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Combined immunotherapy of tumors with different expression of MHC class I molecules
Piataková, Adrianna Julia ; Šmahel, Michal (advisor) ; Krulová, Magdaléna (referee) ; Reiniš, Milan (referee)
Immunotherapy experienced ups and downs before being recognized as a paramount therapy for cancer. Evidence from the latest studies revealed that the tumour microenvironment (TME) plays a decisive role in the outcome of immunotherapeutic treatment. In addition, one of the mechanisms used by cancer cells to evade immunosurveillance is reduction of the expression of major histocompatibility complex class I (MHC-I), by which cancer cells become invisible to cytotoxic T lymphocytes (CTLs). Therefore, cancer immunotherapy should involve combined strategies to target both tumour cells and TME from different sites by activating other immune cells in addition to CTLs, such as tumour-associated macrophages (TAMs). This Ph.D. thesis aimed to investigate combined immunotherapy, composed of DNA immunization, immunostimulatory compounds, and an immune checkpoint inhibitor to activate adaptive and innate immunity and inhibit immunosuppression, respectively. For this purpose, murine models related to HPV-16-induced tumours with either reversibly (TC-1/A9 cell line) or irreversibly (TC-1/dB2m) reduced MHC-I expression were used. The development of the TC-1/dB2m clone was a part of this project and this clone was obtained by deactivating the B2m gene. An important focus of the research was the analysis of TAMs isolated from...
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Checkpoint blockade in cancer immunotherapy
Vacková, Julie ; Šmahel, Michal (advisor) ; Černý, Jan (referee) ; Říhová, Blanka (referee)
The immune checkpoint blockade is a novel approach of cancer therapy, which markedly enhanced treatment efficacy of several cancer types. However, the frequency of cancer patients non-responding to this treatment is high. Establishment of predictive markers to distinguish patients suitable for the immune checkpoint blockade would enhance the number of patients receiving benefit from the therapy. This dissertation thesis focuses on the enhancement of efficacy of immune checkpoint inhibitors (ICIs) and predictive markers in experimental models of mouse tumours induced by TC-1 and TC-1/A9 cell lines and its clones with deactivation of interferon (IFN)-γ signalling (TC-1/dIfngr1 and TC-1/A9/dIfngr1), or CD80 molecule (TC-1/dCD80-1). IFN-γ is presumed to be the main inducer of programmed death ligand 1 (PD-L1) and a major histocompatibility complex I (MHC-I). Moreover, PD-L1 expression may predict sensitivity to PD-1/PD-L1 blockade. Non-functional IFN-γ signalling or downregulated MHC-I expression has been associated with resistance to ICIs in some patients. We found that IFNs type I (IFN-α and IFN-β) induced the expression of PD-L1 and MHC-I on TC-1/A9/dIfngr1 tumour cells with reversible downregulation of both molecules. We also showed that deactivation of IFN-γ signalling in TC-1/A9 cells was not a...
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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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