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Regulation of the B7-H1 (CD274) inhibitory molecule expression on tumour and immune cells.
Hrušková, Veronika ; Reiniš, Milan (advisor) ; Krulová, Magdaléna (referee)
The co-stimulatory/inhibitory molecules of family B7 that are expressed on the surface of antigen-presenting and other cells, including tumour cells, play an important role in immune responses regulations. Owing to binding on the T-lymphocyte receptors, they regulate their positive and also negative responses. An important co-stimulatory member of B7 family is B7-H1 molecule. This molecule is presented in many different tissues, not only on haematopoietic cells. However, it is also expressed on tumour cells. Its two binding receptors are in the cytoplasmic membrane of T-cells. The first receptor is unknown and the second receptor is PD-1 (CD279). The linkage between B7-H1 - PD-1 regulates negatively proliferation, differentiation and cytokine secretion of T-cells. This negative regulation is crucial for the constitution of peripheral immune tolerance and also for escape of tumour cells from T-lymphocyte based anti-tumour responses. Although the mechanisms underlying the B7-H1 cell-surface expression have been intensively studied, and they are not fully understood, especially in tumour cells. IFNγ and TNFα regulate B7-H1 expression on transcription level as well as on post- transcription level. Binding sites for transcription factors IRF-1 and NF-κB are located in B7- H1 promoter region. The IRF-1...
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Construction of various types of vaccines based on the structural proteins of mouse polyomavirus and analysis of immune response after their administration to mice
Hrušková, Veronika ; Forstová, Jitka (advisor) ; Roubalová, Kateřina (referee) ; Reiniš, Milan (referee)
Conclusion Humoral and cellular immune responses developed in mice after intranasal delivery of model mouse polyomavirus derived VLPs carrying epitope of enhanced green fluorescence protein (EGFP) Model chimeric EGFP-VLPs were purified and used for immunization of mice. Immune response of immunized animals was examined. No specific antibodies against EGFP protein, but high titers of specific antibodies against major structural protein VP1 were developed in the sera of immunized animals. Splenocytes derived from immunized animals secreted IL-2 and IFN-γ after their antigen (EGFP or VP1) restimulation. Proliferation of CD4+, but not CD8+ T cells from immunized mice after the stimulation with both EGFP and VP1 was observed. No EGFP specific cytotoxic activity of splenocytes from immunized mice was detected. The presentation of EGFP-VLPs in the context ofMHC class II was blocked by inhibitors of endo-lysosomal as well as proteasomal compartments. Changes in the numbers of CD25+Foxp3+ subpopulation of CD4+ T cells were observed in the spleens if immunized mice. Chimeric VLPs derived from mouse polyomavirus carrying epitopes of human Bcr-Abl fusion protein (Bcr-Abl VLPs) Chimeric Bcr-Abl VLPs carrying 171 amino acids sequence of Bcr-Abl protein (containing Bcr-Abl breakpoint region) were prepared. Chimeric VLPs...
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Genová imunoterapie nádoru: DNA vakcíny proti HPV16
Poláková, Ingrid ; Šmahel, Michal (advisor) ; Hejnar, Jiří (referee) ; Reiniš, Milan (referee)
of PhD. Thesis Gene Immunotherapy of Cancer: DNA Vaccines against HPV 16 Mgr. Ingrid Poláková Cervical carcinoma (CC) represents the second most frequent cancer in women, mostly associated with human papillomavirus (HPV) infection. Nowadays, two prophylactic vaccines, protecting against HPV 16 and HPV 18, are licensed. Nevertheless, development of therapeutic vaccines is desirable to eliminate current HPV infections and to treat progressing tumours. Suitable targets for vaccination are viral E6 and E7 oncoproteins. Since its discovery, DNA vaccination has become an effective strategy for development of vaccines against cancer including CC. Unfortunately, the immunogenicity of DNA vaccines in large animals and particularly in humans is low. Therefore, several ongoing studies are focused on strategies enhancing the efficacy and safety of DNA vaccines. In this work, the immunogenicity of DNA vaccines against HPV 16 delivered by a gene gun was evaluated after the fusion of the E7 and E6 genes with GUS. The increased steady-state level of the E7GGG.GUS deletion mutants and the GUS.E7GGG fusion protein enhanced the production of E7-specific antibodies after immunisation with these vaccines but did not improve the CTL response. Joining of the signal sequence with GUS.E7GGG led to ER-localisation of the...
