何弘能臺灣大學：免疫學研究所吳禹蓉Wu, Yu-JungYu-JungWu2007-11-292018-07-092007-11-292018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/63294胎兒的一半基因來自於父親，為了使胎兒在母親體內不被排斥且能夠順利著床、發育，母親子宮環境中的免疫系統必須經過適當的調節。過去研究指出性賀爾蒙具有調節子宮內膜免疫反應的作用，而人類子宮內膜中的免疫細胞的型態也與一般血液循環系統中的免疫細胞有所差異，趨向於發展成TH2的反應。至於主導免疫調控的調節性T細胞(regulatory T cells)與懷孕之相關性，近期也有一些研究被發表，但調節性T細胞在人類懷孕子宮內膜組織中的分佈情形，目前為止並沒有明確的解釋。我們利用免疫染色法，以FOXP3分子做為調節性T細胞的標誌，找出FOXP+調節性T細胞在懷孕子宮內膜中的分佈位置，並且利用軟體定量，發現相較於早期流產的病人，在正常懷孕的狀況下的確有較高比例的FOXP+調節性T細胞存在。此外我們也注意到人類懷孕子宮內膜中有免疫細胞聚集的現象，此種免疫細胞聚集的結構主要是由CD8+ T細胞組成，在細胞聚集的中央部分有少許的B細胞，CD4+ T細胞數量較少且散佈在細胞聚集的內外，子宮NK細胞有時會包覆在CD8+ T細胞聚集的外圍，而細胞聚集的最邊緣部分則是散佈的CD14+ 細胞。正常懷孕的狀況下會有較大且結構較為完整的免疫細胞聚集。我們也發現，FOXP3+調節性T細胞較易出現在此種免疫細胞聚集的周圍，且FOXP3+調節性T細胞會與CD8+ T細胞有直接接觸，合併針對Granzyme B的染色結果，我們也發現Granzyme B 似乎參與在其中，推測與免疫調節相關。在細胞聚集之中，可發現與CD8+ T細胞功能相關的Granzyme A與Perforin的表現，但是細胞聚集之外的組織部分，正常懷孕相對於早期流產的組別，有明顯較少的Granzyme A與Perforin可被偵測到，並且在這些細胞聚集外的區域也可以發現Granzyme B以及TGF-β的表現。因此，在正常懷孕的狀況下也許是利用此種免疫細胞聚集的機制將活化的CD8+ T細胞限制在特定的區域之內，並且在懷孕子宮內膜中其他部分具有較嚴謹的免疫調控系統，讓發炎反應可以不被大量引發，使胎兒能夠順利成功地著床發育。In order to have successful implantation and development of the fetus, the maternal immune system must be modified by several appropriate regulations in mammal. Besides the immunoregulation of sexual hormones, the immune cells in human decidua (endometrial tissue) during pregnancy have some different characteristics from those in other human tissues. CD4+CD25+ regulatory T cell (Treg) which is a subset of CD4+ regulatory T cells plays a critical role in the modulation of immune response. FOXP3 is a key molecule predominantly restricted to CD4+CD25+ Treg cells. Recently, some human and mice experiments reported that the population of CD4+CD25+ Treg cells increased during pregnancy and these Treg expressed foxp3 mRNA. However, most of these studies investigated the population of CD4+CD25+ Treg cells by flow cytometry. However, the physiological role of FOXP3+ Treg cells and their interactions with other immune cells in the endometrial microenvironment remains unknown. In order to understand this issue in normal and anembryonic human pregnancies, we used immunostaining method to detect FOXP3+ cells and indicated that the percentage of FOXP3+ cells in CD3+ cells was higher in decidua from normal pregnancies compare to those from anembryonic pregnancies. We also noted that the FOXP3+ cells were close to a special structure, CD8+ T cell-rich lymphoid aggregates, in the decdual tissues. Combined with cell markers, CD8, CD4, CD11b, CD19, CD56, we further evidenced the distribution of FOXP3+ cells and other immune cells in lymphoid aggregates. Our data indicate that the CD8+ T cell-rich lymphoid aggregates contain few B cells in the center whereas CD8+ T cells forms the main structure of the lymphoid aggregates. CD4+ T cells are few and scattered, uNK cells surround the CD8+ T cell-rich lymphoid aggregates and CD11b+ macrophages are outside. We also found that FOXP3+ Tregs were around the lymphoid aggregates and sometimes contact to CD8+ T cells. By Granzyme B staining, we suggested that granzyme B may contribute to the regulation between FOXP3+ Treg and CD8+ T cells. Although CD8+ T cells and uNK cells in lymphoid aggregates can express cytotoxic factors such as Granzyme A and Perforin, the Granzyme A and Perforin expressions of CD8+ cells and NK cells outside the CD8+ T cell-rich lymphoid aggregates was less in normal pregnancies compares to that in anembryonic pregnancies. Additionally, there are larger and well organizing CD8+ T cell-rich lymphoid aggregates in normal pregnancies. We speculate that this restriction of CD8+ T cells by lymphoid aggregates may contribute to the immunoregulation in the human decidual tissues. And in other area beside lymphoid aggregates in the normal decidua, there are also some mechanisms to reduce the inflammatory response. By this research, we hope to understand more about the complicated microenvironment during pregnancy and that mechanisms may promote successful implantation and fetus development.Contents 論文口試委員審定書……………………………………………………………………i 授權書...............................................................................................................................ii 致謝..................................................................................................................................iii 中文摘要…………………………………………………………………..