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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 江伯倫 | |
dc.contributor.author | Ling-Hui Hsu | en |
dc.contributor.author | 徐綾徽 | zh_TW |
dc.date.accessioned | 2021-06-13T04:15:02Z | - |
dc.date.available | 2021-07-28 | |
dc.date.copyright | 2011-10-05 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-07-28 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32763 | - |
dc.description.abstract | 研究背景:
先前研究指出,B細胞在免疫系統中能發揮雙向調控的作用。近年許多文獻報告將傳統B-2細胞與T細胞共同培養以誘發調節性T細胞的產生。相對於B-2細胞,B-1細胞是B細胞族群中另一亞群,因能製造大量抑制性的介白素-10 (interleukin-10, IL-10) 在免疫系統中有其獨特的角色。已知在誘導Tr1或Tr1-like調節性T細胞生成的過程中需要介白素-10的參與,但 B-1細胞是否能藉由分泌介白素-10誘發調節性T細胞的生成仍不清楚。本研究欲透過分析B-1與B-2細胞在誘發調節性T細胞生成上的能力,進而闡述B-1與B-2細胞各自透過何種機制達到免疫調控。此外由B-1與B-2細胞所誘導出的T細胞,其特性和抑制免疫反應的機制也會一併釐清。 實驗方法: 於卵蛋白胜肽片段(OVA323-339)加上抗原呈現細胞的抗原特異性刺激系統或anti-CD3/CD28單株抗體刺激系統中,我們將分離出的B-1與B-2細胞分別和CD4+CD25- T細胞共同培養。3天後重新分離B-1與B-2細胞所誘導出出CD4+ T細胞並分析其是否具有抑制能力和免疫特性。我們利用Foxp3-GFP × DO11.10基因轉殖鼠來研究轉錄因子Foxp3於B細胞所誘導出的T細胞上的表達水平;介白素-10基因剔除鼠則被用來研究介白素-10在誘導調節性T細胞或調節性T細胞本身抑制機轉中的作用。 實驗結果: 在所使用的兩種刺激系統中,B-1與B-2細胞均能誘導出具抑制能力的調節性T細胞,並證實誘導過程中不需要介白素-10的參與。我們將B-1細胞與B-2細胞誘導出的調節性T細胞分別命名為“Treg-of-B1”與“Treg-of-B2”細胞。接著,我們發現不同於CD4+CD25+ 天然調節性T細胞 (nTreg),Treg-of-B1與 Treg-of-B2細胞皆不表現Foxp3並各自具有獨特的細胞表面標誌和細胞激素分泌。更進一步地,我們證明Treg-of-B1細胞的抑制機制主要是通過介白素-10之外的其它分泌型因子,而Treg-of-B2細胞的抑制機制則是需要細胞與細胞接觸。 結論: 本篇的實驗結果支持了B-1與B-2細胞可能藉由誘導調節性T細胞產生達到負向調控免疫反應的作用。我們首先釐清了B-1細胞並非藉由分泌介白素-10來誘導Tr1細胞產生。更進一步分析Treg-of-B1與Treg-of-B2細胞的細胞表面標記跟細胞激素分泌情形,顯示由B-1與B-2細胞所誘導出的這兩群細胞分屬兩群獨特的調節性T細胞群,也不同於nTreg或Tr1細胞。此外,這兩群調節性T細胞是藉由截然不同的機制達到抑制CD4+ T細胞的作用。這些研究結果使我們更加了解B-1與B-2細胞如何透過誘導出特性跟抑制機轉不相同的調節性T細胞,反映出各自在免疫調節上所扮演的角色。 | zh_TW |
dc.description.abstract | Background:
B cells have been found to induce both effective as well as tolergenic immune responses. Conventional B-2 cells have been shown to execute immune modulation through induction of Treg cells. The B-1 cell, belongs to another subset of B cell population, is specific and functionally important subset which exert their regulatory role through the production of IL-10. IL-10 has been correlated with the induction of Tr1 or Tr1-like cells, however, whether IL-10 producing B-1 cells are able to induce the generation of Treg cells especially the Tr1 lineage remain poorly understood. In this study, we aimed to study the effect of B-1 and B-2 cells on the inducting ability of Treg cells. Moreover, the properties as well as the effector function of B-1 and B-2 cell-induced Treg cells will be clarified. Methods: To induce Treg cells, B-1 cells and B-2 cells were isolated and co-cultured with naive CD4+CD25- T cells under OVA323-339 pulsed-APCs or anti-CD3/CD28 antibodies stimulation. After 3 days, CD4+ T cells in the co-cultures were purified and their suppressive function was examined. The requirement of IL-10 during the induction or suppression process was investigated using IL-10 knockout mice, and the Foxp3-GFP × DO11.10 mice was used to trace the Foxp3 expression of induced Treg cells. Results: In our two induction systems, we found that B-1 and B-2 cells were both able to convert naive T cells into Treg cell population, which we called “Treg-of-B1 cells” and “Treg-of-B2 cells”. More importantly, dissimilar to