使用腫瘤內電衝法轉染IL-6與IL-15基因可強化CTL及NK細胞毒殺作用並抑制腫瘤的生長

Intratumoral electroporation-mediated IL-6 and IL-15 genes activates CTL and NK cells cytotoxicity and suppresses tumor growth

邱培純(Pei-Chun Chiu)

指導教授 ： 朱瑞民

國立臺灣大學/生物資源暨農學院/獸醫學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.02587

摘要

腫瘤細胞具有逃避免疫系統監測之機制，因而在體內不停增生，使宿主生理功用失常，加上惡性腫瘤細胞的轉移最終造成嚴重後果。腫瘤細胞逃避免疫系統監測機制中主要包括(1)使免疫系統失去辨識能力。最常見的是主要組織相容複合物 (Major histocopatibility complex, MHC)不表現或表現量極低，(2)免疫系統毒殺腫瘤細胞能力下降。抑制免疫細胞之毒殺作用包括T細胞及NK細胞，及 (3)分泌抑制性分子，抑制免疫細胞的作用。
因為犬傳染性花柳性腫瘤(canine transmissible venereal tumor, CTVT)生長期時，TGF-β除了抑制IFN-γ誘導MHC作用，並抑制(tumor infiltrating lymphocyte, TIL)的毒殺能力，造成CTVT在宿主體內快速生長。但實驗接種CTVT於犬隻背部皮下後會自然地進入消退期(R期)。造成消退的主因來自R期時TIL分泌大量的IL-6，拮抗TGF-β抑制IFN-γ之作用，使IFN-γ能誘導CTVT的MHC表現，使T細胞活化。另外IL-6也拮抗TGF-β抑制NK細胞的毒殺能力，腫瘤因此進入R期。
本實驗乃針對CTVT之特性，嘗試以IL-6導入基因解除TGF-β對免疫系統抑制能力，使CTVT細胞之MHC分子表現量上升，更進一步導入IL-15基因活化免疫系統，達到成功治療腫瘤之目的。因此構築IL-6及IL-15基因的質體，用腫瘤內電衝方式治療CTVT，並研究此基因治療的效果與抗腫瘤機制。實驗結果證明合併使用IL-6與IL-15不但能顯著的延緩CTVT的生長更可以成功使腫瘤消退。在複合式療法下，腫瘤中IL-6與IL-15的濃度增加，腫瘤細胞的MHC表現量上昇，MHCⅠ 由治療前5.91%至治療後第四週已上昇至34%；MHCⅡ 由治療前8.86%至治療後第四週已上昇至25.86%。同時發現腫瘤內TIL浸潤程度增加，其中CD8+T細胞佔TIL之百分比有顯著上昇的情形，由5.84%上昇至21.63%。而在腫瘤細胞產生細胞素之能力方面，經治療後，IL-8與TNF-α表現量顯著性上昇，但功用仍不明朗，但CTVT分泌TGF-β的能力則不會因治療而造成影響。而且此療法能有效提升NK細胞與CTL的毒殺能力，然而B細胞受CTVT殺害而數量低落之狀況並未能改善。以上這些結果顯示使用腫瘤內電衝，進行IL-6與IL-15基因治療，在實驗犬模式，可成功破壞腫瘤逃避免疫系統的作用並提昇免疫反應，達到成功治療腫瘤之目的。本研究結果所使用之策略包括：一方面使用IL-6提高MHC的表現，一方面以IL-15強化NK細胞的作用，可做為未來對腫瘤治療上的一個新選擇。

關鍵字

犬傳染性花柳性腫瘤 ； 腫瘤 ； 基因治療 ； 介白素6 ； 介白素15

並列摘要

Malignant cells escape from the surveillance of immune system and proliferate unrestrictedly. They cause severe alternations on host physiological activities.
Malignant cells can metastasize and create further disturbances on normal functions of tissues and organs. There are three major mechanisms for tumor cell to escape: (1)Loss of recognition of foreign antigens. Tumor cells do not express MHCⅠmolecule to escape T lymphocytes recongition. (2) Decrease of cytotoxicity. Decrease the cytotoxiciity of T cells and NK cells (3)Inhibition of immune system. Tumor cells or stromal cells produce some inhibiting factors to suppress the function of immune stystem.
During progression, canine transmissible venereal tumor (CTVT) secretes TGF-β to inhibit the IFN-γ induction of MHC molecules and the cytotoxicity of tumor infiltrating lymphocytes (TIL). In experimentally transplanted CTVT, it exhibits a spontaneous regression (R phase). This is due to the fact that TIL secret high concentration of IL-6 from TIL in R phase, which antagonizes the activity of tumor-derived TGF-β. After the release of TGF-β inhibition, IFN-γ can restore the MHC expression activity and T cells are activated. IL-6 also restore the cytotoxicity of NK cells. That is once inhibited by TGF-β.
This research first used IL-6 gene delivery intratumorly through electroporation to antagonize TGF-β activity to promote MHC expression and then by additional IL-15 gene delivery to activate T and NK cells activities. This combinatize successfully treat this tumor in dogs. Besides, we have studied the mechanism of the combined immunogene. The combined treatment of IL-6 and IL-15 inhibited the growth or eiminated CTVT. The surface expression of MHCⅠmolecules on CTVT cells was significantly increased after gene therapy from 5.9% to 34% and MHCⅡ from 8.86% to 25.86% . This gene therapy induced more infiltrating lymphocytes from in the tumors with significant higher percentage of CD8+ T cell from 5.84% to 21.64%. This treatment also changed the cytokine profile of CTVT in favor of immune response. The mRNA levels of IL-1β, IL-8 and TNF-α were increased. But, there were no difference in TGF-β secreting activity of CTVT cells. NK cells and CTL cytotoxicity were also greatly enhanced. In conclusion, our results demonstrated that intratumoral electroporation-mediated IL-6 and IL-15 gene therapy for canine transmissible venereal tumor in the canine model efficiently tumor escape mechanism and also intensified the host immune responses. This strategy to promote MHC expression and enhance the responses on NK and other immune cells could be additional choice for cancer therapy.

並列關鍵字

CTVT ； tumor ； gene therapy ； IL-6 ； IL-15

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