Sanghee Shin. (2024). T cell-derived extracellular vesicles for cancer immunotherapy. doi: 10.22677/THESIS.200000798011
Type
Thesis
Description
Cancer immunotherapy, Extracellular vesicles, T cells, Macrophages, Immune checkpoint.
Table Of Contents
Ⅰ. Introduction t of contents 1 1.1 Cancer 1 1.2 Cancer immunotherapy 1 1.3 Limitation of cancer immunotherapy 3 1.4 Extracellular vesicles (EVs) 3 1.5 Biogenesis of EVs 3 1.6 Immune cell-derived EVs 4 1.7 Modification of immune cell-derived EVs 5 1.8 Modification strategies of immune cell-derived EVs for cancer immunotherapy 5 1.8.1 Parental cell preconditioning 7 1.8.2 Genetic engineering of EV surface 7 1.9 Research objectives and chapter outlines 8 II. Novel antitumor therapeutic strategy using CD4+ T cell-derived extracellular vesicles 11 2.1 Introduction 11 2.2 Materials and methods 14 2.2.1 Cell culture 14 2.2.2 Human T cell isolation 14 2.2.3 Isolation of T cell-derived EVs 14 2.2.4 Nanoparticle tracking analysis (NTA) for size and concentration analysis of EVs 15 2.2.5 Transmission electron microscope (TEM) analysis 15 2.2.6 Quantitative real-time PCR (qRT-PCR) 15 2.2.7 Western blotting 15 2.2.8 Cell viability assay 16 2.2.9 T cell-mediated cytotoxicity assay (co-culture assay) 16 2.2.10 Flow cytometry 16 2.2.11 Small RNA isolation from EVs 17 2.2.12 miRNA library preparation and sequencing 17 2.2.13 miRNA data analysis 17 2.2.14 Nucleofection of miRNA mimics 18 2.2.15 Immunofluorescence staining 18 2.2.16 In vivo mouse studies 18 2.2.17 Statistical analysis 19 2.3 Results 20 2.3.1 IL2 enhances the release of EV secretion in CD4+ T cells 20 2.3.2 CD4+ T cell-derived EVs increase the proliferation and activity of CD8+ T cells without altering the function of regulatory T cells 23 2.3.3 CD8+ T cells activated by CD4+ T cell-derived EVs show anti-melanoma effect 29 2.3.4 CD4+ T cell-derived EVs suppress tumor growth in the melanoma syngeneic mouse model 32 2.3.5 CD4+ T cell-derived EVs induce antitumor immune response without impacting Treg in vivo 35 2.3.6 CD4+ T cell-derived EVs primarily activate CD8+ T cells in the melanoma syngeneic mouse model 40 2.3.7 miRNA from CD4+ T cell-derived EVs are novel mediator in antitumor response of CD8+ T cells 42 2.4 Discussion 46 III. Engineered T cell-derived extracellular vesicles enhance antitumor effect through dual activation of innate and adaptive immunity 56 3.1 Introduction 56 3.2 Materials and methods 58 3.2.1 Biopanning 58 3.2.2 Enzyme-linked immunosorbent assay (ELISA) 58 3.2.3 Antibody purification 59 3.2.4 Cell culture 59 3.2.5 Preparation and purification of αCD24-tethered T cell-derived EVs 59 3.2.6 Quantitative real-time PCR (qRT-PCR) 60 3.2.7 Nanoparticle tracking analysis (NTA) 60 3.2.8 Transmission electron microscope (TEM) analysis 61 3.2.9 Quantitative evaluation of tag GFP2s employing the photobleaching assay on a single EV 61 3.2.10 Macrophage generation and stimulation 62 3.2.11 Flow cytometry analysis 62 3.2.12 In vitro phagocytosis assay 63 3.2.13 Western blotting 63 3.2.14 Immunofluorescence staining 64 3.2.15 T cell-mediated cytotoxicity assay (co-culture assay) 65 3.2.16 In vivo biodistribution 65 3.2.17 In vivo antitumor efficacy 65 3.2.18 Statistical analysis 66 3.3 Results 67 3.3.1 Generation and validation of αCD24 antibody 67 3.3.2 Design and characterization of αCD24-EVs 71 3.3.3 αCD24-EVs efficiently internalize and specifically bind to CD24 on the surface of tumor cells 74 3.3.4 αCD24 antibody potentiates macrophages-mediated phagocytosis of cancer cells 77 3.3.5 αCD24-EVs increase macrophage-mediated phagocytosis of cancer cells 80 3.3.6 αCD24-EVs inhibit PD-L1 expression in both human and mouse lung cancer cells 83 3.3.7 Tumor-targeting ability of αCD24-EVs in LLC tumor-bearing mouse model 86 3.3.8 αCD24-EVs potentiate the antitumor effect in lung cancer syngeneic mouse model 89 3.4 Discussion 91 IV. Conclusion and future prospects 95 References 97 국문요약 109
