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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊宏志(Hung-Chih Yang) | |
| dc.contributor.author | Pei-Yi Wu | en |
| dc.contributor.author | 吳佩倚 | zh_TW |
| dc.date.accessioned | 2022-11-25T07:31:22Z | - |
| dc.date.available | 2023-08-31 | |
| dc.date.copyright | 2021-08-31 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-08-20 | |
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Karayiannis, New Approaches to the Treatment of Chronic Hepatitis B. J Clin Med, 2020. 9(10). 14. Fernandez, M., J.A. Quiroga, and V. Carreno, Hepatitis B virus downregulates the human interferon-inducible MxA promoter through direct interaction of precore/core proteins. J Gen Virol, 2003. 84(Pt 8): p. 2073-2082. 15. Yu, S., et al., Hepatitis B virus polymerase inhibits RIG-I- and Toll-like receptor 3-mediated beta interferon induction in human hepatocytes through interference with interferon regulatory factor 3 activation and dampening of the interaction between TBK1/IKKepsilon and DDX3. J Gen Virol, 2010. 91(Pt 8): p. 2080-2090. 16. Lang, T., et al., The hepatitis B e antigen (HBeAg) targets and suppresses activation of the toll-like receptor signaling pathway. J Hepatol, 2011. 55(4): p. 762-9. 17. Megahed, F.A.K., X. Zhou, and P. Sun, The Interactions between HBV and the Innate Immunity of Hepatocytes. Viruses, 2020. 12(3). 18. Visvanathan, K., et al., Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein. Hepatology, 2007. 45(1): p. 102-10. 19. Faure-Dupuy, S., J. Lucifora, and D. Durantel, Interplay between the Hepatitis B Virus and Innate Immunity: From an Understanding to the Development of Therapeutic Concepts. Viruses, 2017. 9(5). 20. Shin, E.C., P.S. Sung, and S.H. Park, Immune responses and immunopathology in acute and chronic viral hepatitis. Nat Rev Immunol, 2016. 16(8): p. 509-23. 21. Thimme, R., et al., CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection. J Virol, 2003. 77(1): p. 68-76. 22. Yuen, M.F., et al., Hepatitis B virus infection. Nat Rev Dis Primers, 2018. 4: p. 18035. 23. Bohne, F., et al., T cells redirected against hepatitis B virus surface proteins eliminate infected hepatocytes. Gastroenterology, 2008. 134(1): p. 239-47. 24. Gehring, A.J., et al., Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines. J Hepatol, 2011. 55(1): p. 103-10. 25. Liu, Q., et al., In vivo therapeutic effects of affinity-improved-TCR engineered T-cells on HBV-related hepatocellular carcinoma. J Immunother Cancer, 2020. 8(2). 26. Wisskirchen, K., et al., Isolation and functional characterization of hepatitis B virus-specific T-cell receptors as new tools for experimental and clinical use. PLoS One, 2017. 12(8): p. e0182936. 27. Wisskirchen, K., et al., T cell receptor grafting allows virological control of Hepatitis B virus infection. J Clin Invest, 2019. 129(7): p. 2932-2945. 28. Mosmann, T.R., L. Li, and S. Sad, Functions of CD8 T-cell subsets secreting different cytokine patterns. Semin Immunol, 1997. 9(2): p. 87-92. 29. Zhang, N. and M.J. Bevan, CD8(+) T cells: foot soldiers of the immune system. Immunity, 2011. 35(2): p. 161-8. 30. Krogsgaard, M. and M.M. Davis, How T cells 'see' antigen. Nat Immunol, 2005. 6(3): p. 239-45. 31. Krangel, M.S., Mechanics of T cell receptor gene rearrangement. Curr Opin Immunol, 2009. 21(2): p. 133-9. 32. Fugmann, S.D., et al., The RAG proteins and V(D)J recombination: complexes, ends, and transposition. Annu Rev Immunol, 2000. 18: p. 495-527. 33. De Simone, M., G. Rossetti, and M. Pagani, Single Cell T Cell Receptor Sequencing: Techniques and Future Challenges. Front Immunol, 2018. 9: p. 1638. 