探討 T-bet 於人類臍帶血第一型自然殺手細胞中的調控機制

Regulation of T-bet Expression in Type I NK Cells from Human Umbilical Cord Blood

10.6342/NTU.2009.03210

吳佩玟

type I 極化 ； 後轉錄層面 ； type I polarization ； post-transcriptional level ； T-bet ； IFN-r

臺灣大學病理學研究所學位論文

2009年

碩士

林中梧

英文

在免疫防禦系統中，CD4T (T helper cell, Th cell) 細胞、CD8T (T cytotoxic cell, Tc cell) 細胞以及自然殺手細胞 (Natural killer cell, NK cell) 扮演了重要的角色。入侵的病原體會刺激 CD4T 細胞使其活化並進一步分化為 Th1 及 Th2 兩種族群，各分泌不同的細胞激素，Th1細胞主要分泌 IL-2及IFN-r，而 Th2 細胞則分泌 IL-4及IL-13；近來在 CD8T 和 NK 也發現細胞有趨向 type I 及 type II 極化(polarization) 的現象。 T-bet，一種 T-box 家族的轉錄因子，藉著調控 IFN-r 基因的轉錄以促進細胞趨向 type I 的極化。T-bet 的調控機制先前已於NK淋巴癌細胞株YT (type 0-like T-betweak/IL-4+/IFN-r+) 與 NK92 (type I-like T-bet strong /IL-4-/IFN-r++)中被研究，結果顯示 YT 的 T-bet 表現較低是由於此基因在轉譯成蛋白質時有被抑制的現象，據此推論 T-bet 主要是受到後轉錄層面的調控。為了進ㄧ步探討人類 T 及 NK 細胞中 T-bet 的調控機制，我們自臍帶血中純化出 CD4T, CD8T 及 NK 細胞，利用細胞激素刺激細胞走向 type I 的分化路徑，再以 Flow cytometry 及 Real time PCR 分析這些 type I 極化後細胞其 T-bet 在蛋白質及 mRNA 上的表現程度，藉以研究此基因的調控機制。由於臍帶血 NK 細胞依據其CD56表現強弱而存在 CD56-, CD56dim 及 CD56bright 三種亞群，我們發現 NK 在刺激第0天到第5至第7天的 type I 分化前期中，CD56- 亞群比例將逐漸降低而 CD56bright 則逐漸增多。另外，CD56bright 亞群 T-bet 的 mRNA 表現量約為 CD56- 亞群的兩倍，蛋白質表現量卻也僅有5倍之差，推測 T-bet 於兩個亞群中不同的表現量是由於其在轉錄層面的調控機制；而在完全分化成 NK type I 細胞的第14天也發現 T-bet 蛋白質與 mRNA 表現量皆比尚未分化的第0天高出許多，顯示臍帶血中 type I 極化的 NK 細胞其 T-bet 於 type I 分化初期至第5~7天，亦或分化後期至完全 type I 極化的第14天皆是受到轉錄層面的調控；另一方面，T-bet 在 type I 極化的 CD4 T 細胞是受到轉錄層面的調控，而 CD8 T 細胞則較偏向後轉錄層面。最後，除了依傳統的方式以 IFN-r 及 T-bet 來分析細胞 type I 分化的程度，我們更確立了一些 type I 的細胞標記，例如可專一性作為臍帶血 NK type I 標記的 CD56，以及可作為 type I CD4 和CD8 T 細胞標記的 CD25 或 CXCR3。以上結果顯示，T-bet 於轉錄或後轉錄層面的調控在參與免疫系統中 T 及 NK 細胞的發育與成熟扮演了關鍵的角色。

CD4T (T helper cell, Th cell), CD8T (T cytotoxic cell, Tc cell), and NK (natural killer) cells play important roles in immune defense system. When CD4T cells encounter invading organisms, they will be activated and differentiate into two functionally distinct subsets, Th1 and Th2, with different cytokine profiles. Th1 cells secrete interleukin 2 (IL-2) and interferon r(IFN-r), and Th2 cells secrete the cytokines interleukin 4 (IL-4) and interleukin 13 (IL-13). The phenomenon of type I vs type II polarization was recently found in CD8T cells and NK cells. T-bet, a member of the T-box transcription factor family, may promote type I polarization by regulating transcription of the IFN-r gene. Regulation of T-bet was studied in YT and NK92 lymphoma cell lines of NK-cell origin. YT cells had a type-0 IL-4+/IFN-r+/T-betweak profile, whereas NK92 cells had a type-I IL-4-/IFN-r++/T-betstrong profile. Our data indicated that weak expression of T-bet in the YT cells was due to a translational block in T-bet mRNA, suggesting T-bet regulation at post-transcriptional level. To further investigate T-bet regulation in human primary T and NK cells, we isolated umbilical cord blood (UCB) CD4T, CD8T and NK cells to study the protein and mRNA expression patterns of T-bet in cytokine stimulated type I-polarized cells by flow cytometry and real time PCR. It was reported that there were three distinct populations of human UCB NK cells based on the density of surface expression of CD56, which are CD56-, CD56dim and CD56bright subset, respectively. In the earlier stage of type I polarization, between the period of stimulation day 0 (D0) to day 5-7 (D5-7), the percentage of CD56- subset was decreased and CD56bright subset was increased. There was about 2-fold higher T-bet mRNA and only 5-fold higher expression of T-bet protein in CD56bright subset than in CD56- subset, suggesting different expression of T-bet among the two subsets was due to its transcriptional level regulation. When cells were at totally type I-polarized day 14 (D14), we found T-bet was transcriptional regulated either as both T-bet transcript and protein expression were much higher than day 0 (D0) naïve, undifferentiated NK cell. These data revealed transcriptional regulation of T-bet in UCB type I polarized NK cells from early stage of differentiation, day 0 (D0) to day 5-7 (D5-7) till day 14 (D14), when cell was totally type I polarized. Furthermore, we also showed transcriptional regulation of T-bet in type I polarized CD4T cells but post-transcriptional regulation in CD8T cells. Subsequently, in addition to analyze type I polarization based on IFN-r and T-bet expression traditionally, we also identified several type I surface markers, included CD56, specific for UCB type I polarized NK cells, and CD25 or CXCR3 for CD4T and CD8T cells. Taken together, these data showed T-bet regulation at transcriptional or post-transcriptional level is a critical event in the maturation of immune system and development of T and NK cells.