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Novel methods of treatment of B cell malignancies based on immunotherapy with genetically modified T cells
Novotná, Natálie ; Otáhal, Pavel (advisor) ; Šmahel, Michal (referee)
CAR T cell therapy represents a promising method in treatment of hematological malignancies. Gene immunotherapy uses modified T cells that express a chimeric antigen receptor (CAR) on their surface. Modified T lymphocytes are able to recognize and destroy target cells based on specific surface markers. Although CAR T cell therapy is used in clinical practice, there is a number of limitations that reduce its effectiveness. The aim of this thesis is to explore new possibilities of making the entire therapy more efficient through endogenous secretion of interleukins (IL-7, IL-15, IL-21) under the control of inducible promoters, and thus to strengthen the persistence and expansion of CAR T cells in vivo. For this purpose, inducible expression systems containing the gene for CAR19 receptor specifically recognizing the CD19 molecule and the interleukin gene located under inducible NFAT or NR4A promoters, were constructed. The assembled vectors were electroporated into PBMC cells using the PiggyBac transposon system to achieve stable expression in T lymphocytes. After co-cultivation with RAMOS cell line, data were obtained by measurement on a flow cytometer and the ELISA method. Based on the results, it is evident that stimulated CAR T cells are able to generate higher concentrations of interleukins,...
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Leukemia cell signaling and signaling of non-malignant B-cells
Kanderová, Veronika ; Kalina, Tomáš (advisor) ; Klener, Pavel (referee) ; Otáhal, Pavel (referee)
Acute leukemia (AL) is the most common pediatric cancer. Approximately 90 - 100 children is diagnosed every year in the Czech Republic. Acute leukemia is a complex disease that is pathologically manifested at the DNA, mRNA, protein and cellular level. Leukemic cells aberrantly express molecules that are found in other cell types under physiological conditions and their functional involvement in leukemic cells is unknow. We found that aberrantly expressed CEACAM6 increases the expression and affinity of integrins, increases the phosphorylation of intracellular kinases Akt, p38MAPK and p44/42 MAPK and triggers apoptosis in B-cell precursor acute lymphoblastic leukemia cells. Adaptor molecule NTAL, aberrantly expressed in T-cell acute lymphoblastic leukemia, signals through intracellular kinase p44/42 MAPK and potentiates corticosteroid induced apoptosis. Current leukemia research is focused mainly on monitoring of mutations at the DNA level, however, the functional consequences of these changes on cellular machineries are not straightforward. Since proteome analysis can provide link between gene sequence and cellular physiology, proteomics will contribute to elucidate mechanism of disease, prognosis and response to treatment. Protein microarrays technology is of major interest for basic proteomic research as...
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Differentiation plasticity of hematopoietic cells
Polgárová, Kamila ; Stopka, Tomáš (advisor) ; Otáhal, Pavel (referee) ; Šálek, Cyril (referee)
Hematopoiesis has been for many years seen as a straightforward process based on sequential restriction of cell fate potential leading to production of mature blood cells. In the last decade, however, several works documented an unexpected plasticity of hematopoietic cells with expanded potential of myeloid development from lymphoid progenitors and vice versa. Under physiologic conditions hematopoiesis is tightly controlled and the definite cell fate is denominated by multiple factors that all lead to changes in regulatory networks that include transcription factors, epigenetic changes and post-transcriptional modulations. Any disruption of this strict regulation, caused by mutations or other events, affects the proliferation and lineage fidelity of hematopoietic precursors. This may lead to clonal growth of variable significance or leukemogenesis and may possibly affect the treatment sensitivity of the hematological malignancies. For better understanding of hematopoietic regulation we described gene expression changes during physiological development of lymphoid and myeloid lineages and in leukemic specimens using our own simplified real-time PCR based platform. We investigated expression of 95 genes connected with lymphoid and myeloid differentiation or with leukemogenesis in sorted hematopoietic...