34. Walchli, S., et al., A practical approach to T-cell receptor cloning and expression. PLoS One, 2011. 6(11): p. e27930. 35. Hu, Z., et al., A cloning and expression system to probe T-cell receptor specificity and assess functional avidity to neoantigens. Blood, 2018. 132(18): p. 1911-1921. 36. Kobayashi, E., et al., A new cloning and expression system yields and validates TCRs from blood lymphocytes of patients with cancer within 10 days. Nat Med, 2013. 19(11): p. 1542-6. 37. Aarnoudse, C.A., et al., TCR reconstitution in Jurkat reporter cells facilitates the identification of novel tumor antigens by cDNA expression cloning. Int J Cancer, 2002. 99(1): p. 7-13. 38. Jutz, S., et al., Assessment of costimulation and coinhibition in a triple parameter T cell reporter line: Simultaneous measurement of NF-kappaB, NFAT and AP-1. J Immunol Methods, 2016. 430: p. 10-20. 39. Rosskopf, S., et al., A Jurkat 76 based triple parameter reporter system to evaluate TCR functions and adoptive T cell strategies. Oncotarget, 2018. 9(25): p. 17608-17619. 40. Gaud, G., R. Lesourne, and P.E. Love, Regulatory mechanisms in T cell receptor signalling. Nat Rev Immunol, 2018. 18(8): p. 485-497. 41. Conley, J.M., M.P. Gallagher, and L.J. Berg, T Cells and Gene Regulation: The Switching On and Turning Up of Genes after T Cell Receptor Stimulation in CD8 T Cells. Front Immunol, 2016. 7: p. 76. 42. Guo, X.Z., et al., Rapid cloning, expression, and functional characterization of paired alphabeta and gammadelta T-cell receptor chains from single-cell analysis. Mol Ther Methods Clin Dev, 2016. 3: p. 15054. 43. Spindler, M.J., et al., Massively parallel interrogation and mining of natively paired human TCRalphabeta repertoires. Nat Biotechnol, 2020. 38(5): p. 609-619. 44. Mahajan, S., et al., Epitope Specific Antibodies and T Cell Receptors in the Immune Epitope Database. Front Immunol, 2018. 9: p. 2688. 45. Rosati, E., et al., Overview of methodologies for T-cell receptor repertoire analysis. BMC Biotechnol, 2017. 17(1): p. 61. 46. Scotto-Lavino, E., G. Du, and M.A. Frohman, 5' end cDNA amplification using classic RACE. Nat Protoc, 2006. 1(6): p. 2555-62. 47. Zhu, Y.Y., et al., Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques, 2001. 30(4): p. 892-7. 48. Wulf, M.G., et al., Non-templated addition and template switching by Moloney murine leukemia virus (MMLV)-based reverse transcriptases co-occur and compete with each other. J Biol Chem, 2019. 294(48): p. 18220-18231. 49. Migalska, M., A. Sebastian, and J. Radwan, Profiling of the TCRbeta repertoire in non-model species using high-throughput sequencing. Sci Rep, 2018. 8(1): p. 11613. 50. Lin, Y.H., et al., Dissecting efficiency of a 5' rapid amplification of cDNA ends (5'-RACE) approach for profiling T-cell receptor beta repertoire. PLoS One, 2020. 15(7): p. e0236366. 51. Zong, S., et al., Very rapid cloning, expression and identifying specificity of T-cell receptors for T-cell engineering. PLoS One, 2020. 15(2): p. e0228112. 52. Hamana, H., et al., A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells. Biochem Biophys Res Commun, 2016. 474(4): p. 709-714. 53. von Boehmer, H., et al., Thymic selection revisited: how essential is it? Immunol Rev, 2003. 191: p. 62-78. 54. Siggs, O.M., L.E. Makaroff, and A. Liston, The why and how of thymocyte negative selection. Curr Opin Immunol, 2006. 18(2): p. 175-83. 55. Hogquist, K.A., T.A. Baldwin, and S.C. Jameson, Central tolerance: learning self-control in the thymus. Nat Rev Immunol, 2005. 5(10): p. 772-82. 56. Kerkar, S.P., et al., Genetic engineering of murine CD8+ and CD4+ T cells for preclinical adoptive immunotherapy studies. J Immunother, 2011. 