nTreg cells, we indentified Treg-of-B cells did not necessarily express Foxp3 and acquired unique surface molecule expression as well as cytokine profile. Finally, we showed that the suppressive function of Treg-of-B2 cells required cell-cell interaction whereas Treg-of-B1 cells mainly relied on IL-10-independent but soluble factors mediated suppression. Conclusions: Our results supported the idea that B-1 and B-2 cells might modulate immune responses via the induction of Treg cells. Also, we further clarified that IL-10 producing B-1 cells do not induce Tr1 cells via IL-10 but to induce Treg-of-B1 cells thorough IL-10-independent mechanism. Moreover, we demonstrated that Treg-of-B1 and Treg-of-B2 cells were two unique Treg populations and they interacted with CD4+ T cells through distinct mechanisms. Taken together, these findings might help us in further understanding the role of B-1 and B-2 cells in immune regulation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T04:15:02Z (GMT). No. of bitstreams: 1 ntu-100-R98449003-1.pdf: 1785809 bytes, checksum: e70f3d3cdb9e30b6c8c9869abadd2a6f (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | Chapter I. Introduction………………………………………………………………...1
Part.1 Background…………………………………………………………………...2 1. Characteristics of B-1 and B-2 cell subsets………………………………...2 2. The immune regulatory roles of B cells…………………………...2 2.1 B cells modulate the function of T cells via antigen presentation and co-stimulation……………....………………………………………....3 2.2 B cells modulate the function of T cells via cytokine production…….4 3. Treg cell populations ………………………………………………………4 3.1 Naturally occurring regulatory T (nTreg) cells………………………..5 3.1.1 Cell contact-dependent suppression…………………………...6 3.1.2 Soluble factors-dependent suppression……………………..7 3.2 Inducible Treg (iTreg) cells..……………………………………….…8 3.2.1 Type 1 regulatory T (Tr1) cells………………………………...8 3.2.1.1 The induction of Tr1 and Tr1-like cells………………….9 4.1 Conventional B-2 cells and the induction of Treg cells………….……….9 4.2 B-1 cells and the induction of Treg cells……….………………………..10 Part.2: Statement of the Motive………………………………………………...…..12 Part.3: Experimental Aims………………………………………………………….14 Chapter II. Materials and Methods……………………………………………...…..15 Part.1: Materials…………………………………………………………………….16 1. Animals……………………………………………………………………....16 2. Cell culture………………………………………………………………......16 2.1 Culture medium and buffer……………………………………………..16 2.2 Antigens, mitogens, and monoclonal antibodies (mAbs) used in cell culture……………...…………………………………………………...17 3. Flow cytometry………………………………………………………………18 4. Enzyme-linked immunosorbent assay (ELISA)……………………………..19 5. BD IMagTM cell purification…………………………….…………………...19 6. CFSE staining………………………………………………………………..20 7. Proliferation Assay…………………………………………………………..20 Part.2: Methods……………………………………………………………………..21 1. Cell culture………………………………………………………………......21 1.1 Preparation of peritoneal wash-out cells (PWCs) and splenocytes… …21 2. Cell isolation…………………………………………………………………21 3. Induction of Treg cells by B-1 and B-2 cells…………………………….......22 4. Flow cytometic assays and cell sorting……………………………………...23 4.1 Staining of cell-surface markers……………………………………..