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研究生:林士鈺
研究生(外文):Shr-Yu Lin
論文名稱:研究B-1a與B-2細胞免疫調節的機轉
論文名稱(外文):Studies on the Immune Regulatory Mechanisms of B-1a and B-2 Cells
指導教授:江伯倫江伯倫引用關係
指導教授(外文):Bor-Luen Chiang
口試委員:繆希椿曾賢忠
口試委員(外文):Shi-Chuen MiawShiang-Jong Tzeng
口試日期:2013-06-28
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:免疫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:英文
論文頁數:118
中文關鍵詞:B-1細胞B-2細胞調節性B細胞介白素-10調節性T細胞免疫調節
外文關鍵詞:B-1 cellsB-2 cellsregulatory B cellsIL-10regulatory T cellsimmunoregulation
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研究背景:
一群有功能性的B細胞,稱為調節性B細胞,被證明可以維持免疫系統的平衡。它們有能力分泌介白素-10 (interleukin-10, IL-10) 並且表現CD5和CD1d。另一群B細胞,B-1a細胞擁有調節性B細胞的特色,且經過刺激後分泌介白素-10的能力會增加。然而,傳統的B-2細胞在刺激後只能分泌少量介白素-10。根據這兩種B細胞亞群的特色,我們推測B-1a細胞會在免疫反應中扮演調節的角色。因此,我們想要研究B-2細胞和B-1a細胞的調節作用的機轉。

實驗方法:
在共同培養或分隔培養的系統中,我們將分離出的B-2細胞或B-1a細胞和以塗層形式的anti-CD3及anti-CD28單株抗體刺激的CD4+ T細胞培養在一起。經過3天培養,T細胞被分離出來且其增生能力可用[3H]-thymidine incorporation程度及CFSE染色來決定。在抑制過程中是否需要介白素-10的作用是使用中和介白素-10的單株抗體、阻斷介白素-10的單株抗體或介白素-10基因剔除鼠而證明。此外,T細胞和B細胞的特性由FACS分析而細胞激素的分泌由ELISA偵測。

實驗結果:
是B-1a細胞而非B-2細胞藉由介白素-10且不需要細胞之間的接觸有調節作用進而抑制T細胞的增生;然而,從野生型或介白素-10基因剔除鼠分離的B-1a和B-2細胞都可以誘導產生調節性T細胞。經過再刺激,T of B-2細胞產生較多的介白素-2 (interleukin-2, IL-2) 及介白素-10而T of B-1a細胞會分泌較多的干擾素-γ (interferon-γ, IFN-γ) 及介白素-17 (interleukin-17, IL-17)。

結論:
本篇研究結果證明B-1a細胞會藉由分泌介白素-10而執行調節功能且B-2及B-1a細胞都可以藉由誘導調節性T細胞的產生而調控免疫反應。因此,未來更需要進一步研究B-2及B-1a細胞在調控免疫疾病上的應用。

關鍵字: B-1細胞、B-2細胞、調節性B細胞、介白素-10、調節性T細胞、免疫調節


Background:
A functional B cell subset, regulatory B cells, has recently been shown to contribute to maintain the balance of the immune system. They are IL-10 competent and express CD5 as well as CD1d. Another B cells subset, B-1a cells, has the features of regulatory B cells and the basal levels of IL-10 increase in response to stimulation. However, conventional B-2 cells secrete only small amount of IL-10 with or without stimulation. Based on the different characters of these two B cell populations, we speculated that B-1a cells might play a regulatory role in immune responses. Hence, we aimed to study the regulatory mechanisms compared between B-2 and B-1a cells.
Methods:
B-2 and B-1a cells were isolated and cultured with CD4+ T cells which stimulated with coating form of anti-CD3 and anti-CD28 in co-culture or transwell system. After 3 days incubation, T cells were purified and the proliferation ability was determined by [3H]-thymidine incorporation and CFSE stain. The requirement of IL-10 during the suppression process was investigated by using IL-10 neutralizing or blocking antibodies as well as IL-10 knockout mice. Moreover, the features of T and B cells were analyzed by FACS and the cytokines production was determined by ELISA.
Results:
B-1a cells but not B-2 cells had the regulatory function to suppress the proliferation of T cells through IL-10 dependent but cell-cell contact independent pathway; however, both of B-1a and B-2 cells purified from wild type or IL-10 knockout mice were able to induce regulatory T cells from naive CD4+ T cells. T of B-2 cells produced more IL-2 and IL-10 while T of B-1a cells secreted more IFN-γ and IL-17 after restimulation.
Conclusions:
Our results demonstrated that B-1a cells executed the regulatory function by secretion of IL-10 and both B-2 as well as B-1a cells might modulate immune responses through the induction of regulatory T cells. In the future, B-2 and B-1a cells should be studied more for further application to modulate immunological diseases via distinct strategies.

