探討介白質-15異構蛋白對於介白質-15調節訊息傳遞、T細胞存活及與介白質-15受器α結合的影響

Abstract

介白質-15 (Interleukin-15, IL-15) 是一個多效型的細胞激素，能夠調節記憶性CD8 T細胞 (Memory CD8+ T cell)、自然殺手細胞 (Natural killer cell, NK cell) 及自然殺手T細胞 (Natural killer T cell, NKT cell) 的複製和存活，同時也能夠促進其他細胞激素的表現。本實驗室過去的研究中發現，一種缺少了部分的第七外顯子的IL-15選擇性剪接異構體，（以下簡稱IL-15ΔE7），無法促進IL-2和 IL-15依賴性細胞HT-2細胞的STAT5的磷酸化以及抗細胞凋亡基因bcl-2的轉錄。在本論文研究，我成功建立酵母菌蛋白表現與純化系統製造重組IL-15以及IL-15ΔE7二種蛋白，並且經過西方墨點法及快速液態層析儀 (Fast Protein Liquid Chromatography, FPLC) 確認該蛋白的活性與生化特性。為了更進一步研究IL-15ΔE7對於細胞存活的影響，利用了流式細胞儀分析分別經過IL-15以及IL-15ΔE7處理後的HT-2 細胞，藉以偵測細胞膜上磷脂絲胺酸 (phosphotidylserine, PS) 外翻及DNA斷裂的程度以檢驗細胞凋亡的情況。在以IL-15培養的情況下，大部分HT-2細胞存活並具有增殖性；然而，有很高比例的IL-15ΔE7處理過後的HT-2細胞在第12小時進行細胞凋亡，而全部的細胞在第24小時死亡，這樣的情況與去除細胞激素培養的控制組細胞類似。以IL-15ΔE7預處理接著以IL-15處理或者同時以IL-15ΔE7和IL-15處理的HT-2細胞，這兩種處理方式都不會抑制IL-15促進細胞存活與增殖的作用；此外，高濃度的IL-15ΔE7刺激也不會引起IL-15培養的細胞死亡。這些實驗闡述了雖然IL-15ΔE7無法維持HT-2細胞的存活，但是外部添加IL-15ΔE7也無法抑制IL-15調節的細胞增生。利用Micro-Western進行訊號體 (Signalosome) 分析的實驗指出，IL-15ΔE7能夠相較於IL-15引起更多的P70 S6 kinase、SrcY416、SrcY527、Syk以及GSK3的磷酸化，同時我們也觀察到更多IκB的蛋白質累積；更進一步地，我們使用傳統的西方墨點法以檢驗MAPK和SrcY416的訊息傳導並確認了IL-15ΔE7無法造成ERK的磷酸化，卻能夠造成更多的JNK、P38和SrcY416的磷酸化，我們也發現IL-15引起的IKK磷酸化在IL-15ΔE7刺激的細胞中卻是被抑制的，然而，P65的磷酸化程度在IL-15ΔE7和IL-15刺激的細胞中卻是相當的，也因此IL-15ΔE7在NF-κB訊息傳遞當中所扮演的角色需要更深入的研究。從IL-15或者IL-15ΔE7結合至IL-15Rα的情況分析及結合後對於細胞表面IL-15Rα表現的影響結果當中指出，相對IL-15而言，IL-15ΔE7以非常低的親和力結合上IL-15Rα；同時，IL-15所引起的細胞表面IL-15Rα的表現量下降，在IL-15ΔE7處理的細胞則不顯著。已知藉由非淋巴性白血球轉位呈現 (trans-presentation) IL-15至T細胞和NK細胞是對於這兩類細胞能夠發展出活化後功能的重要機制；因此，未來的實驗應該加強探討IL-15ΔE7如何藉由調控轉位呈現得以調節IL-15的功能。

IL-15 mediates the proliferation and survival of memory CD8+ T cells, natural killer (NK) and NKT cells as well as enhancing cytokine production. We have previously shown that an alternatively spliced IL-15 isoform that has partial deletion in exon 7 of the IL-15 gene (IL-15ΔE7) failed to activate phosphorylation of STAT5 and blocked transcription of anti-apoptotic gene bcl-2 in an IL-2/IL-15 dependent T cell line (HT-2 cells). In this thesis, I have successfully generated recombinant IL-15ΔE7 and IL-15 proteins from yeast expression system and confirmed by Fast Protein Liquid Chromatography (FPLC) and Western Blot analyses. To further investigate the effects of IL-15ΔE7 on cell survival, IL-15ΔE7-treated or IL-15-treated HT-2 cells were examined for cell apoptosis by detecting the expression of phosphatidylserine (PS) on the plasma membrane and DNA fragmentation by flow cytometric analysis. HT-2 cells were proliferative and survived in IL-15 culture. However, high percentage of cells underwent apoptosis at 12 h and all cells died at 24 h in IL-15ΔE7-treated HT-2 cells. The patterns were similar to cytokine-deprived control cells. Pretreatment of HT-2 cells with IL-15ΔE7 followed by IL-15 treatment or co-treatment of the cells with a mixture of IL-15 and IL-15ΔE7 did not blocked IL-15-mediated cell survival and proliferation. High concentration of IL-15ΔE7 treatment did not promote cell death in IL-15 treated cells. These experiments demonstrated that while IL-15ΔE7 failed to support HT-2 cell survival, exogenous addition of IL-15ΔE7 did not exert inhibitory effects on IL-15-mediated cell proliferation. Signalosome analysis by Micro-Western assay showed that IL-15ΔE7 treatment resulted in increased levels of phosphorylation of p70 S6 kinase, SrcY416, SrcY527, Syk and GSK3 compared with those in IL-15 treated cells. Increased protein level of IκB was also observed. Further analysis of the protein phosphorylation in MAPK pathways and SrcY416 by conventional Western blot confirmed that IL-15ΔE7 failed to phosphorylate ERK but enhanced phosphorylation of JNK, P38 and SrcY416. IL-15 induced phosphorylation of IKK was suppressed in IL-15ΔE7 treated cells. Since the phosphorylation levels of p65 were comparable in IL-15-treated and IL-15ΔE7-treated cells, the role of IL-15ΔE7 in NF-κB signaling pathways requires further investigation. Analysis of the binding of IL-15 or IL-15ΔE7 to IL-15Rα and the effects on the surface expression of IL-15Rα after ligand binding demonstrated that IL-15ΔE7 bound to IL-15Rα with very low affinity compared with IL-15. IL-15 triggered downregulation of surface expression of IL-15Rα was less significant in IL-15ΔE7-treated cells. Since trans-presentation of IL-15 by IL-15Rα on myeloid cells is known as an important mechanism for developing effector functions of T cells and NK cells, the role for IL-15ΔE7 in regulating IL-15 function via trans-presentation is worth of further investigation.