34(4): p. 343-52. 57. Baumann, J.G., et al., Murine T cells potently restrict human immunodeficiency virus infection. J Virol, 2004. 78(22): p. 12537-47. 58. Delville, M., et al., A Nontoxic Transduction Enhancer Enables Highly Efficient Lentiviral Transduction of Primary Murine T Cells and Hematopoietic Stem Cells. Mol Ther Methods Clin Dev, 2018. 10: p. 341-347. 59. Hagani, A.B., et al., Activation conditions determine susceptibility of murine primary T-lymphocytes to retroviral infection. J Gene Med, 1999. 1(5): p. 341-51. 60. Lee, J., M. Sadelain, and R. Brentjens, Retroviral transduction of murine primary T lymphocytes. Methods Mol Biol, 2009. 506: p. 83-96. 61. Kurachi, M., et al., Optimized retroviral transduction of mouse T cells for in vivo assessment of gene function. Nat Protoc, 2017. 12(9): p. 1980-1998. 62. Lai, M.J., et al., Distributions of human leukocyte antigen-A, -B, and -DRB1 alleles and haplotypes based on 46,915 Taiwanese donors. Hum Immunol, 2010. 71(8): p. 777-82. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82457 | - |
| dc.description.abstract | "雖然抗病毒藥物（包含nucleos(t)ide analogues (NAs) 和 interferon-α）已大量減少B型肝炎病毒（Hepatitis B virus, HBV）的負面風險，但慢性B型肝炎（chronic hepatitis B，CHB）患者即使在有效的抗病毒治療之後，HBV的共價閉合環狀DNA（closed circular DNA，cccDNA）仍能存在在受HBV感染的肝臟細胞中。欲使慢性B型肝炎痊癒需完全破壞cccDNA或清除所有受感染的肝臟細胞，而胞殺性T cell可藉由其表面的T細胞接受器（T cell receptor，TCR）和受感染肝臟細胞所表現的三聚體肽／主要組織相容性複合物（trimeric peptides/major histocompatibility complex, pMHCs）相互作用，特異性地以HBV感染的肝臟細胞為標的，且也可分泌interferon-γ (IFN-γ) 抑制病毒複製。因此，目前備受期待的治療方案為帶有HBV特異性TCR的工程性T細胞（engineered T cells with TCRs, TCR-T），它不僅具有完整的抗病毒能力，也可以用來特異性的辨認並殺死受HBV感染的肝臟細胞。然而，此方法需有效的辨認並選殖出經VDJ重組後具有功能性的TCR，目前針對尋找高親和力HBV-specific TCR-α/β pairs的方法 需要進行單細胞定序或選殖，不僅耗時、耗費人力，且通常需要特定儀器方可操作，故使功能性TCR的探尋更加複雜。為了解決這個困難，我們設計了一個新穎的選殖表現策略，可藉由reporter-Jurkat cells篩選HBV特異性T細胞，也利用Enrichment of Antigen-Specific T cells (EAST) ，例如：多聚體的分選方法（multimer-based sorting method），事先降低TCR library的複雜度，避開繁瑣的單細胞定序或選殖的方法，有機會能在很短時間內更有效率地找到具功能性的HBV特異性TCR，此法將可促進工程性T細胞用於慢性B型肝炎治癒治療的發展。" | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T07:31:22Z (GMT). No. of bitstreams: 1 U0001-1908202112061200.pdf: 5792211 bytes, checksum: bfa5047af6ef246761578143b44f2e87 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "論文口試委員審定書 i 致謝 ii 摘要 iii Abstract v 1 Introduction 4 1.1 Hepatitis B virus 4 1.1.1 Epidemiology of HBV infection 4 1.1.2 HBV replication cycle 4 1.1.3 Current treatment of chronic hepatitis B 5 1.1.4 Immune response to HBV 6 1.1.5 Role of engineered T cells in chronic hepatitis B 7 1.2 TCR cloning strategy 7 1.2.1 The overview of general TCR cloning strategy 8 1.2.2 The importance of reporter T cells 9 1.2.3 Limitations of current TCR cloning strategies 9 1.3 Expression cloning of HBV-specific CD8+ TCRs by Enrichment of Antigen-Specific T cells 10 1.3.1 Principles of Enrichment of Antigen-Specific T cells (EAST) 10 1.3.2 Application of cap-switching RACE to amplify comprehensive TCRs 11 2 Specific aims 13 3 Materials and methods 14 3.1 Plasmid 14 3.2 Cell lines and culture conditions 15 3.3 Lentiviral transduction of Jurkat cells 16 3.4 Flow cytometry 17 3.5 FACS sorting 17 3.6 RNA extraction, cap-switching RACE and nested PCR 18 3.7 TCR cloning by Gibson Assembly 20 3.8 Mice immunized with HBV surface (HBs) peptide 21 4 Results 23 4.1 Development of an efficient TCR cloning approach by EAST 23 4.1.1 Successful amplification of pooled TCRs by cap-switching 5’RACE and nested PCR 24 4.1.2 Low efficacy of TCRs cloning into the expression vector by Gibson Assembly 26 4.1.3 Trouble shooting for the low efficiency of TCR cloning by Gibson Assembly 27 4.2 Application for cloning HBs-specific TCRs from HBs-immunized