Genetic Engineering a TGF-beta1 Tolerant Human Natural Killer Cell Line for Effective Anticancer Immunotherapy

• Other Authors: Wang, Qingming (author.)
• Format: Others
• Language: English; Chinese
• Published: 2016
• Online Access: http://repository.lib.cuhk.edu.hk/en/item/cuhk-1292209

自然殺傷(NK)細胞在人類腫瘤固有免疫中扮演極為重要的角色。儘管如此，由於其所處腫瘤微環境中存在大量轉化生長因子b1 (TGF-b1)，而該因子已被廣泛證實能極大程度地抑制自然殺傷細胞的癌症殺傷效應。腫瘤細胞通過分泌大量轉化生長因子b1從而協助其逃避宿主免疫反應。我們前期試驗發現，TGF-b1/Smad3 信號是腫瘤進展過程中的關鍵性促進信號，荷瘤小鼠體內試驗證實抑制或敲除Smad3 能夠促進NK細胞發育及其抗腫瘤效應，並能有效阻止腫瘤的浸潤及轉移。儘管如此，通過全身注射Smad3抑制劑等方法來抑制Smad3 可能導致免疫系統的損傷。因此，此研究旨在通過特異性敲除NK細胞內的Smad3 來建立一種全新的抗腫瘤免疫療法。 === 本研究將特異性靶向Smad3的短發卡狀RNA(shRNA)通過慢病毒載體成功導入NK-92 細胞，并建立Smad3穩定低水平表達的NK-92 細胞系（NK-92-S3KD），選擇NK-92作為本實驗的基因改造細胞主要因為其已進入二期臨床試驗，有利於將來新型改造細胞的臨床應用，具體過程列入本論文中第三章詳訴。我們體外檢測了NK-92-S3KD的細胞毒性及細胞因子的分泌水平，發現下調Smad3極大地提升了干擾素（IFN-ɣ）、颗粒酶B（Granzyme B）等细胞因子的水平。我們隨後在聯合免疫缺陷（NOD/SCID）小鼠上建立了侵襲性肝癌（HepG2）和黑色素瘤(A375)模型，并通過向荷瘤小鼠輸注NK-92-S3KD细胞以驗證該細胞的體內殺瘤效應，试验发现，NK-92-S3KD细胞与对照组相比能够极大的抑制肿瘤的生长。 === 眾所周知，E4BP4在NK細胞發育過程中起著相當重要的作用。研究發現，TGF-b1可下調NK-92細胞E4BP4的表達。相反，下調Smad3可導致E4BP4表達上調。對NK-92-S3KD細胞進行E4BP4敲落的試驗結果表明，E4BP4的缺失可部份抵消由於Smad3敲落所致的IFN-ɣ提升的效應。進一步研究表明，Smad3通過直接作用於E4BP4的3’端非翻譯區（3’UTR） 對其轉錄水平進行調控。E4BP4繼而通過上調IFN-ɣ的啟動子活性促進IFN-ɣ的表達。因此，我們可以推斷，在腫瘤微環境中癌症細胞可能利用TGFβ1/Smad3/E4BP4這條信號傳遞軸來逃避NK細胞介導的免疫殺傷效應。 === 除此之外，我們通過細胞因子抗体阵列發現Smad3 缺失上調了NK-92細胞中GM-CSF的表達水平。進一步研究發現，Smad3可能通過直接抑制基因啟動子活性下調GM-CSF表達。 === 總之，TGF-b1/Smad3信號通過抑制NK細胞介導的抗腫瘤效應促進了癌症的進展。作為Smad3介導的NK細胞抑制效應的下游分子，E4BP4接受Smad3在基因轉錄水平的直接調控。更為重要的是，本研究通過基因改造技術成功建立了一Smad3穩定敲落的人源NK細胞株NK-92-S3KD。通過阻滯TGFβ1/Smad3/E4BP4 信號途徑，這種新型細胞株獲得了高效的抗腫瘤免疫效應。因此，NK-92-S3KD細胞免疫治療可望成為一可靠及安全的抗癌新方案。 === Natural killer (NK) cell is one of the major components of human innate immunity, but can be largely suppressed by the tumor microenvironment rich in cancer-derived transforming growth factor beta 1 (TGF-b1). Our previous work revealed that TGF-b1/Smad3 signaling is a curial promoter for cancer progression. Inhibition or deletion of Smad3 largely restored the TGF-b1 suppressed NK cell development and anti-cancer activities in tumor-bearing mice, thereby effectively preventing cancer invasion and metastasis. However, targeting Smad3 systemically may induce severer side effects by impairing the normal immune system. Thus, we aim to develop a NK cell specific Smad3-targeted anticancer immunotherapy by a genetic engineering approach in this project. === As described in Chapter 3, a stable Smad3-knockdown human NK cell line (NK-92-S3KD) was established on a phase II clinical trial enrolled cell line NK-92 by transduction of lentivirus containing Smad3-shRNA sequence. Disruption of Smad3 alleviated the TGF-b1-suppressed cancer-killing activities of NK-92 cells against human invasive hepatoma HepG2 and melanoma A375 cells in vitro. Additionally, the TGF-b1-suppressed cytokine production (i.e. IFN-ɣ, granzyme B, perforin) was also restored by knockdown of Smad3. The therapeutic potential of NK-92-S3KD was further examined on two xenograft models by using HepG2 or A375 tumor bearing NK-deficient NOD/SCID mice. Tumor growth was largely inhibited by the adoptive transfer of NK-92-S3KD cells compared to the mice received empty vector control NK-92 (NK-92-EV) cells, enhanced production of NK cell derived cytokines were detected in the serum and tumor microenvironment. === Mechanistic study revealed that deletion and inhibition of Smad3 significantly up-regulated the expression of an essential transcription factor for NK cell development, E4BP4, which was dramatically down-regulated in the NK-92-EV cells by TGF-b1. We found that TGF-b1 could reduce transcription of E4BP4 via triggering physical binding of Smad3 to the 3’ untranslated region (3’UTR) of E4BP4. Unexpectedly, IFN-ɣ production in NK-92 cells was largely enhanced by Smad3-knockdown but partially abrogated by the double knockdown of E4BP4 and Smad3, we eventually discovered that E4BP4 can directly bind to the promoter region and facilitate the transcription of IFN-ɣ in NK cells by performing luciferase reporter assay. Furthermore, using cytokine array we found that not only expression of IFN-ɣ but also GM-CSF, a cytokine important in restoration of immune function of leukocytes under inflammatory events, was also enhanced in the NK-92-S3KD cells, where Smad3 directly down-regulated GM-CSF transcription via suppressing the activity of promoter. Thus, cancer may utilize a TGFβ1/Smad3/E4BP4 axis to circumvent the NK cell-mediated immunity in the tumor microenvironment. === In conclusion, TGF-b1/Smad3 signaling promotes cancer by suppressing the NK cell mediated anti-cancer immunity. E4BP4 is a direct Smad3 target gene and serves as the downstream effector for TGF-b1/Smad3-mediated inhibition of NK cell immunity against cancer. Most importantly, we have successfully developed a genetically engineered human NK cell line NK-92-S3KD to effectively promote the anti-cancer immunity of NK cells by blocking the TGFβ1/Smad3/E4BP4 axis in the tumor-friendly microenvironment. Thus, our novel TGF-b1 tolerant cell line NK-92-S3KD may represent a promising immunotherapy for cancer. === Wang, Qingming. === Thesis Ph.D. Chinese University of Hong Kong 2016. === Includes bibliographical references (leaves ). === Abstracts also in Chinese. === Title from PDF title page (viewed on …). === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only.