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Lineage plasticity in normal and malignant lymphocyte precursors
Rezková Řezníčková, Leona ; Froňková, Eva (advisor) ; Otáhal, Pavel (referee)
Klasické schéma vývoje hematopoetických buněk předpokládá časné oddělení lymfoidního a myeloidního prekurzoru. V poslední době jsou navrhovány složitější modely, které předpokládají větší flexibilitu hematopoezy a navrhují existenci progenitorů s lymfoidním i myeloidním potenciálem. Akutní hybridní leukémie jsou malignity, které podle různých kritérií nelze jednoznačně zařadit k lymfoidní nebo k myeloidní linii a jejichž chování spíše dává za pravdu novým modelům hematopoezy. Předkládaná práce se zabývala především výzkumem dětských leukémií s přesmykem z lymfoidní do myeloidní linie během indukční léčby. Jedná se o rozsáhlý projekt, v jehož rámci si diplomová práce si kladla za úkol určit liniové zařazení leukemických blastů pomocí detekce přestaveb genů pro imunoglobuliny a T-buněčné receptory (TCR). Potvrdili jsme, že myeloidní buňky derivované v průběhu léčby pochází u všech pacientů z původního lymfoidního klonu. Dále jsme u těchto případů zkoumali expresi vytipovaných genů ve srovnání s běžnými druhy leukémií. Třetí částí práce byl výzkum prognostického významu přítomnosti přestaveb TCR (a tedy příslušnosti k lymfoidní linii) u leukémií z T-lymfoidní řady.
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Signaling pathways of leukocyte membrane receptors, their regulation and deficiencies
Fabišik, Matej ; Brdička, Tomáš (advisor) ; Dobeš, Jan (referee) ; Otáhal, Pavel (referee)
Because of the profound effects of signal transduction on cell behaviour, the activity of signalling pathways must be carefully regulated. Otherwise, dysregulation of signalling might harm the organism by not responding to danger or by an excessively strong reaction. Therefore, various regulatory mechanisms became essential parts of signal transduction pathways. They affect these pathways at all levels, including ligands, receptors, signalling enzymes, adaptor proteins and other signalling mediators, as well as transcription factors further downstream. In this thesis, I present the results of the research on the role of transmembrane and membrane associated adaptor proteins LST1, SCIMP, PSTPIP2 and WBP1L in the regulation of leukocyte signalling and homeostasis. Transmembrane adaptor protein LST1 is a short protein expressed in the cells of the myeloid lineage. Observation of LST1-/- mice revealed that these animals are overall healthy without visible phenotype, with the exception of mild reductions in myeloid, NK and NKT cell populations at the steady state. On the other hand, LST1 deficiency had significant protective effect during acute colitis induced by dextran sodium sulphate, suggesting the role of LST1 in the regulation of gut inflammation. Studies on PSTPIP2 and SCIMP presented in this...
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Optimizing chimeric antigenic receptors (CARs) T-cells for immunotherapy of hematological malignancies
Mucha, Martin ; Otáhal, Pavel (advisor) ; Zadražil, Zdeněk (referee)
Immunotherapy based on chimeric antigen receptor (CAR)-expressing T lymphocytes has proven to be highly successful in the treatment of acute lymphoblastic leukemia (ALL), leading to development of CAR-based immunotherapies for other hematologic malignancies. Currently, efforts are underway to refine T cell modifications to make patient treatment more effective. Each time, this modification then needs to be empirically validated in in vitro experiments. We decided to study the effect of the cytokine IL-21 on the antitumor function of CD19-specific CAR T cells using in vitro assays. A construct that co-expressed IL-21 under the control of the inducible NFAT promoter together with CARs against CD19 was introduced into T cells. In a series of experiments, the properties of these cells were compared after coculture with tumor B cell lines and CLL cells obtained from patients. The results showed that CAR T cells that express IL-21 proliferate and activate better, even after repeated stimulation with leukemia cells. In addition to CARs specific against the CD19 molecule, we also investigated CARs specific against the CLL1 molecule, which has been described in the literature as one of the promising targets for the treatment of AML. We prepared CAR T cells against CLL1 producing IL-21. For this purpose, we...