mice 30 4.3 Establishment of the Reporter-Jurkat cells 32 4.3.1 The expression of NFAT reporter induced by TCR stimulation in Jurkat cells 32 4.3.2 Overexpression of mouse CD8 on the reporter Jurkat cells 34 5 Discussion 35 5.1 Establishment of an efficient cloning method for comprehensive TCRs 35 5.2 The proposed method for expression cloning of HBs-specific TCRs using the reporter Jurkat cells 38 5.3 Challenges on transduction efficiency of primary mice T cells and further tests 40 6 Figures 42 Figure 1. Schematic illustration of the flowchart for expression cloning of TCR and function test. 42 Figure 2. Amplification of the TCRα/β V(D)J regions by cap-switching 5’RACE and nested PCR. 43 Figure 3. Design and validation of the TCR cloning 45 Figure 4. Optimized results of Gibson Assembly with PCR-1 product cleaned up by Sera-Mag™ Select 47 Figure 5. Enrichment of HBs-specific T cells by HBs Dextramer 48 Figure 6. Application on HBs-immunized mice T cells 49 Figure 7. Establishment of the TCR activation-reporter Jurkat cells 51 Figure 8. CD8 construct design and CD8+ reporter Jurkat cells clonal selection 52 7 Tables 53 Table 1. Sequencing results of PCR products (gel extraction) TOPO cloning 54 Table 2. Sequencing results of Gibson Assembly (PCR-2 product gel extraction) 56 Table 3. Sequencing result of PCR products with increased cDNA input 58 Table 4. Sequencing results of PCR products purified by beads cleaned up and gel extraction 60 Table 5. Sequencing results of Gibson Assembly products (PCR products beads clean up and gel extraction) 62 Table 6. Sequencing result of HBs specific T cells PCR products purified by beads cleaned up and gel extraction 64 Table 7. Sequencing result of HBs specific TCRs Gibson Assembly products (beads clean up and gel extraction) 66 8 Reference 67 9 Supplementary Information 74 Supplementary Table 1. Primers and Adaptor used in cap-switching RACE and NGS 74 Supplementary Table 2. Higher PCR cycles may amplify short DNA fragments 75 Supplementary Table 3. DNA sizes from 500-1000 bp were all TCR V regions 76 Supplementary Table 4. Abbreviation list 78 Supplementary Figure 1. Higher PCR cycles may amplify short DNA fragments 79 Supplementary Figure 2. Electrophoresis graph of TCRα/β after PCR amplification 80 Supplementary Figure 3. Proposed method for HBs specific TCR expression on reporter Jurkat cells and further tests 81" | |
| dc.language.iso | en | |
| dc.subject | T細胞受體 | zh_TW |
| dc.subject | T細胞治療 | zh_TW |
| dc.subject | 慢性B型肝炎 | zh_TW |
| dc.subject | B型肝炎病毒 | zh_TW |
| dc.subject | 工程性T細胞 | zh_TW |
| dc.subject | chronic hepatitis B | en |
| dc.subject | Hepatitis B virus | en |
| dc.subject | T cell therapy | en |
| dc.subject | engineered T cells | en |
| dc.subject | T cell receptor | en |
| dc.title | 藉EAST發展表現選殖具B型肝炎病毒專一性的CD8+T細胞接受器 | zh_TW |
| dc.title | Development of expression cloning for HBV-specific CD8+ T cell receptors by Enrichment of Antigen-Specific T cells (EAST) | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 牟昀(Hsin-Tsai Liu),邱彥霖(Chih-Yang Tseng),曾岱宗,繆希椿 | |
| dc.subject.keyword | B型肝炎病毒,慢性B型肝炎,T細胞受體,工程性T細胞,T細胞治療, | zh_TW |
| dc.subject.keyword | Hepatitis B virus,chronic hepatitis B,T cell receptor,engineered T cells,T cell therapy, | en |
| dc.relation.page | 81 | |
| dc.identifier.doi | 10.6342/NTU202102506 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2021-08-20 | |
| 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 | 2023-08-31 | - |
| 顯示於系所單位： | 微生物學科所 | |
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| U0001-1908202112061200.pdf | 5.66 MB | Adobe PDF | 檢視/開啟 |
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