GM-CSF-Transduced Tumor Vaccine:Application in an Animal Model of Cancer Therapy
| Autor: | Chia-Ling Hsieh, 謝嘉玲 |
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| Rok vydání: | 1999 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 87 In this study, we evaluated the feasibility of employing the cytokine-transduced tumor cells as a tumor vaccine for cancer treatment. The strategy needs a powerful gene transfer technique to deliver gene efficiently into cancer cells and results in a high level expression of transgene. For this purpose, the retroviral vector was employed as a high-efficiency gene-delivery system in this study. We have recently constructed a bicistronic retroviral vector, S1, and the transcriptional efficiency of the S1 bicistronic vector was as good as the single gene vector. However, in some cases, the relatively high level of transcripts produce only low amounts of protein. Therefore, in the first part of this study, a new bicistronic retroviral vector, S2, was constructed to improve the expression efficiency by removing a viral gag initiation codon that is retained in the S1 vector at 5'' to the first initiation codon of transduced gene. It was shown that the new bicistronic vector, S2, was able to increase the level of protein yield by 4~14 folds when compared with that of S1. Since the RNA levels and splicing patterns from these two vectors remained similar, the improvement was most likely resulted from the increased translational efficiency of the S2 vector. Thus, the modified S2 retroviral vector was then utilized for cancer therapy because of its high levels of expression. Previous studies on tumor vaccine made containing granulocyte-macrophage colony-stimulating factor (GM-CSF) were all based on solid tumors. In the second part of this study, I used a murine T leukemia as a model to evaluate the efficacy of the GM-CSF-transduced tumor vaccine for the treatment of solid tumors as well as leukemias. By using the S2 bicistronic retroviral vector, mouse GM-CSF cDNA was transduced into a highly tumorigenic T leukemia cell line, RL♂1. Injection of GM-CSF-secreting RL♂1 cells into syngeneic BALB/c mice elicited a systemic and long-lasting immunity that prevented the tumor formation. The tumor vaccine also inhibited the growth of pre-established tumors that were introduced subcutaneously (solid tumor status) or intravenously (leukemia status) in animals. However, the therapeutic effects were less prominent in mice inoculated with a large tumor load or in mice treated later. Further basic immunological analysis supports the proposition that CD8+ T lymphocytes might have played an important role in rejecting the tumors in vivo. However, the CD8+ T lymphocytes of the animals bearing large tumors seemed to be suppressed or anergized. Systematic histological analysis for 2 weeks provided further insight into various infiltrates at the vaccine sites and at the tumor sites in response to the inoculation of GM-CSF-secreting tumor vaccine. Despite the detection of tumor-specific CTL activities and CD8+ T cells in the large tumor-bearing mice treated with GM-CSF-transduced tumor vaccine, the immune cells did not confer protective effects upon the animals. Therefore in the third part of this study, the question how tumors manage to grow in the vicinity of activated CD8+ T cells was addressed. Firstly, it was found that adoptive transfer of a large number of activated CD8+ T cells into the tumor sites could regress tumors that had already been present for 7 days. Secondly, it was noted that the expression levels of immunosuppressive cytokines, TGF-b and IL-10, at the tumor region were correlated with the size of tumors. TGF-b and IL-10 were also detected in the culture supernatant of RL♂1 tumor cells. In vitro assay showed that co-cultivation of T cells with the culture supernatant of RL♂1 cells inhibited the tumor specific T cell proliferation. Inhibition TGF-b and IL-10 activity in the supernatant by antibody blocking abolished the immunosuppressive effects. Furthermore, pre-administration of the RL♂1 cultured supernatant or recombinant IL-10 into mice receiving the (1, 4)-treatment protocol greatly reduced the therapeutic effects of tumor vaccine. Taken altogether, these results suggest two possibilities that might explain how tumors escape from immune attract. The first possibility is that CTLs to tumor (C/T) ratio at the tumor location does not reach the threshold required for large tumor regression. The second is that the CTL activity in large tumor-bearing animal is suppressed by abundant immunosuppressive factors secreted by the tumors. Furthermore, the expression of surface maker on antibody-stimulated T cells or monocytes in the presence of tumor cell cultured supernatant was analyzed. The results showed that secretion of IL-10 by RL♂1 tumor cells may reduce T cell activation directly through inhibition of IL-2 receptor expression on T cells, or indirectly by down-regulation of the antigen presentation function of APC. As a result, how to improve current tumor vaccine efficacy and overcome the immunosuppressive effects of tumor cells seem to be urgent tasks before the cytokine gene therapy can be applled as a treatment strategy for the patients bearing large tumor burdens |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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