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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊宏志(Hung-Chih Yang) | |
| dc.contributor.author | Yu-Hua Yang | en |
| dc.contributor.author | 楊郁華 | zh_TW |
| dc.date.accessioned | 2023-03-19T22:16:26Z | - |
| dc.date.copyright | 2022-10-13 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-09-21 | |
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Abelin, J.G., et al., Mass Spectrometry Profiling of HLA-Associated Peptidomes in Mono-allelic Cells Enables More Accurate Epitope Prediction. Immunity, 2017. 46(2): p. 315-326. Nagler, A., et al., Identification of presented SARS-CoV-2 HLA class I and HLA class II peptides using HLA peptidomics. Cell Rep, 2021. 35(13): p. 109305. Steinberg, J., T. Wadenpohl, and S. Jung, The Endogenous RIG-I Ligand Is Generated in Influenza A-Virus Infected Cells. Viruses, 2021. 13(8): p. 1564. Hong, C.H., et al., Antigen Presentation by Individually Transferred HLA Class I Genes in HLA-A, HLA-B, HLA-C Null Human Cell Line Generated Using the Multiplex CRISPR-Cas9 System. J Immunother, 2017. 40(6): p. 201-210. Zhou, F., Molecular mechanisms of IFN-gamma to up-regulate MHC class I antigen processing and presentation. Int Rev Immunol, 2009. 28(3-4): p. 239-60. Boehm, U., et al., Cellular responses to interferon-gamma. Annu Rev Immunol, 1997. 15: p. 749-95. Rein, T., M. Müller, and H. Zorbas, In vivo footprinting of the IRF-1 promoter: inducible occupation of a GAS element next to a persistent structural alteration of the DNA. Nucleic Acids Res, 1994. 22(15): p. 3033-7. Baldeón, M.E., et al., Interferon-gamma independently activates the MHC class I antigen processing pathway and diminishes glucose responsiveness in pancreatic beta-cell lines. Diabetes, 1997. 46(5): p. 770-8. Drew, P.D., et al., Regulation of MHC class I and beta 2-microglobulin gene expression in human neuronal cells. Factor binding to conserved cis-acting regulatory sequences correlates with expression of the genes. J Immunol, 1993. 150(8 Pt 1): p. 3300-10. Ma, W., et al., Interferon-gamma rapidly increases peptide transporter (TAP) subunit expression and peptide transport capacity in endothelial cells. J Biol Chem, 1997. 272(26): p. 16585-90. Seliger, B., et al., IFN-gamma-mediated coordinated transcriptional regulation of the human TAP-1 and LMP-2 genes in human renal cell carcinoma. 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Chong, C., et al., High-throughput and Sensitive Immunopeptidomics Platform Reveals Profound Interferonγ-Mediated Remodeling of the Human Leukocyte Antigen (HLA) Ligandome. Mol Cell Proteomics, 2018. 17(3): p. 533-548. Javitt, A., et al., Pro-inflammatory Cytokines Alter the Immunopeptidome Landscape by Modulation of HLA-B Expression. Frontiers in Immunology, 2019. 10. Parker, R., et al., The Choice of Search Engine Affects Sequencing Depth and HLA Class I Allele-Specific Peptide Repertoires. Mol Cell Proteomics, 2021. 20: p. 100124. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/84581 | - |
| dc.description.abstract | 直至今日，流行性感冒病毒仍然是人類全球健康的重大威脅，每年都會導致病患輕度至重度呼吸道疾病。由於抗原漂變(antigenic drift)經常發生在抗體的辨識位點上，使得疫苗誘導產生的抗體對於流感病毒不同亞型或病毒株的交叉保護能力很差。因此，開發既長效又廣效的疫苗是有必要的。 CD8+ T細胞與抗體的作用機制不同，是通過辨識第一型主要組織相容性複合體(major histocompatibility complex class-1 [MHC-I])所呈獻的病毒蛋白衍生之序列具有保守性的胜肽(conserved virus-derived peptides)，來提供交叉保護的作用。第一型主要組織相容性複合體，在人類又稱為白血球抗原(human leukocyte antigen [HLA])，辨識它即是CD8+ T細胞活化的第一步。有鑒於第一型主要組織相容性複合體和病毒的抗原決定區(epitope)有著緊密的關聯，鑑定人類第一型白血球抗原限制性(HLA class I-restricted)保守抗原決定區對於T 細胞疫苗的開發至關重要。 目前已發表的許多關於流感病毒的抗原決定區鑑定之研究，但關於台灣人常見的第一型白血球抗原限制性保守抗原決定區的資訊知之甚少。因此，本研究旨在鑑定由CD8+ T細胞辨識之台灣人常見第一型白血球抗原所呈獻的流感病毒保守抗原決定區，這這是開發通用的流感T細胞疫苗重要的一環。為了無誤差的鑑定特定第一型白血球抗原限制性保守抗原決定區，我們使用表現單一白血球抗原基因型的細胞株(monoallelic HLA cell lines)，利用轉染或感染的方式送入病毒蛋白至細胞中，再以免疫胜肽組(immunopeptidomics)搭配逆相高效液相層析法(reversed phase high performance liquid chromatography [RP-HPLC])與液相層析串聯質譜儀(LC-MS/MS)來分析白血球抗原限制性保守抗原決定區。 目前我們已建立八種分別表達單一台灣人常見的第一型白血球抗原的細胞株，且也已經使用建立之表現HLA-A* 11:01的293T細胞鑑定出流感病毒蛋白M1、NP與PB1衍生之胜肽。除此之外，為了提高胜肽鑑定的敏感度，我們也運用抗原呈獻細胞(antigen presenting cells)外加γ干擾素(IFN-γ)的方式提升293T細胞第一型白血球抗原的表達量，來達到提高鑑定出的胜肽數量。目前發現以48小時的γ干擾素可造成表達量上升至原本的兩倍之效果。若能夠結合增加使用的細胞數以及添加γ干擾素，對於提升抗原決定區之胜肽鑑定是很有幫助的。 | zh_TW |
