許秉寧臺灣大學：免疫學研究所楊政遠Yang, Cheng-YuanCheng-YuanYang2007-11-292018-07-092007-11-292018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/63338TRAIL屬於腫瘤壞死因子家族之中的一員，具有引發多種轉型細胞株進行細胞凋亡的能力，但是對於一般正常的細胞並沒有細胞毒殺的能力。而在我們實驗室之前的研究中顯示出，同時給予T細胞抗CD3抗體和DR4的刺激之下，可以引起T細胞的活化增生和誘導干擾素-gamma的產生。這樣的結果指出，當TRAIL與其對應的死亡受體結合之後，除了會造成細胞的凋亡之外，TRAIL本身也會傳遞一個反向的訊息導致T細胞的活化。然而，在我們之前的研究更進一步發現到，在TRAIL所引起T細胞的活化當中可以觀察到PI3K/Akt的活化，顯示PI3K/Akt可能參與在其中。然而，由TRAIL所傳遞的反向訊息路徑的詳細機制目前仍然未被研究清楚。目前已知，NF-kappaB訊息傳導路徑參與在CD28所引起的細胞共活化之中，顯示NF-kappaB訊息傳導路徑對於T細胞共活化相當重要。在最近的研究中也發現到，在T細胞共活化的情況之下，CARMA1, BCL10, 以及MALT1對於調控NF-kappaB訊息傳導路徑扮演著相當重要的角色。並且也有報導指出，Akt會透過與CARMA1的結合去調控NF-kappaB訊息傳導路徑。在本篇研究中，我們利用西方墨點法去研究在TRAIL所引起T細胞共活化的情況之下，NF-kappaB訊息傳導路徑所扮演的角色。我們證明了在TRAIL所引起T細胞共活化之情況下，NF-kappaB從細胞質轉移到細胞核的量會有所增加。並且這個現象會伴隨著細胞質中IkappaB-alpha所被分解量的增加同時發生。更進一步的研究顯示，TRAIL所引起T細胞活化增生和介白素-2的產生可以藉由加入NF-kappaB抑制劑而達到顯著的抑制。並且，我們也發現到，在只以TRAIL的死亡受體DR4的刺激情況下，即可以造成PKC-theta的磷酸化而造成NF-kappaB的活化，使NF-kappaB從細胞質轉移到細胞核中，這樣的結果顯示出，TRAIL所引起的反向訊息會透過增加NF-kappaB的活性而來增加活化T細胞的能力。Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a member of the TNF superfamily which is capable of inducing apoptosis in transformed cells but not in most of the primary cells. In our previous study, we demonstrated that TRAIL stimulated with immobilized DR4, in conjunction with suboptimal anti-CD3, induced T cell proliferation and enhanced IFN-gamma production. This indicates that TRAIL can transduce reverse signal to induce T cell activation. Furthermore, we found that PI3K/Akt activity was enhanced after TRAIL-mediated T cell activation. However, the reverse signaling pathway transduced by TRAIL is still not clear.It has been demonstrated that NF-kappaB was involved in CD28 costimulation signaling pathway, suggesting that NF-kappaB signaling pathway is important in costimulation of T cells. Recent studies have shown that the CARMA1, Bcl10, and MALT1 proteins are critical for the NF-kappaB signaling pathway to the TCR and CD28. Recently studies have confirmed that Akt plays a modulatory role and CARMA1 is required for Akt-mediated in NF-kappaB induction by TCR and CD28. In our previous study, we found that PI3K/Akt activity was enhanced after TRAIL-mediated T cell activation, suggest that PI3K/Akt signaling pathway is involved in TRAIL reverse signaling pathway. In this study, we use Western blotting to identify the role of NF-kappaB signaling pathway in TRAIL-induced costimulation of T cells. Our results showed that NF-kappaB activation and translocation into nucleus is enhanced in TRAIL-mediated T cell activation. In accordance with this observation is the concomitant enhanced degradation of IkappaB-alpha. Furthermore, TRAIL-mediated T cell proliferation and IL-2 production was significantly decreased by NF-kappaB inhibitor, indicating that NF-kappaB signaling pathway is involved in TRAIL-induced activation of T cells. Moreover, we directly induced PKC-theta phosphorylation and NF-kappaB translocation with DR4-Fc stimulation alone. These results indicated that TRAIL-reversed signaling in T cells transduce via enhancing NF-kappaB activation.Acknowledgement i Abstract (Chinese) ii Abstract iii Chapter I. Introduction 1 1. TRAIL and TRAIL receptors 1 2. Reverse signal transduction in TNF superfamily 2 3. T cell activation 2 4. NF-κB signaling pathway 4 5. Lipid raft in T cell activation 5 6. Rationale of the study 6 Chapter II. Materials and methods 8 Part 1. Experimental Materials 8 Part 2. Experimental Procedures 12 Purification of soluble TRAIL receptor DR4-Fc 12 Jurkat cell activation 12 Cytosolic and nuclear extract separation 13 SDS-PAGE and Western blotting 13 Human primary T cell purification 14 Human primary T cell proliferation assay 14 Mouse IL-2 production ELISA 15 Luciferase reporter assay 15 Statistical analysis 16 Chapter III. Results 17 Part 1. NF-κB is a possible downstream molecule in the signaling pathway of TRAIL-induced T cell activation. 17 Part 2. The NF-κB activity is enhanced by DR4-TRAIL engagement 18 Part 3. IκBα degradation and NF-κB nuclear translocation after TRAIL-induced costimulatory signal 19 Part 4. Akt maybe a modulator in TRAIL-induced costimulation 20 Part 5. NF-κB pathway is directly involved in the TRAIL reverse signal 21 Part 6. TRAIL-induced NF-κB activation is through PKC-θ 23 Chapter IV. Discussion 25 Part 1. NF-κB pathway is involved in the TRAIL-induced reversed signaling in T cell activation. 25 Part 2. The role of Akt in NF-κB signaling pathway in TRAIL-induced costimulation 27 Part 3. NF-κB is activated by TRAIL reverse signal 28 Reference 30 Figures 34 Figure 1. TRAIL-induced IL-2 production in EL4 cells could be blocked by NF-κB activation inhibitor (QNZ) 34 Figure 2. TRAIL-induced IL-2 production in human primary T cells could be blocked by NF-κB activation inhibitor (QNZ) 35 Figure 3. TRAIL-induced human primary T cell proliferation could be blocked by NF-κB activation inhibitor (QNZ) 36 Figure 4. After DR4-TRAIL engagement in conjunction with anti-CD3 enhanced NF-κB luciferase activity in Jurkat T cells 37 Figure 5. DR4-TRAIL engagement in conjunction with anti-CD3 enhanced NF-κB nuclear translocation in Jurkat T cells 38 Figure 6. DR4-TRAIL engagement in conjunction with anti-CD3 enhanced cytosolic IκBα degradation in Jurkat T cells 40 Figure 7. The DR4-induced NF-κB activation was slightly suppressed by Akt inhibitor 42 Figure 8. DR4 directly enhanced NF-κB activity in a dose-depend manner in Jurkat T cells 43 Figure 9. DR4-Fc directly induce NF-κB nuclear translocation and sustained nuclear translocation for 2 hours in Jurkat T cells 44 Figure 10. DR4-Fc directly induced PKC-θ phosphorlation in Jurkat T cells 45 Figure 11. DR4-Fc-induced NF-κB nuclear translocation could be inhibited by PKC-θ inhibitor 46en-US訊息傳導路徑signaling pathwayother