自然殺手細胞過繼免疫療法延長低度遠端轉移乳癌小鼠的生存期及增強肺部樹突狀細胞和 T 細胞的活化

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研究生：	廖浩廷 Hao-Ting Liao
論文名稱：	自然殺手細胞過繼免疫療法延長低度遠端轉移乳癌小鼠的生存期及增強肺部樹突狀細胞和 T 細胞的活化 Adoptive natural killer cell therapy prolongs overall survival in mice with low-burden metastases breast cancer and enhances dendritic cell and T cell activation in metastatic lungs
指導教授：	廖南詩 Nan-Shih Liao 羅月霞 Yueh-Hsia Luo
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
論文出版年：	2024
畢業學年度：	112
語文別：	英文
論文頁數：	65
中文關鍵詞：	自然殺手細胞、樹突細胞、幹細胞樣T細胞
外文關鍵詞：	Natural killer cell, Dendritic cell, Stem cell-like CD8+ T cell
相關次數：	點閱：22 下載：0
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先前的研究表明，在沒有後天性免疫細胞參與的情況下，自然殺手細胞可通過釋放細胞顆粒和細胞激素來對抗癌細胞的轉移。早期抗腫瘤免疫反應中，在 1 型輔助 T 細胞 (TH1) 和具有細胞毒殺性的 CD8+ T 細胞生成和活化之前，自然殺手細胞是主要 IFN-γ 的生產者，對於腫瘤微環境中免疫反應的調控相當重要。此外，IFN-γ 能促進樹突狀細胞 (DC) IL-12 的釋放，並誘導 T 細胞分化成 TH1 細胞。IFN-γ 還可增強活化 CD8+ T 細胞的增殖和細胞毒性，例如 granzyme B 和 TNF 相關凋亡誘導配體的表達。然而，自然殺手細胞也可以產生免疫抑制性激素 IL-10，該細胞激素通過抑制 T 細胞的共同刺激路徑來控制 T 細胞的激活，此外，也能抑制 DC 的成熟和抗原呈現的功能。
由於我們體外擴增的自然殺手細胞表達 IFN-γ 和 IL-10。因此透過使用野生型、Ifng-/- 和 Il-10-/- 自然殺手細胞，探討自然殺手細胞衍生的 IFN-γ 和 IL-10 是否影響後天性免疫反應來控制肺部轉移的癌細胞。使用自發性 EO771 乳腺癌轉移模型，我們發現，在原發腫瘤切除後進行自然殺手細胞治療可以顯著延長低程度的癌細胞轉移小鼠的長期存活率。這種自然殺手細胞療法的效果需要 CD8+ T 細胞的幫助。此外，自然殺手細胞治療增強癌症轉移肺部中 1 型和 2 型傳統樹突狀細胞 (cDCs)、Foxp3- CD4+ T 細胞和幹細胞樣 (SCL) CD8+ T 細胞的活化。然而，未觀察到肺引流縱隔淋巴結中的 T 細胞活化。這些結果表明，自然殺手細胞活化傳統樹突細胞，隨後傳統樹突細胞促進肺中的 T 細胞活化。此外，幹細胞樣 CD8+ T 細胞增殖的增加暗指肺中的功能性 T 細胞得到了補充。我們發現自然殺手細胞中的 IFN-γ 缺失會削弱這些效果，並降低低程度的癌細胞轉移小鼠的生存率。綜合來說，自然殺手細胞療法可以透過 IFN-γ 活化傳統樹突細胞與 T 細胞和其他方式來影響功能性 T 細胞的功能以治療低程度的乳腺轉移。

