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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 楊宏志(Hung-Chih Yang) | |
dc.contributor.advisor | 楊宏志(Hung-Chih Yang | hcyang88@ntu.edu.tw | ), | |
dc.contributor.author | Chia-Yi Chao | en |
dc.contributor.author | 趙佳儀 | zh_TW |
dc.date.accessioned | 2023-03-19T23:34:46Z | - |
dc.date.copyright | 2022-10-13 | |
dc.date.issued | 2022 | |
dc.date.submitted | 2022-09-16 | |
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Dupont, J., et al., Artificial Antigen-Presenting Cells Transduced with Telomerase Efficiently Expand Epitope-Specific, Human Leukocyte Antigen–Restricted Cytotoxic T Cells. Cancer Research, 2005. 65(12): p. 5417-5427. 13. Butler, M.O., et al., Long-Lived Antitumor CD8+ Lymphocytes for Adoptive Therapy Generated Using an Artificial Antigen-Presenting Cell. Clinical Cancer Research, 2007. 13(6): p. 1857-1867. 14. Sun, S., et al., Dual function of Drosophila cells as APCs for naive CD8+ T cells: implications for tumor immunotherapy. Immunity, 1996. 4(6): p. 555-64. 15. Turtle, C.J. and S.R. Riddell, Artificial antigen-presenting cells for use in adoptive immunotherapy. Cancer J, 2010. 16(4): p. 374-81. 16. Steenblock, E.R., et al., Antigen presentation on artificial acellular substrates: modular systems for flexible, adaptable immunotherapy. Expert Opinion on Biological Therapy, 2009. 9(4): p. 451-464. 17. Oelke, M., et al., Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells. Nat Med, 2003. 9(5): p. 619-24. 18. Butler, M.O. and N. Hirano, Human cell-based artificial antigen-presenting cells for cancer immunotherapy. Immunol Rev, 2014. 257(1): p. 191-209. 19. Kim, J.V., et al., The ABCs of artificial antigen presentation. Nature Biotechnology, 2004. 22(4): p. 403-410. 20. Schmidts, A., et al., Cell-based artificial APC resistant to lentiviral transduction for efficient generation of CAR-T cells from various cell sources. Journal for immunotherapy of cancer, 2020. 8. 21. Rosskopf, S., et al., Creation of an engineered APC system to explore and optimize the presentation of immunodominant peptides of major allergens. Scientific Reports, 2016. 6(1): p. 31580. 22. 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. 23. Rudolf, D., et al., Potent costimulation of human CD8 T cells by anti-4-1BB and anti-CD28 on synthetic artificial antigen presenting cells. Cancer Immunology, Immunotherapy, 2008. 57(2): p. 175-183. 24. Maus, M.V., et al., Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB. Nature Biotechnology, 2002. 20(2): p. 143-148. 25. Yu, X., et al., Artificial antigen-presenting cells plus IL-15 and IL-21 efficiently induce melanoma-specific cytotoxic CD8+ CD28+ T lymphocyte responses. Asian Pacific Journal of Tropical Medicine, 2013. 6(6): p. 467-472. 26. Steenblock, E.R., et al., An Artificial Antigen-presenting Cell with Paracrine Delivery of IL-2 Impacts the Magnitude and Direction of the T Cell Response *<sup></sup>. Journal of Biological Chemistry, 2011. 286(40): p. 34883-34892. 27. Melichar, B., et al., Expression of costimulatory molecules CD80 and CD86 and their receptors CD28, CTLA-4 on malignant ascites CD3+ tumour-infiltrating lymphocytes (TIL) from patients with ovarian and other types of peritoneal carcinomatosis. Clin Exp Immunol, 2000. 119(1): p. 19-27. 28. Croft, M., et al., The significance of OX40 and OX40L to T-cell biology and immune disease. Immunol Rev, 2009. 229(1): p. 173-91. 