초록 ▼

본 과제에서 우리는 마우스 모델을 이용하여 종양 발달 과정에서의 면역학적 변화를 관찰하였다. 특히 NKG2D 리간드를 형질도입한 세포주를 확립하여 이들 세포주가 주입된 종양에서의 면역학적 변화를 살폈다. NKG2D 리간드의 발현 여부는 특히 복강내의 NK 세포의 침윤 정도에 크게 영향을 주는 것을 확인하였다. 이어 우리는 두경부종양세포주를 통하여 면역저해물질 TGF-${\beta}1$의 능동적 과발현 기전을 살펴보았고, 최종적으로 NK 세포를 이용한 항암면역요법을 항암화학요법과 병행 가능성을 얻기 위해 NKG2D

본 과제에서 우리는 마우스 모델을 이용하여 종양 발달 과정에서의 면역학적 변화를 관찰하였다. 특히 NKG2D 리간드를 형질도입한 세포주를 확립하여 이들 세포주가 주입된 종양에서의 면역학적 변화를 살폈다. NKG2D 리간드의 발현 여부는 특히 복강내의 NK 세포의 침윤 정도에 크게 영향을 주는 것을 확인하였다. 이어 우리는 두경부종양세포주를 통하여 면역저해물질 TGF-${\beta}1$의 능동적 과발현 기전을 살펴보았고, 최종적으로 NK 세포를 이용한 항암면역요법을 항암화학요법과 병행 가능성을 얻기 위해 NKG2D 리간드의 항암제에 대한 발현 변화를 살펴보았다. glioblastoma 세포주에서 항암제 VPA가 직접적인 세포독성 외에 NKG2D 리간드의 발현을 높임으로서 NK 세포를 이용한 항암면역요법을 병행의 근거를 얻었다. 또한 multiple myeloma 세포주에서도 특히 NF-kB inhibitor가 이러한 작용을 하고 있는 것을 밝혔다.
이 결과는 1) NKG2D 리간드와 복강내 면역세포를 이용한 이러한 마우스 모델은 지속적으로도 NK 특이적 면역학적 모니터링 방법이 될 것을 보였으며 2) 기존의 항암치료법, 특히 NF-kB inhibitor와 항암면역요법의 병행에 대한 가능성을 제시했다.

Abstract ▼

I. Research Mission
It has been reported in cancer patients that as the extent of the disease grows so does the extent of immune deficiency, especially in terms of NK cell function. Various immunotherapeutical strategies have been attempted, but a clear beneficial effect has not been demonstrated