| dc.description.abstract | Influenza virus remains a significant threat of human global health, causing mild to severe respiratory diseases annually. Vaccine-induced antibodies have poor cross-protectivity against different viral strains or subtypes because of antigenic drift and shift of antibodies. It is necessary to develop a long-lasting, universal vaccine. Unlike antibodies, CD8+ T cells can provide cross-protectivity by recognizing conserved virus-derived peptides presented by major histocompatibility complex class I (MHC-I), also named human leukocyte antigen (HLA) in human. It is essential to identify conserved HLA-restricted epitopes for development of T cell vaccine. There have been many studies published which identified influenza virus-derived epitopes, but little information is known about those restricted by common HLA class I alleles of Taiwanese. Therefore, this study aimed to identify Taiwanese common HLA class I restricted influenza A virus CD8+ T cell conserved epitopes, which are a critical stepping stone for development of universal influenza T cell vaccine. To unbiasedly identify the epitopes for a particular HLA allele, we utilized monoallelic HLA cells with the overexpression of viral antigens and infection approaches, and then analyzed the HLA-restricted peptides by mass spectrometry-based immunopeptidomics. Currently, we have generated 8 individual single-HLA allele cell lines that express 8 common Taiwanese HLA alleles, and we identified one M1- and NP-derived peptide, and two PB1-derived peptides using monoallelic HLA-A* 11:01 expressing 293T cells. To improve epitope mapping sensitivity, we treated antigen presenting cells with IFN-γ for 48 hours and the HLA expression levels increased two times. By simultaneous increase of the input cell number and IFN-γ treatment, we achieved more sensitive epitope mapping. | en |
| dc.description.provenance | Made available in DSpace on 2023-03-19T22:16:26Z (GMT). No. of bitstreams: 1 U0001-2109202202563600.pdf: 5582547 bytes, checksum: 81a99b65a8efa16bffc2571e382bfef5 (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | 口試委員審定書 I 誌 謝 II 中文摘要 III Abstract V 1. Introduction 1 1.1 Epidemiology of influenza in humans 1 1.2 Genome and structure of influenza viruses 1 1.3 The antigenic drift and shift of influenza virus genome 3 1.4 Current influenza vaccines 3 1.5 CD8+ T cells and T cell vaccine 4 1.6 Influenza virus conserved CD8+ T cell epitopes 5 1.7 T cell epitope mapping strategies 5 1.8 Mass spectrometry-based identification of HLA-bound peptides 7 1.9 Monoallelic HLA class I cells enable more accurate epitope identification and prediction 7 1.10 Establishment of the HLA class I null artificial antigen presenting cells (AAPCs) 8 1.11 IFN-γ up-regulate MHC class I antigen processing and presentation 10 2. Specific Aims 12 3. Materials and Methods 13 3.1 Cell lines and culture 13 3.2 Plasmids 13 3.3 Lentiviral transduction 14 3.4 Flow cytometry 14 3.5 FACS sorting 15 3.6 Single cell polymerase chain reaction (PCR) and sequencing 15 3.7 Immunoblotting assay 16 3.8 DNA transfection for CRISPR editing 16 3.9 Large-scale DNA transfection for immunopeptidome analysis 17 3.10 Purification of HLA class I molecules 17 3.11 Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis 18 3.12 Identification of Influenza A virus-derived HLA peptides 18 4. Results 