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研究生:吳玟璇
研究生(外文):Wen-Hsuan Wu
論文名稱:介白素15與介白素15受體之交互作用在CD11cintCD45RBhigh樹突細胞所誘發的第一型調控T細胞生成及周邊免疫耐受反應上所扮演的角色
論文名稱(外文):The role of IL-15/IL15R interaction in CD11cintCD45RBhigh dendritic cell dependent generation of type 1 regulatory T cell and peripheral tolerance
指導教授:吳宗舜
指導教授(外文):Tzong-shoon Wu
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
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:2007
畢業學年度:95
語文別:英文
論文頁數:60
中文關鍵詞:免疫耐受性樹突細胞介白素15第一型調節性T細胞
外文關鍵詞:tolerancedendritic cellIL-15type 1 regulatory T cell
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誘發免疫耐受性對於維持免疫系統的恒定是很重要的一環。近幾年有一些研究指出,身體裡有一群在自然生理情況下即存在的樹突細胞亞群,其細胞表面表現中等程度的CD11c,高程度的CD45RB (CD11cintCD45RBhi),具有誘導第一型調節性T細胞的分化的能力,而且能進一步調控周邊免疫耐受性。然而目前尚未清楚這群樹突細胞會受到哪些因素而影響其分化及功能。在本研究中,我們先是發現,小鼠在缺乏介白素15或是缺乏其受體的次單元的情況下,會導致此種小鼠的脾臟及淋巴節中的一群表現型為CD11cintCD45RBhi的樹突細胞亞群消失,而這群消失的樹突細胞具有不成熟的特徵,例如其細胞表面第二類主要組織相容複合物,細胞共同活化分子的表現都很低,恰好與具有誘發免疫耐受性的樹突細胞亞群(tDC)之表徵相似。並且我們排除了這群受到IL-15/IL-15R影響的樹突細胞是所謂的胞漿性樹突細胞的可能性。然而在以LPS刺激的實驗中,我們並未發現這群細胞會分泌大量的介白素10,而這是tDC所具有的功能性特徵。進一步我們測試其是否具有抗原呈現細胞的特性。在測試其是否能夠吞噬,處理,以及表現抗原的實驗中,相較於傳統的大量表現CD11c的樹突細胞,我們發現這群CD11cintCD45RBhi DCs表現出較弱的抗原吞噬及表現的能力。 此外,若使用這群CD11cintCD45RBhi DCs來刺激T細胞,在外加介白素15的情況下,則此T細胞再度受到刺激時會傾向分泌大量介白素10;同時在顯微鏡下觀察會發現,這些T細胞分裂的程度較低。 總結:在這個研究中,我們提供了一些證據支持介白素15與其受體的交互作用會影響一群樹突細胞之分化或是存活,而這群樹突細胞能誘使T細胞分泌大量IL-10,具有誘發免疫耐受性的潛力。因此,介白素15與其受體的交互作用對於調控周邊免疫耐受性具有直接或間接的影響力。
The induction of tolerance is critical for the maintenance of immune homeostasis. In a recent study, a natural DC subset with a distinct CD11cintCD45RBhi phenotype has been identified and characterized by their abilities to induce type 1 regulatory T cell (Tr1) differentiation and peripheral tolerance. However, factors involved in the differentiation and function of the tolerogenic DC (tDC) have not been clarified. In this study, we showed that mice deficient in IL-15R or IL-15 had a significant reduction in a CD11cintCD45RBhi DC subset in their spleens and lymph nodes. In phenotypical characterization, the results indicated these CD11cintCD45RBhi DCs displayed immature phenotypes, e.g. low expression in MHC II and costimulatory molecules, which were reminiscent of tDCs. Moreover, the DCs reduced in IL-15R-/- mice were not B220+mPDCA-1+CD11cint plasmacytoid DCs. However, CD11cintCD45RBhi DCs were not able to produce abundant IL-10 upon LPS stimulation, which is functional characteristic of tDCs. In the function assay in determining the activity of professional antigen presenting cells, compared to the conventional CD11chi DCs, CD11cintCD45RBhi DCs displayed a relatively low capacity in antigen phagocytosis, processing, and presenting. In addition, T cells primed by the CD11cintCD45RBhi DC subset in the presence of IL-15 tended to produce copious amount of IL-10 with a low proliferation rate during restimulation. In conclusion, we provided evidence supporting the essential role of IL-15/IL-15R interaction in the development or survival of a DC subset which is capable of priming CD4+ T cells into IL-10 secretion and potentially tolerogenic. Hence, IL-15/IL-15R interaction plays a direct and indirect role in regulation of the peripheral tolerance.
Contents
指導教授推薦書……………………………………………………..
口試委員會審定書…………………………………………………..
授權書………………………………………………………………... iii
誌謝…………………………………………………………………... v
中文摘要……………………………………………………………... vi
英文摘要………….………………………………..………...……... viii