…23 4.2 Cell sorting……………………………………………………………..23 4.3 Detection of Foxp3 expression by intracellular staining……………….23 5. Cytokine assay……………………………………………………………….24 6. In vitro suppression assay……………………………………………….…...25 7. Transwell experiment………………………………………………………..25 8. CFSE labeling………………………………………………………………..26 9. Statistical analysis..…………………………………………………………..27 Chapter III. Results…………………………………………………………………...28 1. The B cell population in peritoneal wash-out cells of BALB/c mice……………29 2. Isolation of B-1, B-2, CD4+CD25- T and CD4+CD25+ nTreg cells………....29 3. Part I: B-1 and B-2 cells are capable of inducing Treg cells under antigen-specific stimulation……………………………………………………………………….30 3.1 Induction of Treg cells by B-1 and B-2 cells under antigen-specific stimulation………………………………………………………………….30 3.2 The suppressive function of Treg-of-B cells induced under antigen-specific stimulation…………………..……………………………………………...31 4. Explore the suppressive mechanisms of Treg-of-B cells in antigen-specific induction system…………………………………………………………………32 4.1 Suppressive function of Treg-of-B2 cells is cell contact-dependent……….32 4.2 Reduction of the intrinsic proliferation of Treg-of-B1 cells unmasked the suppressive effect…………………………………………………………...33 4.3 Suppressive function of Treg-of-B1 cells is mediated by soluble factors.....34 5. Foxp3 expression of Treg-of-B cells induced under antigen-specific stimulation……………………………………………………………………….34 6. Characteristics of Treg-of-B cells………………………………………………..35 7. Cytokine profiles of Treg-of-B cells……………………………………………..36 8. Part II: B-1 and B-2 cells are capable of inducing Treg cells under anti-CD3/CD28 antibodies stimulation………………………………………….37 8.1 Induction of Treg cells under anti-CD3/CD28 antibodies stimulation …….37 8.2 The suppressive function of Treg-of-B cells induced under anti-CD3/CD28 antibodies stimulation………………………………………………………38 9. IL-10 was not necessary for the induction of Treg-of-B cells……………..…….38 10. Explore the suppressive mechanisms of Treg-of-B cells in anti-CD3/CD28 induction system………………………………………………………………....39 10.1 Suppressive function of Treg-of-B1 cells is mediated by soluble factors other than IL-10………………………...…………………………………39 10.2 Suppressive function of Treg-of-B2 cells is cell contact-dependent but IL-10-independent……………………………………………………...…40 11. Foxp3 expression of Treg-of-B cells induced from wild-type or IL-10-/- co-cultures under anti-CD3/CD28 stimulation…………………,……………….41 Chapter IV. Discussion………………..………………………………………………43 Figures…………………………………………………………………………………56 References………………………………………………………………………….......81 | |
dc.language.iso | en | |
dc.title | B-1與B-2細胞誘導調節性T細胞之功能性探討 | zh_TW |
dc.title | Study on The Effect of B-1 and B-2 Cell Subsets on Induction of T cells with Regulatory Function | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 繆希椿,曾賢忠 | |
dc.subject.keyword | B-1細胞,B-2細胞,調節性T細胞,介白素-10,免疫調節, | zh_TW |
dc.subject.keyword | B-1 cells,B-2 cells,Treg cells,IL-10,immune regulation, | en |
dc.relation.page | 86 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-07-28 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 免疫學研究所 | zh_TW |
顯示於系所單位: | 免疫學研究所 |
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