19 4.1 Establishment of monoallelic HLA 293T cell lines 19 4.2 Establishment of HLA class I null A549 cell lines 19 4.3 Confirmation of influenza M1, NP, and PB1 protein expression in 293T cells 21 4.4 Purification of HLA class I molecules using immunoprecipitation 22 4.5 Identification of HLA-A*02: 01 or HLA-A* 11: 01-restricted IAV M1-derived CD8+ T cell epitopes by immunopeptidome analysis 23 4.6 Determining the optimal ratio of cell lysate and antibodies utilized for immunoprecipitation 24 4.7 Identification of HLA-A*11: 01-restricted IAV NP- and PB1-derived CD8+ T cell epitopes by immunopeptidome analysis 25 4.8 Enhancing the HLA presentation of monoallelic HLA 293T cells by IFN-γ treatment 26 5. Discussion 29 5.1 The establishment of monoallelic HLA 293T cells and its limitation 29 5.2 Monoallelic HLA epithelial cell-based immunopeptidome analysis enables more accurate epitope identification 29 5.3 The effect of HLA expression level of monoallelic HLA 293T in immunopeptidome analysis 31 5.4 The number of identified human endogenous peptides were much lower than others using C1R cells 34 6. Figures 37 Figure 1. The expression levels of HLA class I molecules in monoallelic HLA 293T cell lines 37 Figure 2. Screening and identification of HLA class I-null A549 cell lines 38 Figure 3. The design of viral protein M1, NP and PB1 with FLAG-tag and protein expression detected by western blot 40 Figure 4. Western blot analysis of HLA class I complexes purify efficiency by using immunoprecipitation. 41 Figure 5. Identification of HLA-A* 02: 01/ HLA-A* 11: 01-restricted M1-derived peptides for influenza A virus strain A/Taiwan/126/2009. 42 Figure 6. Western blot analysis of determining the most proper ratio of cell lysate and antibodies utilized for immunoprecipitation 44 Figure 7. Identification of HLA-A* 11: 01-restricted NP-/PB1-derived peptides for influenza A virus strain A/Taiwan/126/2009. 45 Figure 8. The enhancement of HLA class I presentation in monoallelic HLA 293T cell lines under IFN-γ treatment 47 Figure 9. Identification of HLA-A* 11: 01-restricted NP -derived peptides for influenza A virus strain A/Taiwan/126/2009 with IFN-γ treatment. 48 7. References 50 8. Supplementary Information 54 Supplementary figure 1. Screening of HLA class I-null A549 cells 54 Supplementary table 1. gRNA sequences for HLA-A, -B, and -C target editing 55 Supplementary table 2. gRNA target site of A549 HLA-A, -B, and -C alleles 55 Supplementary table 3. Sequences of HLA-A, -B, and -C specific primers 56 Supplementary table 4. Primer sequences for cloning M1, NP, and PB1 56 | |
| dc.language.iso | en | |
| dc.subject | 免疫胜肽組 | zh_TW |
| dc.subject | T細胞疫苗 | zh_TW |
| dc.subject | 保守抗原決定區 | zh_TW |
| dc.subject | 流行性感冒病毒 | zh_TW |
| dc.subject | 人類第一型白血球抗原 | zh_TW |
| dc.subject | T cell vaccine | en |
| dc.subject | CD8+ T cell conserved epitopes | en |
| dc.subject | monoallelic HLA cells | en |
| dc.subject | immunopeptidomics | en |
| dc.subject | Influenza virus | en |
| dc.title | 台灣人常見第一型白血球抗原所呈獻流感病毒CD8+ T細胞保守抗原決定區之鑑定 | zh_TW |
| dc.title | Identification of influenza virus CD8+ T cell conserved epitopes presented by common HLA class I alleles of Taiwanese | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 胡哲銘(Che-Ming Hu),林妙霞(Miao-Hsia Lin) | |
| dc.subject.keyword | 流行性感冒病毒,T細胞疫苗,保守抗原決定區,人類第一型白血球抗原,免疫胜肽組, | zh_TW |
| dc.subject.keyword | Influenza virus,T cell vaccine,CD8+ T cell conserved epitopes,immunopeptidomics,monoallelic HLA cells, | en |
| dc.relation.page | 56 | |
| dc.identifier.doi | 10.6342/NTU202203696 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-09-21 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2022-10-13 | - |
| 顯示於系所單位： | 微生物學科所 | |
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