29. Bansal-Pakala, P., et al., Costimulation of CD8 T Cell Responses by OX40. The Journal of Immunology, 2004. 172(8): p. 4821-4825. 30. Cheuk, A.T., G.J. Mufti, and B.A. Guinn, Role of 4-1BB:4-1BB ligand in cancer immunotherapy. Cancer Gene Ther, 2004. 11(3): p. 215-26. 31. Bagheri, S., et al., Targeting the 4-1BB costimulatory molecule through single chain antibodies promotes the human T-cell response. Cellular & Molecular Biology Letters, 2020. 25(1): p. 28. 32. Zeng, Q., Y. Zhou, and H. Schwarz, CD137L-DCs, Potent Immune-Stimulators—History, Characteristics, and Perspectives. Frontiers in Immunology, 2019. 10. 33. Nilvebrant, J. and J. Rockberg, An Introduction to Epitope Mapping. Methods Mol Biol, 2018. 1785: p. 1-10. 34. Gershoni, J.M., et al., Epitope mapping: the first step in developing epitope-based vaccines. BioDrugs, 2007. 21(3): p. 145-56. 35. Kaseke, C., et al., HLA class-I-peptide stability mediates CD8<sup>+</sup> T cell immunodominance hierarchies and facilitates HLA-associated immune control of HIV. Cell Reports, 2021. 36(2). 36. Vergote, V., et al., Development of peptide receptor binding assays: methods to avoid false negatives. Regul Pept, 2009. 158(1-3): p. 97-102. 37. Lorente, E., R. García, and D. López, Allele-dependent processing pathways generate the endogenous human leukocyte antigen (HLA) class I peptide repertoire in transporters associated with antigen processing (TAP)-deficient cells. J Biol Chem, 2011. 286(44): p. 38054-38059. 38. Koch, J., et al., Functional Dissection of the Transmembrane Domains of the Transporter Associated with Antigen Processing (TAP)*. Journal of Biological Chemistry, 2004. 279(11): p. 10142-10147. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/86056 | - |
dc.description.abstract | T細胞在後天性免疫中扮演重要的角色,現今有許多癌症免疫療法,即是希望可藉由刺激更多的T細胞,使其活化、擴增,最後有效清除癌細胞而得到治療的成效。而刺激更多的T細胞有許多方法,其中一項就是分離人體內的樹突細胞,讓此抗原呈現細胞在體外受到抗原刺激,再將活化的抗原呈現細胞打回體內,因而可在體內達到刺激 T 細胞的效果,但此方法十分耗時且昂貴,因此,逐漸發展出利用人造的抗原呈現細胞在體外刺激T細胞。 而抗原呈現細胞刺激T細胞需要三種訊號,其一就是需要主要組織相容性複合體將抗原的抗原表位呈現給T細胞的受體;其二,需要一些協同刺激的分子放大刺激 T 細胞的訊號及延長刺激T細胞的時間,這些分子包含 CD86、CD70、4-1BBL 等等。其三,需要細胞激素如白血球介素-2 (IL-2)等的協助,以增加T細胞的擴增、活化。因此,本研究目的即是要做出一種人造的抗原呈現細胞,在人造的抗原呈現細胞上給與刺激T細胞所需的主要組織相容性複合體,並利用帶有4-1BBL、CD86 等協同刺激分子的質體使此細胞過度表現這些分子,最後在體外給予細胞激素作為額外的刺激,期望能在體外更有效率、快速地刺激並活化T細胞。 除了好的抗原呈現細胞可以更有效率刺激T細胞外,一個好的抗原表位(Epitope) 也很重要。抗原表位被T細胞受體辨識後即可活化T細胞,因此對於了解哪些抗原表位會引起T 細胞反應是必要的。 目前較常使用T2細胞作為抗原表位定位的工具,由於此細胞缺乏抗原加工相關轉運體(transporter associated with antigen processing; TAP protein)而無法將內源性的胜肽運至表面與主要組織相容性複合體結合,因此可利用外加胜肽來測試此抗原表位是否會與細胞表面的主要組織相容性複合體結合而穩定其結構,若偵測到的主要組織相容性複合體表現量有上升則代表可成功結合。但T2細胞表面並不是表現單一等位基因的主要組織相容性複合體,因此即使胜肽結合上去使偵測到的主要組織相容性複合體表現量有升高,也不清楚此胜肽是與哪個主要組織相容性複合體的等位基因結合。基於此,我們利用群聚且有規律間隔的短回文重複序列(CRISPR)技術建立一株缺乏抗原加工相關轉運體的細胞,並只表達單一等位基因主要組織相容性複合體,相比於以往使用的T2細胞株,可以更精確知道此抗原表位會與哪個主要組織相容性複合體的等位基因結合,因此可使用此細胞株作為抗原表位定位的工具。 有了人造抗原呈現細胞可以更有效率地刺激毒殺性T細胞,再加上可以更精確知道抗原表位的主要組織相容性複合體基因型,對於未來免疫療法會有極大的貢獻,希冀可以造福更多需要被治療的病患們。 | zh_TW |
dc.description.abstract | T cells play a vital role in adaptive immunity. With enough functional T cells, cancer cells can be effectively eliminated to achieve cancer cure. One strategy to stimulate more functional T cells is to isolate patients’ dendritic cells, a kind of antigen presenting cells, which are then activated in vitro and subsequently transferred to patients. However, this approach is very time-consuming and expensive; therefore, artificial antigen presenting cells (APCs) have gradually gained wide interest, and are also used to stimulate T cells in vitro. APCs activate antigen-specific T cells require three distinct