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Gene Therapy of CML: Experimental Vaccines against Bcr-abl-transformed Cells
Lučanský, Vincent ; Vonka, Vladimír (advisor) ; Roubalová, Kateřina (referee) ; Reiniš, Milan (referee)
Chronic myeloid leukemia is malignant disease characterized by myeloproliferative clonal expansion of hematopoietic stem cell. It is causally associated with the formation of the so called Philadelphia chromosome and production of its specific product, the chimeric BCR-ABL protein. The amino acid sequence of the fusion region is unique, implying that the BCR-ABL protein carries tumor specific antigen. Currently imatinib mesylate dominates the treatment of CML. It is well tolerated and when compared to the other drugs used, it prolongs the life expectancy significantly. Unfortunately, it is not capable to cure the disease. The only potentially curative approach nowadays is the bone marrow transplantation; however, it is connected with a relatively high morbidity and mortality. Moreover, it is available only to a minority of the patients. Under these circumstances the need for the development of a relatively safe and generally available treatment is understandable. Immunotherapy could be such a treatment. Several experimental vaccines based on BCR-ABL sequence were developed and tested in mice in our institute. The DNA vaccines used were carrying sequences coding for the whole BCR-ABL protein, or for 25 amino acids long junction region (these DNA sequences were fused with adjuvant genes such as...
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Regulation of the expression of MHC class I molecules and other immunoactive molecules on tumor cells
Moravcová, Simona ; Vopálenský, Václav (referee) ; Reiniš, Milan (advisor)
4 Abstract Regulation of the expression of MHC class I molecules and other immunoactive molecules on tumor cells The aim of this master thesis project was to characterize the effects of IFNγ, TNFα and of the epigenetic agents 5-azacytidine (5AC) and 5-aza-2'-deoxycytidine (5AZAD) on the expression of molecules important for antigen presentation and modulation of the immune responses against tumors (MHC class I molecules and other immunoactive molecules CD54, CD80, B7-H1, B7-H2 and CD1d) on tumor cells. The experimental model used for this purpose were the HPV16-associated murine tumor cell lines, either MHC class I positive or negative. The goal was to determine the changes in surface expression of these molecules after treatment by FACS studies and also the expression at the mRNA level using qPCR. Expression of these proteins was also compared in the experiments in vitro on tumor cell lines and ex vivo on tumor cells explanted from animals. The most interesting result is the observation that 5AZAD increases the expression of B7-H1 which inhibits T cell mediated immune response and that 5AZAD and IFNγ act synergistically in the induction of the expression of MHC class I, CD54 and B7-H1 molecules. Klíčová slova: imunoeditace, protinádorová imunita, HVP16, MHC glykoproteiny I. třídy, B7-H1, IFNγ, TNFα,...
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Induction of the immune response against HPV16-associated tumours with experimental vaccines
Kalenská, Romana ; Krulová, Magdaléna (referee) ; Reiniš, Milan (advisor)
5 Induction of the immune response against HPV16-associated tumours with experimental vaccines The E6/E7 oncoproteins of human papillomaviruses are expressed in most trans- formed cells of cervical carcinoma and, therefore, are attractive targets for T cell-mediated immunotherapy. We have investigated the capacity of vaccines based on E7 oncoprotein- derived peptides to induce cellular immune responses and their therapeutic potential for treatment of minimal residual disease after surgery in a murine experimental model mi- micking human HPV16-associated carcinomas. We compared the effect of E749-57 peptide (RAHYNIVTF) exhibiting immunodominant epitope recognized by cytotoxic T lymphocy- tes with E744-62 peptide (QAEPDRAHYNIVTFCCKCD = 8Q) exhibiting CTL, TH and B- cell epitopes. Immune responses were compared in healthy mice and in mice after surgery or chemotherapy of tumours with ifosfamide derivative CBM-4A. Cellular immune re- sponses were monitored in spleen cells of C57BL/6 mice using ELISPOT-IFN-γ and 51 Cr- release assay. Flow cytometry was used for the detection of CD4+ , CD8+ , CD4+ CD25+ , Gr-1+ CD11b+ and CD3+ NK1.1+ populations. Vaccination with 8Q peptide and synthetic CpG oligodeoxynucleotide as adjuvant induced stronger antitumour immune responses than immunodominant CTL epitope alone in both...
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Epigenetic regulation of expression of immunoactive genes on tumor cells
Hejhal, Tomáš ; Reiniš, Milan (advisor) ; Šmahel, Michal (referee)
The aim of this master thesis was to identify epigenetically silenced genes and to determine molecular effects of DNA methyltransferases inhibitor (5-aza-2'-deoxycytidine) and histon deacetylases inhibitor trichostatin A (TSA) on gene expression in cancer cell lines TC-1/A9 and RVP3. Epigenetic events play an important role in tumorigenesis and also in escape of cancer cells from immune surveillance. I analyzed global changes in gene expression profiles of two cell lines by microarray analysis with a special attention paid to immunoactive genes. The experimental model used for this purpose were murine tumor cell lines, MHC class I deficient. I identified epigenetic regulation of several genes that are involved in cancer and immune system interactions. These data demonstrate that epigenetic agents are capable to activate expression of genes that are important for antigen presentation, cell adhesion and apoptosis. Powered by TCPDF (www.tcpdf.org)
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