I. Research Mission
It has been reported in cancer patients that as the extent of the disease grows so does the extent of immune deficiency, especially in terms of NK cell function. Various immunotherapeutical strategies have been attempted, but a clear beneficial effect has not been demonstrated yet. Our study aims 1) to delineate the mechanism behind tumor immune evasion and 2) to develop therapeutical strategies to overcome such immune evasion, specifically investigating :he therapeutic feasibility of combining chemotherapy and immunotherapy.
II. Research Content
A. Investigation of the mechanism of tumor immune evasion during tumor progression through animal model studies.
1. Establishment of animal models with Rae1 gene transfected cell lines
2. Investigation of changes in immune factors upon tumor progression
B. Development of NK immunotherapy regimen through activation of NKG2D and/or NKG2D ligands(NHG2DL).
1. Development of methods to overcome NKG2D downregluation through in vitro experimentation
2. Investigation of expression level of NKG2DL upon treatment with chemo agents, in order to determine the feasibility of combinatorial therapy
3. Delineation of the molecular mechanism of NKG2DL expression, in order to develop effective strategies of combinatorial chemo/immunotherapy.
III. Research Results
1) Confirmed of changes in the immune system upon tumor progression in animal tumor models
Upon s.c. inoculation of CT26 cancer cells in mice, spleen NK cell number was observed to be significantly decreased.
2) Established a mouse cancer cell lines with expression of NKG2DL, established in vitro.
To investigate the mechanism of interaction between NKG2D and NKG2DL during tumor progression, we obtained the RMA-rae1(rae1 transfected) and RMAS(MHC I null) mouse cancer cell lines.
3) Established animal models inoculated with cell line.
Aforementioned cells were inoculated i.p, in mice, and were collected after growth. No difference in rae1 and MHC I expression level was confirmed.
4) Observed changes in tumor infiltration levels during tumor progression in animal model.
Only RMA-rae1 cells inoculated mice showed tumor infiltration of NK cells. No difference was observed in immune cells isolated from the spleen.
5) Mechanism of TGF-${\beta}1$ overexpression in cancer cells upon immune evasion
To investigate whether TGF-${\beta}1$ overexpression by cancer cells occur actively as a defense mechanism, HNSCC cell lines were cocultured with IL-2 activated NK cells. TGF-${\beta}1$ expression was shown to be increased in cancer cells, which was demonstrated to be an effect by IFN-$\gamma$. Such effect was also shown in TGF-$\alpha$ treatment. Overall, we demonstrated that cancer cells actively overexpress TGF-${\beta}1$ upon interaction with anti-tumor immunity, causing immune suppression. 6) Effect of chemo agent VPA on NKG2DL MICA expression in glioblastoma
MICA expression level was observed to be increased upon treatment with VPA as compared to control cells. This result suggests a strategy to upregulate MICA in cancer cells as a means to induce anti-tumor immunity. Additionally, MICA expression was increased after treatment with NAC, an NF-kB inhibitor, and this increase was shown to be higher when treated with both NAC and VPA.
7) Effect of NF-kB inhibitor on NKG2D ligand expression in multiple myeloma cells
NKG2D ligands induction upon treatment with chemo agents is most likely dependent on NF-kB. In multiple myeloma cells both protein and mRNA expression of NKG2DL MICA/B were induced by NF-kB inhibitor treatment. We thereby concluded that NF-kB suppresses NKG2DL expression.
IV. Application of Research Outcome
1) By determining the mechanism of immune evasion during tumor progression, clinical therapy methodologies using NK cells can be developed.
2) By determining the molecular biology of certain chemo agents that can boost the effect of immunotherapy, specific methods of combinatorial therapy can be developed.

목차 Contents

• II. 총괄연구과제 연구결과...29
• 1. 연구개발과제의 배경 및 필요성...30
• 2. 국내외 기술개발 현황...32
• 3. 연구개발과제의 추진체계...32
• 4. 연구개발수행 내용 및 결과...33
• 1) 동물암모델에서 종양 진행에 따른 면역 시스템 변화 여부 확인...33
• 2) in vitro 모델에서 NK 수용체의 리간드를 형질도입한 마우스 암세포주의 확립...34
• 3) NK 수용체의 리간드를 형질도입한 암세포주를 이용한 동물암모델의 확립...35
• 4) 동물암모델에서 종양의 형성과정에 따른 면역세포인자의 변화...35
• 5) 면역회피기전으로서의 암세포에서의 TGF-${\beta}1$의 과발현 기전...36
• 6) 항암제 VPA가 Glioblastoma에서 NKG2D 리간드인 MICA의 발현에 미치는 영향...38
• 7) multiple myeloma에서 NF-kB inhibitor가 NKG2D 리간드 발현에 미치는 영향...40
• 5. 목표달성도 및 관련분야 기여도...42
• 5.1. 총괄연구개발과제의 목표달성도...42
• 5.2. 관련분야 기여도...42
• 6. 향후 연구성과 추진 계획...42
• 7. 연구개발결과의 파급효과...42
• 7.1. 기술적 파급효과...42
• 8. 연구개발결과의 활용계획...42
• 8.1. 연구개발결과의 활용계획 및 추가연구의 필요성...42
• 9. 연구개발과정에서 수집한 해외과학기술정보...43
• 10. 참고문헌...43
• IV. 첨부서류...44
• 1. 자체평가의견서...45
• 2. 연구성과 실적 증빙서류...48
• 3. 기타 요청서류...48