探討表觀遺傳治療對肺癌中腫瘤反應T細胞的免疫調節作用

Immunomodulatory Effects of Epigenetic Therapy on Tumor Reactive T Cells in Lung Cancer

吳怡潔(Yi-Chieh Wu)

指導教授 ： 蔡幸真

國立臺灣大學/醫學院/毒理學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803261

摘要

肺癌在全球的癌症死亡率排名第一，有40%的肺癌患者於診斷時已是晚期階段，導致治療效果有限，因此發展新的治療方式是當前研究的一大重點。近年來癌症免疫療法的研究，為晚期肺癌患者帶來一線曙光。癌細胞利用免疫檢查點(immune checkpoint)的機制抑制腫瘤微環境中的T細胞活性，使T細胞功能減弱或喪失，因而無法有效的清除癌細胞，稱之為T細胞耗竭(T cell exhaustion)。目前針對肺癌的免疫療法，即是通過免疫檢查點抑制劑(immune checkpoint inhibitor)如anti-PD-1和anti-PD-L1單株抗體，重新活化對抗腫瘤的T細胞，使其恢復對癌細胞之毒殺作用。然而，臨床結果顯示只有少部分的肺癌患者對免疫檢查點抑制劑療法有良好反應，且免疫檢查點抑制劑只能短暫恢復T細胞功能，並無法全面持久地改變T細胞的耗竭狀態。目前研究已知，表觀遺傳(epigenetic)調控在T細胞耗竭中扮演重要角色，耗竭性T細胞其基因上的修飾及染色質的構造不同於功能性T細胞，其中耗竭性T細胞相關基因的DNA甲基化也限制了免疫檢查點抑制劑的療效。因此本研究希望能探討表觀遺傳治療對於肺癌中腫瘤反應T細胞的免疫調節作用。
我們自肺癌患者的腫瘤組織與惡性胸水(malignant pleural effusion, MPE)中分離出初代T細胞和肺癌細胞，建立自體腫瘤免疫模型(autologous model)，以及系統性地利用流式細胞儀評估腫瘤及惡性胸水中T細胞之表型，並觀察Ｔ細胞在表觀遺傳藥物處理後，是否可造成功能性的改變。結果顯示，惡性胸水中有大量T細胞，相較於健康受試者的血液，惡性胸水中T細胞的免疫檢查點有較高表現，呈現耗竭性T細胞的特性。經由DNA去甲基化藥物decitabine(DAC)處理後，發現10nM DAC具有較低的細胞毒性且明顯地提升T細胞的多功能性(T cell polyfunctionality)，我們深入探討DAC對於T細胞中PD-1+和PD-1-亞群之影響，發現PD-1+亞群之多功能性T細胞較易受到DAC提升，其中干擾素-γ(IFN-γ)為主要增加的細胞激素。另外，我們利用高通量表觀遺傳藥物篩選平台(epigenetic drug library)，初步發現幾種組蛋白修飾的抑制劑藥物會影響T細胞的多功能性，如PCI 34051、Scriptaid、Suberohydroxamic Acid等。
本研究探討肺癌患者的惡性胸水中T細胞特性，並了解DAC對肺癌中的PD-1+ T細胞亞群具有較佳之免疫調節作用，此外，也藉由表觀遺傳藥物篩選平台找出其他可能恢復或提升T細胞功能的藥物，為將來的臨床應用奠定基礎。未來我們將會分析更多樣本中的腫瘤反應T細胞經表觀遺傳藥物處理後的功能性變化，並透過惡性胸水中抽取出的初代細胞，所建立的autologous model，進行T細胞對癌細胞的毒殺分析(immune cell killing assay)。

關鍵字

肺癌 ； 癌症免疫治療 ； 表觀遺傳 ； T細胞 ； 惡性胸水

並列摘要

Lung cancer is the leading cause of cancer death worldwide. Nearly 40 % of people with lung cancer were diagnosed at later stages, which results in limited therapeutic options and poor prognosis. Therefore, development of new treatment for lung cancer is very important. In the tumor microenvironment (TME), cancer cells evade immune responses by expressing immune checkpoint to inhibit effector functions of tumor infiltrating lymphocytes (TILs). These functionally impaired T cells were known as exhausted T cells. Currently, immune checkpoint blockade therapy, such as anti-PD-1 and anti-PD-L1, has demonstrated promising responses in advanced lung cancer patients through reinvigorating dysfunctional tumor-reactive T cells. Nevertheless, many patients still do not have favorable responses to this therapy. Besides, T cell rejuvenation may be transient. Recent evidence has suggested that epigenetic regulation plays an important role in T cell exhaustion. Several reports demonstrated that dysfunctional T cells display specific alterations in chromatin accessibility. Dysfunctional T cells also acquire abnormal DNA methylation during tumor development, which restricts the ability of exhausted T cells to respond to PD-1 blockade therapy. Therefore, we aim to investigate immunomodulatory effects of epigenetic therapy on tumor-reactive T cells in lung cancer. In this study, we isolated T cells and lung cancer cells from malignant pleural effusion (MPE) or tumor tissues of lung cancer patients to establish an autologous model of tumor and tumor-reactive lymphocytes. We utilized flow cytometry to characterize the phenotype of T cells from MPE or tumor tissues. We evaluated the effects of decitabine (DAC), a DNA methyltransferase inhibitor, on T cell polyfunctionality. We discovered that T cells in MPE had higher expression of inhibitory immune checkpoints that represent the exhausted phenotype. We also found that PD-1+ T cell subpopulation tended to be more responsive to 10nM DAC in terms of augmentation of polyfunctinality. Among these effector cytokines, IFN-γ is the major functional marker increased by DAC. Furthermore, we utilized epigenetic drug library screening to identify potential drugs that may enhance T cell polyfunctionality. We found some histone modification-targeting drugs that could enhance T cell polyfunctionality, such as PCI 34051、Scriptaid、Suberohydroxamic Acid. Collectively, we characterized the immunophenotypes of tumor reactive T cell in MPE, and the effect of DNA demethylating agents, DAC, on T cell polyfunctionality. In addition, we identified other epigenetic drugs that could potentially be used for modulating T cell functions. In the future, we will study more patient samples to comprehensively investigate the association between epigenetic regulation and tumor-reactive T cells in lung cancer. Furthermore, we will conduct immune cell killing assays using the autologous model we established from MPE to further validate the effects of epigenetic drugs on direct T cell cytotoxicity.

並列關鍵字

Lung cancer ； Immunotherapy ； Epigenetics ； T cell ； Malignant pleural effusion

參考文獻

1. Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2018. CA. Cancer J. Clin. 68, 7–30 (2018).

2. Lung Cancer Symptoms, Signs, Stages, Treatment & Types. MedicineNet Available at: https://www.medicinenet.com/lung_cancer/article.htm. (Accessed: 21st May 2018)

3. Lung Cancer: Stages, Survival Rates, and More. Healthline (2016). Available at: https://www.healthline.com/health/lung-cancer-stages-survival-rates. (Accessed: 16th June 2018)

4. Carter, B. W., Halpenny, D. F., Ginsberg, M. S., Papadimitrakopoulou, V. A. & de Groot, P. M. Immunotherapy in Non–Small Cell Lung Cancer Treatment: Current Status and the Role of Imaging. J. Thorac. Imaging 32, 300–312 (2017).

5. Crespo, J., Sun, H., Welling, T. H., Tian, Z. & Zou, W. T cell anergy, exhaustion, senescence, and stemness in the tumor microenvironment. Curr. Opin. Immunol. 25, 214–221 (2013).