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Construction and characterization of chimeric antigen receptors
Ptáčková, Pavlína ; Otáhal, Pavel (advisor) ; Kuželová, Kateřina (referee) ; Havránek, Ondřej (referee)
Background: The CD19 chimeric antigen receptor (CAR) adoptive T-cell therapy for B-cell leukemia is a promising treatment for relapsed or refractory malignities. The overall response rate of CD19 CAR-T cells in clinical trials was greater than 80% for patients with B-cell acute lymphoblastic leukemia (B-ALL) and non-Hodgkin's lymphoma (NHL). However, CAR-T cell therapy of leukemias and solid tumors has been limited by a lot of factors such as antigen loss of tumor escape variants, reduced proliferation, persistence and tumor-infiltration of CAR-T cells in vivo, immunosuppressive tumor environment, absence of ideal antigens and on-target, off-tumor toxicities. Therefore, new strategies improving the safety and efficacy of CAR-T cells, including further T-cell modification to overcome the immune suppression, are tested. Aims: (i) Bispecific CARs designed to express two antigen-binding domains prevent of antigen escape. (ii) T-cells were genetically modified to express CAR along with an inducible IL-21 gene cassette driven by NFAT-responsive promoter. IL-21 directly enhances CAR-T cell activity and anti-tumor effects. (iii) Applying suicide epitope modification in CAR enables significantly increasing the therapeutic safety of CAR-T cells. Methods: CARs were constructed by using molecular biology...
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Epigenetic Aspects of normal and malignant hematopoiesis: role of chromatin remodeling ISWIATPase.
Zikmund, Tomáš ; Stopka, Tomáš (advisor) ; Dráber, Peter (referee) ; Otáhal, Pavel (referee)
Chromatin remodeling protein Smarca5 participates on many cellular processes, which are important for tissue development and tumorigenesis. Among these processes utilizing ATPase activity of Smarca5 belong also transcription, replication and DNA repair. We hypothesized that Smarca5 represents essential molecule for chromatin modulation primarily at early developmental stages at the level of fast-dividing progenitors of many origins, in whose the ATPase is highly expressed. To such tissues may belong also hematopoiesis, in which the Smarca5 has highest expression. The subject of my doctoral thesis is therefore analysis of the effect Smarca5 depletion on proliferation and differentiation of hematopoietic progenitors in vivo and a search for mechanisms behind the resulted developmental defects. We utilized conditionally knockout allele of Smarca5 in blood precursors to study in a mouse model how depletion of the ISWI ATPase causes accumulation of earliest progenitors inhibited from further maturation to erythroid and other myeloid lines. The proerythroblasts became dysplastic and the majority of basophilic erythroblasts ceased cycling around the G2/M stage. An expected mechanism for observed changes appeared the activation of stress pathway of protein p53 that is often associated with unrepaired DNA...
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Differentiation plasticity of hematopoietic cells
Polgárová, Kamila ; Stopka, Tomáš (advisor) ; Otáhal, Pavel (referee) ; Šálek, Cyril (referee)
Hematopoiesis has been for many years seen as a straightforward process based on sequential restriction of cell fate potential leading to production of mature blood cells. In the last decade, however, several works documented an unexpected plasticity of hematopoietic cells with expanded potential of myeloid development from lymphoid progenitors and vice versa. Under physiologic conditions hematopoiesis is tightly controlled and the definite cell fate is denominated by multiple factors that all lead to changes in regulatory networks that include transcription factors, epigenetic changes and post-transcriptional modulations. Any disruption of this strict regulation, caused by mutations or other events, affects the proliferation and lineage fidelity of hematopoietic precursors. This may lead to clonal growth of variable significance or leukemogenesis and may possibly affect the treatment sensitivity of the hematological malignancies. For better understanding of hematopoietic regulation we described gene expression changes during physiological development of lymphoid and myeloid lineages and in leukemic specimens using our own simplified real-time PCR based platform. We investigated expression of 95 genes connected with lymphoid and myeloid differentiation or with leukemogenesis in sorted hematopoietic...
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