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Establishment and characterization of mouse tumor cell line with irreversible downregulation of MHC class I molecules
Lhotáková, Karolína ; Poláková, Ingrid (advisor) ; Černý, Jan (referee)
Malignant diseases are one of the major causes of death worldwide and the most common reason is the ability of tumor cells to escape the immune system. One of the main mechanisms used by tumors to avoid the immune response is downregulation of MHCI expression. In this thesis, the preparation of tumor cell line with irreversible downregulation of B2m is described. Inactivation of B2m was confirmed by flow cytometry. However, MHCI heavy chain H2-Db were expressed at the cell surface in a small amount after stimulation with IFNγ. In vitro proliferation of this clone was decreased. The tumor formation was delayed, and this effect was mediated by NK cells that played the major role in antitumor immunity. Metastatic activity of these cells was not affected after inactivation of B2m. TC-1/dB2m cells are not sensitive to DNA immunization. In combined immunotherapy experiments with DNA vaccinaton and ODN1826 adjuvant, a weak deceleration of tumor growth was achieved repeatedly. TC-1/dB2m induced tumors contained more CD4+ T lymphocytes, Treg, γδ T lymfocytes and plasmacytoid dendritic cells, and fewer NK cells and macrophages than tumors induced by original TC-1 cells. Moreover, combined immunotherapy did not increase the infiltration of the TC-1/dB2m tumors with immune cells. The TC-1/dB2m tumor cell line...
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Establishment and characterization of mouse tumor cell line with irreversible downregulation of MHC class I molecules
Lhotáková, Karolína ; Poláková, Ingrid (advisor) ; Černý, Jan (referee)
Malignant diseases are one of the major causes of death worldwide and the most common reason is the ability of tumor cells to escape the immune system. One of the main mechanisms used by tumors to avoid the immune response is downregulation of MHCI expression. In this thesis, the preparation of tumor cell line with irreversible downregulation of B2m is described. Inactivation of B2m was confirmed by flow cytometry. However, MHCI heavy chain H2-Db were expressed at the cell surface in a small amount after stimulation with IFNγ. In vitro proliferation of this clone was decreased. The tumor formation was delayed, and this effect was mediated by NK cells that played the major role in antitumor immunity. Metastatic activity of these cells was not affected after inactivation of B2m. TC-1/dB2m cells are not sensitive to DNA immunization. In combined immunotherapy experiments with DNA vaccinaton and ODN1826 adjuvant, a weak deceleration of tumor growth was achieved repeatedly. TC-1/dB2m induced tumors contained more CD4+ T lymphocytes, Treg, γδ T lymfocytes and plasmacytoid dendritic cells, and fewer NK cells and macrophages than tumors induced by original TC-1 cells. Moreover, combined immunotherapy did not increase the infiltration of the TC-1/dB2m tumors with immune cells. The TC-1/dB2m tumor cell line...
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Modification of murine tumor cell lines with CRISPR/Cas9 system and their characterization
Lhotáková, Karolína ; Poláková, Ingrid (advisor) ; Brábek, Jan (referee)
MHCI molecules are constitutively expressed in all nucleated cells and play a key role in antigen presentation to CD8+ T lymphocytes. One of the tumor immune evasion strategies is MHCI expression downregulation. This leads to an impaired recognition of tumor antigens by CD8+ T lymphocytes that are unable to start the immune response. Since the MHCI expression downregulation occurs in up to 90 % of some tumors it is neccesary to have a clinical relevant tumor model without a MHCI surface expression that would be used for testing of immunotherapeutic approaches. This thesis describes a production of new model cell lines of TC-1 tumor cells with irreversibly downregulated MHCI. That was achieved by an inactivation of B2m, which is a part of MHCI, by gene editing using CRISR/Cas9. The B2m inactivation was confirmed by flow cytometry, western blot and sanger sequencing of single alleles. The inactivation slowed down the cell growth for both in vitro and in vivo. The cell metastatic activity was not affected. The tumors established by cells without the B2m expression are not sensitive to DNA vaccine against HPV16 E7 oncoprotein by a pBSC/PADRE.E7GGG vaccine. The main effector function against these tumors possess the NK1.1+ cells. In a therapeutic vaccination experiment it was repeatedly achieved of...
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Significance of MHC class I molecules in antitumor immunity
Lhotáková, Karolína ; Poláková, Ingrid (advisor) ; Horníková, Lenka (referee)
The main function of the major histocompatibility complex class I (MHC I) glycoproteins is to present antigenic peptides to CD8+ T lymphocytes. Majority of the peptides displayed by this complex come from cell protein degradation and CD8+ T lymphocytes do not respond to them. Tumor development leads to alterations in protein production and new epitopes are generated which impacts peptide repertoire presented by MHC I glycoproteins on the cell surface. Peptides originated from tumor antigens activate CD8+ T lymphocytes and induce anti-tumor immune responses. Decreased surface expression of MHC I molecules is a common phenomenon in tumor cells that prevents effective immune response. As appropriate MHC I expression level on tumor cells is needed for their effective killing by T cell-mediated immune response, downregulation of the MHC I expression may lead to the selection of tumor cells with decreased MHC I level. This downregulation can be either reversible or irreversible, affecting not only the expression of genes encoding the light and heavy chains of MHC I molecules, but also genes of the antigen-processing machinery (APM). Immunotherapeutics focused on the induction of surface expression of MHC I molecules often have other unfavorable impacts on the immune system. Therefore, a new approach is...
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Orthopedagogic Music Therapy
Lhotáková, Karolina ; Leontovyčová, Jana (advisor) ; Váňová, Miroslava (referee)
In this work I'm describing the genesis of music therapy, the types and forms. The work is focused on orthopedagogic music therapy. You can find information how to access to clients with disabilities and techniques and games for use in orthopedagogic music therapy practically. Most knowledge comes from my own experience, all the techniques I have already applied in use. I was underpinned by the professional literature. The aim is to present a comprehensive view of orthopedagogic music therapy focused on the use of that knowledge and methods of work with handicapped clients.
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