…………....iv Abstract…………………………………………………………………….………….....v Contents…………………………………………………………...……….…………...vii Figure and Table of Contents…………….………………………….………...………...ix Chapter I Introduction……………………………………….………………………...1 1.1 Immune system modification in the human pregnancy………………….......2 1.2 Regulatory T cell and pregnancy…………………………..………………...5 1.3 CD8+ T cell-rich lymphoid aggregate in human endometrium………....…...8 1.4 Effector activity of CD8+ T cells in human endometrium……………...……9 1.5 Purpose of our study…………………………………………………………9 Chapter II Materials and Methods…………………………………………...……...12 2.1 Sample collection and preparation…………………………….…………...13 2.2 Fixation………………………………………………………...…………...13 2.3 Immunohistochemistry (IHC) ……………………………………………...14 2.4 Immunofluorescence staining (IF)……………………......………………...15 2.5 H & E staining………………………………………………………....…...17 2.6 Data Analysis……………………………………………………..………...18 2.7 Statistical Analysis…………...……………………………………………..18 Chapter III Results……………………………………………………………………19 3.1 Distribution of FOXP3+ cells in human decidual tissues..............................20 3.2 Population of FOXP3+ cells in human normal decidual tissues was significantly higher than in human anembryonic decidual tissues…………21 3.3 Composition of CD8+ T cell-rich lymphoid aggregates……………………22 3.4 Effector function of CD8+ T cells and uNK cells inside and outside the lymphoid aggregates…………………...…………………………………...24 3.5 Cytokines and chemokines around the CD8+ T cell-rich lymphoid aggregates…………………….…………………………………………….26 Chapter IV Discussion……………………………….……………………......……...27 References……………………………………………..……………………………...34 Figures and Tables……………………………………………………………………...43 Figure and Table of Contents Figure 1. H&E or IHC staining of FOXP3+ cells in normal human decidua………...44 Figure 2. Immunostaining of FOXP3+ cells in normal human decidua……...………45 Figure 3. More FOXP3+ cells appear around the CD3+ cell lymphoid aggregates in normal human decidual tissues.…………………...…………………...…...46 Figure 4. Phenotype of FOXP3+ cells in human decidual tissues. FOXP3+ cells are CD4+ CD25+ Tregs and physical contact with CD8+ T cells...…..…..…......47 Figure 5. Proportion of FOXP3+ cells in CD3+ cell population from human normal decidual tissues was significantly higher than human anembryonic decidual tissues.……………………….……………………………...……………....48 Figure 6. Composition of CD8+ T cell-rich lymphoid aggregates.…………...…........49 Figure 7. Schematic illustrating the composition of CD8+ T cell-rich lymphoid aggregates……………….…………….………………………..…………..51 Figure 8. Expressions of Granzyme A, Granzyme B and Perforin in human normal and anembryonic decidual tissues.…………………...…………..………...52 Figure 9. TGF-β localizes in the stroma of human decidual tissues.…………..……..56 Figure 10. Our model of suggested regulatory mechanisms about CD8+ T cell-rich lymphoid aggregates, FOXP3+ Tregs, and uNK cells in decidual tissues from normal human pregnancies...………………...…………………….…57 Table 1. Anti-human primary antibodies used for immunofluorescence stainig……..58 Table 2. Secondary antibodies used for immunofluorescence staining………………59 Table 3. The medium, 25%, and 75% of FOXP3+ cell percentage in CD3+ cell population from human normal and anembryonic decidual tissues...……...60 Table 4. Characteristics of CD8+ T cell-rich lymphoid aggregates in decidua from normal and anembryonic pregnancies……………………………..….........61 Table 5. Characteristics of Gramzyme A, Granzyme B, Perforin, and TGF-β in decidua from normal and anembryonic pregnancies…………….…………625546949 bytesapplication/pdfen-US調節性 T 細胞CD8 T 細胞聚集子宮內膜FOXP3CD8+ T Cell-Rich Lymphoid AggregatesDeciduaotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/63294/1/ntu-96-R94449008-1.pdf