signals. Signal one is antigen-specific signaling mediated by T-cell receptor engagement of pathogenic peptides presented by major histocompatibility complex (MHC) molecules, also named human leukocyte antigens (HLA) in humans. Signal two is costimulatory molecules expressing on APCs, which mainly function to amplify signals and prolong the time of T cell stimulation and activation. Signal 3 is polarizing signaling mediated by various cytokine milieus to enhance T cell activation and expansion. Accordingly, in my thesis study, the first research purpose is to establish an artificial APC with robust ability for T cell stimulation. First of all, we used the technique of clustered, regularly interspaced, short palindromic repeats (CRISPR) to knock out the endogenous class I MHC and then transferred mono-allelic one on this artificial APC. Second, we generated the cells overexpress the costimulatory molecules that are critical for T cell activation. Last, we supple the cells with cytokine in vitro additionally as another stimulator. The ability of this artificial APC in activating and proliferating T cells will be examined and optimized. On the other hand, APCs can present epitopes to T cells and stimulate them. As a result, epitopes are important for stimulating T cells. However, it remains a time-consuming and labor-intensive work for epitope mapping. It is known that T2 cells lack the transporter associated with antigen processing (TAP) protein, so can be utilized for epitope mapping. Sometimes, the results of epitope mapping are ambiguous due to the presence of multiple HLA alleles. For the unambiguous identification of class I HLA-restricted epitopes, the cells expressing mono-allelic class I HLA that we established previously were used to knock out the TAP genes by CRISPR. After generating these mono-allelic HLA expressing TAP Knock out cell lines, we can precisely confirm the class I HLA-restricted epitopes to T cells. In the end, it is possible to develop powerful T cell-based immunotherapies against many diseases using epitope mapping and artificial APCs. | en |
dc.description.provenance | Made available in DSpace on 2023-03-19T23:34:46Z (GMT). No. of bitstreams: 1 U0001-1509202212043900.pdf: 4762685 bytes, checksum: 79d47b07180ad1ba95b32ad4eb7592d1 (MD5) Previous issue date: 2022 | en |
dc.description.tableofcontents | 口試委員審定書 i 致謝 ii 中文摘要 iii 英文摘要 v Contents vii 1. Introduction 1 1.1 Cancer immunotherapy 1 1.2 Benefits of artificial APCs 2 1.3 Cell-based APCs 3 1.4 Signals required for T cell activation 4 1.5 Mono-allelic HLA-expressing HEK 293T 5 1.6 Desired costimulatory molecules 6 1.7 The importance of epitope identification 8 1.8 Strategies for epitopes identification 8 1.9 Peptide binding assay (MHC binding assay) 9 1.10 Generate TAP genes knock out cell lines 10 2 Specific aim 11 3 Materials and methods 12 3.1 Plasmids 12 3.2 Cell lines and culture conditions 13 3.3 HLA gRNA / TAP genes gRNA design and cloning 15 3.4 DNA transfection 16 3.5 Gibson Assembly 16 3.6 Lentivirus production 17 3.7 Lentiviral transduction of HEK 293T and K562 cells 18 3.8 RNA extraction and RT-qPCR assay 18 3.9 Intracellular cytokine staining 19 3.10 Flow cytometry 19 3.11 FACS sorting 20 3.12 Single-cell polymerase chain reaction (PCR) and sequencing 21 3.13 Peptide binding assay 22 3.14 Peripheral blood mononuclear cells (PBMCs) isolation 24 3.15 aAPC functional test 24 4 Results 26 4.1 Establish an HLA null cell line by CRISPR 26 4.2 Genetic