Previous studies indicated that Natural killer (NK) cells exert effective anti-metastases activity through direct release of cytotoxic granules and cytokines without the involvement of adaptive immunity. NK cell is the sole producer of IFN-γ, an important regulator of immune cells within the tumor microenvironment (TME), in the early phase of an anti-tumor immune response prior to the generation and activation of type 1 T helper (TH1) cells and cytotoxic CD8+ T cells. Moreover, IFN-γ promotes DC to produce IL-12 and triggers T cell differentiation into TH1 cells. It also enhances the proliferation and cytotoxicity of activated CD8+ T cells, such as the expression of granzyme B and TNF-related apoptosis-inducing ligand (TRAIL). However, NK cells can produce immunosuppressive IL-10, which inhibits the T cell co-stimulatory pathway to control T cell activation and suppresses the maturation and antigen-presenting functions of DCs.
Our ex-vivo expanded NK cells express IFN-γ and IL-10. Therefore, I performed comparative study on wild type, Ifng-/- and Il-10-/- NK cells to examine whether NK-cell-derived IFN-γ and IL-10 influences adaptive immune response to control metastatic cancer in the lung. Using a spontaneous metastasis model of EO771 breast cancer, we found that transfer of our NK cells after primary tumor resection prolong overall survival in mice bearing low-burden metastases. This effect of NK cell therapy depends on CD8+ T cells. Furthermore, the NK cell treatment enhances the activation of type 1 and type 2 conventional dendritic cells (cDCs) and the activation of Foxp3- CD4+ T cells and stem cell-like (SCL) CD8+ T cells in the metastatic lung. Whereas no enhancement of T cell activation in the lung-draining mediastinal lymph nodes was observed. These results imply that NK cells activate DCs, and subsequently, cDC promote T cells activation in the lung. Furthermore, the increase SCL CD8+ T cells proliferation suggest a replenishment of effector T cells in the lung. Importantly, we found that IFN-γ deficiency in NK cells diminishes these effects and attenuate survival rate in mice with low-burden metastases. Collectively, NK cell therapy can effectively treat low-burden metastatic breast cancer by activating the cDC-T cell axis and other effector T cell.

中文摘要    i
Abstract    iii
誌謝    v
Contents    vi
Figure contents    viii
Table contents    ix
Chapter 1. Introduction    1
1.1 Breast cancer    1
1.2 Tumor microenvironment (TME)    1
1.3 Natural killer (NK) cells    2
1.3.1 NK cell development    2
1.3.2 NK cell activation and anti-tumor function    3
1.4 The role of dendritic cell (DC) in cancer    4
1.5 NK-DC-T cell axis    6
1.6 The states of CD8+ T cells in TME    7
Chapter 2. Materials and methods    9
2.1 Mice    9
2.2 Tumor cell lines    9
2.3 Ex vivo expansion of murine NK cells and sorting    9
2.4 E0771 resection and metastasis model and NK cells adoptive transfer    10
2.5 T cell depletion    10
2.6 Flow cytometer    11
Chapter 3. Result    13
3.1 E0771-resected lung metastasis model    13
3.2 Syngeneic NK cell therapy is effective in treating low-burden metastases.    13
3.3 The effect of syngeneic NK cell therapy depends on CD8+ T cells.    15
3.4 NK cell transfer promotes T cell activation and increases SCL CD8+ T cells in metastatic lung.    15
3.4 NK cell transfer promotes T cell activation and increases SCL CD8+ T cells in metastatic lung.    15
3.5 NK cell transfer increases MHC-IIhi cDC1 subsets and upregulates cDC1 activation in metastatic lung.    17
3.6 NK cell transfer upregulates cDC2 activation in metastatic lung.    19
3.7 NK cell transfer leads to an increase of activated cDCs but does not affect the state of T cell activation in the mLN.    20
3.8 IFN-γ of transferred NK cells is not the sole determinant of OS but enhances early survival of mice bearing low-burden metastases.    21
Chapter 4. Discussion    23
Chapter 5. Conclusion    27
Chapter 6. References    28
Figure 1. Syngeneic NK cell therapy is effective in E0771 resection mice with low metastatic burden.    34
Figure 2. Depletion of CD8+ cells abolish the efficacy of NK cell therapy.    35
Figure 3. Transferred of NK cell promotes T cell activation and proliferation, as well as increases SCL CD8+ T cells in metastatic lung.    38
Figure 4. NK cell transfer increases MHC-IIhi cDC1 subsets and modulates the expression of APC functional molecules by cDC1 subsets in metastatic lung.    41
Figure 5. NK cell transfer increases MHC-IIhi cDC2 subsets and modulates the expression of APC functional molecules by cDC2 subsets in metastatic lung.    44
Figure 6. NK cell transfer auguments the activation of migratory cDC1 in mLN.    47
Figure 7. NK cell transfer does not influence the state of T cell activation in mLN.    49
Figure 8. IFN-γ deficiency of transferred NK cells affects the early survival of E0771 resection mice with low metastatic burden.    50
Antibodies used for FACS analysis of murine cells    51
                                

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