Keywords: B-1 cells, B-2 cells, regulatory B cells, IL-10, regulatory T cells, immunoregulation


口試委員審定書 I
致謝 II
中文摘要 IV
ABSTRACT VI
CONTENTS VIII
CONTENTS OF FIGURES XIII


CHAPTER I INTRODUCTION 1
Part 1. Background 2
1. Overview of B cells 2
1.1. Characterization of B-1 cells 2
1.2. Characterization of B-2 cells 4
1.3. Characterization of regulatory B cells 5
1.4. Overview of surface markers on B cells 7
1.4.1. The general surface markers on B cells 7
1.4.1.1. Characterization of CD80/CD86 7
1.4.1.2. Characterization of FcγRIIB 8
1.4.2. The surface markers on B-2 cells 9
1.4.2.1. Characterization of CD23 9
1.4.3. The surface markers on B-1 cells 9
1.4.3.1. Characterization of CD5 9
1.4.3.2. Characterization of CD9 10
1.5. Effects of B cells on T cells 11
2. The role of interleukin-10 in regulatory function 12
2.1. Characterization of interleukin-10 12
2.1.1. Effects of interleukin-10 on T cells 13
2.1.2. Effects of interleukin-10 on B cells 13
2.2. Interleukin-10-competent B cells 13
3. B cell therapy and immune diseases 14
3.1. Intrinsic IL-10-competent B cells 14
3.2. Induced regulatory B cells 15
3.3. B-1 cells 15
Part 2. Statement of the Motive 16
Part 3. Experimental Aims 18
CHAPTER II MATERIALS AND METHODS 19
Part 1. Materials 20
1. Animals 20
2. Cell culture 20
2.1. MACS cell purification 20
2.2. Culture medium and buffers 21
2.3. Mitogens, monoclonal antibodies, and cytokines used in cell culture 22
2.4. Proliferation assay 22
2.5. CFSE assay 22
3. Flow cytometry 23
4. Enzyme-linked immunosorbent assay (ELISA) 26
5. RNA extraction 27
6. Reverse transcription-polymerase chain reaction (RT-PCR) 27
7. Quantitative real time polymerase chain reaction (qPCR) 27
Part 2. Methods 30
1. Cell culture 30
1.1. Preparation of peritoneal wash-out cells (PWCs) and splenocytes 30
1.2. Cell isolation 30
1.3. General cell culture process 31
2. IL-10 expression culture 31
3. Suppression assay 31
3.1. Suppressive function of supernatants 31
3.2. Suppressive function of T of B cells 32
4. Transwell experiment 32
5. CFSE labeling 33
6. Flow cytometic assay 33
7. Enzyme-linked immunosorbent assay (ELISA) 34
8. RNA extraction 35
9. Reverse transcription-polymerase chain reaction (RT-PCR) 36
10. Quantitative real time polymerase chain reaction (qPCR) 36
11. Statistical analysis 37
CHAPTER III RESULTS 38
1. Pattern, isolation, and phenotypical characterization of B cells 39
2. The optimization of stimulation conditions 40
3. Effect of supernatants from B cells on anti-CD3/CD28-induced T cells proliferation 40
4. Suppressive function of B-1a cells is IL-10 dependent but cell-cell contact independent 41
5. B-1a cells can induce IFN-γ and IL-17 production in T─B culture system 42
6. B-1a cells express more co-stimulatory molecules after culture with T cells 43
7. Both co-culture induced T of B-2 and T of B-1a cells could inhibit proliferation of T cells 43
8. T of B-1a cells express higher ICOS but lower PD-1 44
9. T of B-1a cells secreted more IFN-γ and IL-17 45
10. Genes profiles of cytokines from B-2 and B-1a cells 45
CHAPTER IV DISCUSSION 47
FIGURES 56
REFERENCES 110


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