Chapter I Introduction………………………….………………….. 1
1.1 Background and significance....……………………….…… 1
1.1.1 Peripheral tolerance and type 1 regulatory T cells……..… 1
1.1.2 Tolerogenic DCs………………………………………….. 3
1.1.3 IL-15, IL-15Rand biological functions on DCs............... 4
1.1.4 A DC subset was reduced in IL-15R-/- mice…………..... 5
1.2 Specific aims……………………...………………...……… 7

Chapter II Materials and methods…………………………………. 8
2. 1 Mice………………………….………………………………… 8
2.2 Medium, antibodies, and reagents…..…...........…….…………. 8
2.3 Cell isolation………………………………………………….. 10
2.3.1 DC isolation……………………………......……………. 10
2.3.2 OT-II CD4+ T isolation ……............................................ 11
2.4 VIP administration to induce tDCs in vivo..……….………… 12
2.5 Functional assay of DCs……….……………...……………… 12
2.5.1 LPS stimulation…………………………………………. 12
2.5.2 Phagocytosis…………………………………………….. 13
2.5.3 Ag processing and presenting…………………………... 13
2.6 In vitro Tr1 induction……….…………………….………….. 14
2.7 Flow cytometry…………….………………….……………… 14
2.7.1 Surface marker staining………………………………..... 14
2.7.2 Intracellular cytokine staining……………………….….. 15
2.8 Sandwich ELISA……………..…………....……………......... 15
2.9 RT-PCR…………....…........................…………………..….... 15

Chapter III Results………….…………………………….………. 17
3.1 Determination of the DC profile in IL-15R deficient mice…. 17
3.2 Phenotypical characterization of the IL-15/IL-15Rdependent CD11cintCD45RBhigh DCs in wt mice…………………….………. 18
3.2.1 Determination of surface marker expressions…………... 18
3.2.2 To investigate the capability of VIP to enrich the splenic CD1cintCD45RBhi DC subset…………………………………. 19
3.3 Functional characterization of the IL-15/IL-15Rdependent CD11cintCD45RBhighDCs in wt mice ……..…………….……...… 20
3.3.1 Determination of cytokine profile………………………. 20
3.3.2 Assessment of abilities in antigen phagocytosis, presenting, and processing…………………………………………. 22
3.4 Investigation of the activity in Tr1 induction of wt CD11cintCD45RBhigh DCs………………………………………… 22

Chapter IV Discussion……………….……..………….........……. 25

Figures.……………………………..………………………………... 30
Fig. 1 Identification of the CD11cintCD45RBhiMHC IIlo DC subset reduced in IL-15R-/- mice……………………………………….. 30
Fig. 2 The DC subset reduced in IL-15R-/- mice was not plasmacytoid DC subset..………………………………………… 32
Fig. 3 Splenic DCs isolated by modified purification procedure displayed enhanced purity………………...……………………… 34
Fig. 4 CD11cintCD45RBhi DCs were reduced significantly in spleens and lymph nodes of IL-15R-/- mice………………………….….. 36
Fig. 5 CD11cintCD45RBhi DCs displayed immature phenotypes.... 38
Fig. 6 CD11cintCD45RBhi DCs were reluctant to LPS activation... 39
Fig. 7 The population of CD11cintCD45RBhi DCs could be enriched by VIP treatment…………………………………………….……….. 40
Fig. 8 Treatment of VIP did not change the phenotypes of each DC subset……………………………………………………………... 41
Fig. 9 VIP administration did not induce CD11cintCD45RBhi DCs significantly………………………………………………………. 43
Fig. 10 The cytokine profiles of LPS-stimulated CD11cintCD45RBhi DCs were determined by I.C. cytokine staining………………….. 44
Fig. 11 The kinetics of I.C. cytokine production in LPS-stimulated CD11cintCD45RBhi DCs and CD11chi DCs………………………. 46
Fig. 12 The cytokine profiles of LPS-stimulated CD11cintCD45RBhi DCs were determined by ELISA…………………………………. 47
Fig. 13 The cytokine profiles of LPS-stimulated CD11cintCD45RBhi DCs were determined by RT-PCR………………………………... 48
Fig. 14 CD11cintCD45RBhi DCs phagocytosed, processed, and presented antigens, but with relatively low capability.……...……. 49
Fig. 15 T cells primed by the CD11cintCD45RBhi DC subset didn’t display a Tr1 cytokine profile.………………………………..…... 51
Fig. 16 Exogenous IL-15 enhanced the IL-10 production in T cells generated by the CD11cintCD45RBhi DC subset…………………. 52
Fig. 17 CD11cintCD45RBhi DC displayed a higher capability in inducing IL-10/TGF-1-producing T cells……………………….. 54

References..……………………………….…………………………. 55
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