell activation receptor NKR- P1C and searching for its binding partner. The soluble form of the protein NKR-P1C was prepared by recombinant expression using the transient transfection of HEK293 cell line (human embryonic kidney 293) with wild type or homogenous glycosylation as IgG - Fc fusion protein, from which was it possible to obtain pure dimer of NKR P1C, after process of affinity purification, TEV protease cleavage and HPLC chromatography. The fusion protein was bound to protein A labeled with a fluorescent probe DyLight 488. Mice tissues and cell lines were labeled by this complex for purpose of seeking ligand.

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...

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...

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...

Molecular dynamics simulations are a theoretical method enabling to trace the movement of atoms within a system. The system studied is usually treated on the atomistic level, however its overall properties can be also described satisfactory if several atoms are handled as one particle (coarse-grained molecular dynamics). This thesis presents molecular modeling and (coarse-grained) molecular dynamics as tools for the description of different biologically relevant systems. The coarse-grained force field parameters had to be developed prior to characterization of the thylakoid membrane from cyanobacterium Synechocystis PCC6803. Two different compositions of the membrane were studied in order to reveal differences in their behavior. The PsbI subunit of photosystem II was embedded into the thylakoid membrane and its behavior, both as an isolated protein and as a cluster of several units, was described. The last system examined was the C-type lectin-like domain of NKR-P1, a surface receptor of natural killer cells. Attention was payed to its structural characterization.

Natural killer (NK) cells represent a crucial component of the innate immune system and are primarily regulated by the interactions of their activation and inhibitory receptors with ligands available on target cells. The genetically linked Ly49 and NKR-P1 family of receptors constitute two of the major regulatory receptor systems used by NK cells and have been shown to bind different ligands. Whereas the Ly49 receptors survey MHC-I ligands on target cells, the NKR-Pl receptor family members bind to various members of the C-type lectin-related (Clr) family. Interestingly, NKR-P1 and Clr haplotypes possess a stable genomic polymorphism across multiple mouse strains, suggesting that this inhibitory receptor:ligand relationship has an important role in the maintenance of host cellular cognate specificities. The NKR-P1 and Clr receptor-ligand pairs identified in mice include the NKR-P1B:Clr-b and the NKR-P1G:Clr-f interacting pairs. Previous RT-PCR and in situ RNA hybridization data generated by our laboratory determined that kidney tubular epithelium as well as the small and large intestinal epithelial cells specifically and highly expresses the Clr-f transcripts. Contrarily, the Clr-b transcripts were only detected on hematopoietic cells of various lymphoid organs and kidneys. Moreover, foregoing studies revealed that the loss of Clr-b following viral or chemical induced stress mediates NK cell killing of the target cell, suggesting a tissue-specific immune-surveillance mechanism in parallel with the global MHC-I-dependent missing-self model. However, the role of the NKR-P1B:Clr-b recognition-system have never been examined in the intestine. Additionally, the role of Clr-f in the kidney and intestines, where they are highly expressed, has not been investigated. For these reasons, I aimed in my thesis to provide a better understanding of the functional aspect of the NKR-P1B:Clr-b and NKR-P1G:Clr-f recognition systems in mediating gut mucosal and renal homeostasis, respectively. First, in order to determine the role of NKR-P1B and Clrb receptor:ligand pair as a “missing-self” immunosurveillance system in the gut, I started by identifying the expression pattern of both the receptor and ligand on various intestinal cells. My results demonstrate that NK cells do not represent the major NKR-P1B-expressing cells in the gut lamina propria. Instead, ILC3 subsets constituted the predominant cell population expressing the receptor, whereas γδT cells composed a small fraction of NKR-P1B+ lymphocytes. In addition, the NKR-P1B expression on myeloid cells was exclusive to colon macrophages and DC subsets. Interestingly, the highest percentage of NKR-P1B+ immune cells was found in the gut, which suggests the dominant role of NKR-P1B in regulating immune functions at the level of intestinal mucosa. As expected, the expression of the NKR-P1B ligand, Clr-b, appeared on all innate immune cell types in the gut. Next, using oral infection models of Salmonela typhimurium and Citrobacter rodentium, I showed that NKR-P1B-deficient NK cells, ILC3 and γδ T cells are hyporesponsive compared to their WT counterparts. In particular, gut NKR-P1B-deficient NK cells and γδT cells secreted low levels of IFNγ cytokine while infected with S.typhimurium. Importantly, the decreased IFNγ secretion by NK and γδT cells was associated with an increased dissemination of the bacterium into the knockout spleens at day 5 post-infection. Likewise, I detected a significant decrease in IL-22 cytokine production by NKR-P1B-deficient ILC3 compared to their WT counterparts at both steady state and following C.rodentium infection. Next, I address the potential role of Clr-f in the kidney. Renal tubular epithelial cells have been shown to express high levels of Clr-f transcripts. Epithelial cells constitute the major cellular component of kidney tubules and are well known to mediate metabolic waste excretion, reabsorption of essential molecules as well as other physiological functions, such as ions exchange and water retention. To determine the role of Clr-f in renal epithelial cells, I generated a Clr-f-deficient mouse with the help of two of my previous lab colleagues. Importantly, chemical analysis on urine and serum samples from knockout and WT littermates indicated that Clr-f-deficient kidneys display a decreased filtration capacity. In particular, higher creatinine levels were detected in the Clr-f deficient serum. In addition, Clr-f-deficient mice appeared to have a lower fractional excretion of sodium (FENa) in their urine filtrates in comparison to WT excreted urine. Blood pressure measurements on the same mice at 12 and 24 weeks of age revealed a hypotensive phenotype in the Clr-f-deficient mice. Furthermore, pathological assessment of Clr-f-deficient kidneys exhibited moderate and aggravated lesions of the tubular epithelium along with marked glomerular mesangiolysis. Lastly, flow cytometry analysis on isolated lymphocytes from Clr-f-deficient and WT mice demonstrated comparable immune infiltrates between the two mouse genotypes. Altogether, our data shows that the absence of Clr-f results in the development of glomerular and tubular lesions in an immune-independent manner leading to an abnormal kidney function. Additionally, the disruption of NKR-P1B:Clr-b recognition system results in abnormal innate immune cell number and function in the mouse intestine. These novel findings sheds light on the important role of Clr-f in maintaining healthy kidney morphology and function, as well as the crucial role for NKR-P1B:Clr-b interactions in mediating intestinal homeostasis at steady and infected states.