analysis of HLA class I-negative cell clones 27 4.3 Establishment of a mono-allelic HLA expressing cell line 29 4.4 Generation of a mono-allelic HLA-expressing artificial APC 30 4.5 Functional test of HEK 293T-based artificial APC 30 4.6 Establishment of a TAP genes knock out cell line 31 4.7 Peptide binding assay of TAP knock out cell line 32 5 Discussion 34 5.1 Generation of HLA class I-null expressing HEK 293T cells by CRISPR editing and its application 34 5.2 The homologous recombination occurs in HLA-A of HLA-null expressing HEK 293T cells 35 5.3 HEK 293T-based and K562-based mono-allelic HLA expressing artificial APC ……………………………………………………………………………….36 5.4 Functional test of HEK 293T-based artificial APC 37 5.5 Future application of artificial APC 38 5.6 Establishment of a TAP genes knock out cell line 38 5.7 TAP-deficient mono-allelic HLA expressing HEK 293T cells 39 5.8 Future application of TAP-deficient mono-allelic HLA expressing HEK 293T cells ……………………………………………………………………………….40 6 Figures 40 Figure 1. Schematic illustration of the flowchart for generation of a functional artificial APC 42 Figure 2. Schematic illustration of the flowchart for generation of HLA-null HEK 293T cells 43 Figure 3. Screening and identification of class I HLA-negative HEK 293T cell lines ……………………………………………………………………………….45 Figure 4. Mapping of the CRISPR-induced deletion of the HLA-A locus in HLA null #54-1-4 47 Figure 5. Selecting mono-allelic HLA expressing HEK 293T #54-1-4 49 Figure 6. Functional test of HEK 293T-based aAPC 51 Figure 7. Schematic illustration of the flowchart for generation of TAP-deficient mono-allelic HLA expressing HEK 293T cells. 52 Figure 8. gRNA editing efficiency to knock out TAP genes by RT-qPCR. 53 Figure 9. Screening and identification of TAP-knockout HEK 293T #54-1-4 cells. 54 Figure 10. Peptide binding assay of HLA-A*11:01 HEK 293T TAP-knockout cells 55 7 References 56 8 Supplementary informations 59 Supplementary table 1. gRNA sequences for HLA-A, B, C target editing 59 Supplementary table 2. Sequences of HLA-A, HLA-B, HLA-C, HLA-H specific primers ……………………………………………………………………………….59 Supplementary table 3. gRNA primers design 60 Supplementary table 4. gRNA sequences for TAP genes knock out 60 Supplementary table 5. Sequences of TAP1 and TAP2 specific primers 61 Supplementary figure 1. Confirmation of homologous recombination (HR) in #54-1-4……...……………………………………………………………………………….61 Supplementary figure 2. Mono-allelic HLA expressing K562 cells 63 Supplementary figure 3. Alignment between HLA-H and HLA-A2 64 Supplementary figure 4. HLA expression level of K562-based artificial APC will decrease 65 | |
dc.language.iso | en | |
dc.title | 建立表達單一第一型白血球抗原的人造抗原呈現細胞以刺激T細胞及抗原表位定位 | zh_TW |
dc.title | Establishment of the mono-allelic HLA class I expressing artificial antigen presenting cells for T cells stimulation and epitope mapping | en |
dc.type | Thesis | |
dc.date.schoolyear | 110-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 凌嘉鴻(Chia-Hung Ling),陶秘華(Mi-Hua Tao),曾岱宗(Tai-Chong Tseng) | |
dc.subject.keyword | 群聚且有規律間隔的短回文重複序列,主要組織相容性複合體,人造抗原呈現細胞,缺乏抗原加工相關轉運體,免疫療法, | zh_TW |
dc.subject.keyword | CRISPR,human leukocyte antigens,artificial APC,TAP deficient knock out,immunotherapy, | en |
dc.relation.page | 66 | |
dc.identifier.doi | 10.6342/NTU202203428 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2022-09-16 | |
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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