초록 ▼

Regulatory T 세포를 활용한 효율적인 면역치료전략의 수립을 위하여는 regulatory T 세포의 면역억제 기전에 대한 근본적인 이해가 필요한데 세포사이의 복잡한 상호작용을 조절할 수 없는 현행 in vitro 배양방법으로는 그 연구에 한계가 명확함. 본 연구에서는 이러한 기존연구의 한계점을 극복하고 1) 세포간의 복잡하고 다양한 상호작용을 제어할 수 있으며 2) 염증반응 관련 사이토카인의 시공간적인 구배를 조절할 수 있으며 3) 단일세포수준에서 세포간의 상호작용을 분석할 수 있는 새로운 도구를 미세가공 및 미세유체 기술을

Regulatory T 세포를 활용한 효율적인 면역치료전략의 수립을 위하여는 regulatory T 세포의 면역억제 기전에 대한 근본적인 이해가 필요한데 세포사이의 복잡한 상호작용을 조절할 수 없는 현행 in vitro 배양방법으로는 그 연구에 한계가 명확함. 본 연구에서는 이러한 기존연구의 한계점을 극복하고 1) 세포간의 복잡하고 다양한 상호작용을 제어할 수 있으며 2) 염증반응 관련 사이토카인의 시공간적인 구배를 조절할 수 있으며 3) 단일세포수준에서 세포간의 상호작용을 분석할 수 있는 새로운 도구를 미세가공 및 미세유체 기술을 활용하여 개발하였음. 개발한 미세가공/미세유체 기반의 도구를 활용하여 regulatory T 세포와 effector T 세포의 상호작용 및 이로 인한 증식, 분화, 사멸 등을 두 종류의 다른 세포의 공간적 배치의 함수로 분석할 수 있었으며, 이를 통하여 regulatory T 세포의 면역억제 관련 다양한 가설들을 비교검증할수 있었음.

Abstract ▼

Ⅰ. Objectives of the research
The objective of this research is to develop novel in vitro model systems, which enables systematic and quantitative study of regulatory T cell-effector T cell interactions, and to study the immunosuppressive mechanisms of regulatory T cells in single cell levels usi

Ⅰ. Objectives of the research
The objective of this research is to develop novel in vitro model systems, which enables systematic and quantitative study of regulatory T cell-effector T cell interactions, and to study the immunosuppressive mechanisms of regulatory T cells in single cell levels using the developed systems.

Ⅱ. Scope of the research
Regulatory T cells play a key role in modulating the strength of immune responses by suppressing the activation of T cells and downregulating effector functions of activated T cells. Therefore, it is expected that many immune-related diseases such as autoimmue diseases and asthma can be treated by harnessing regulatory T cells. For the establishment of efficient therapeutic stratigies, we first need to better understand the mechanism of immunosuppression by regulatory T cells, but current in vitro culture systems have many limitations due to lack of controls of complex cell-cell communication. To overcome these limitations, we first developed new in vitro model systems which enables 1) precise control of complex and diverse cell-cell communication 2) control over spatio-temporal distribution of inflammation-related cytokines, and 3) analysis of the outcome of cell-cell communication in single cell level. Then, we utilized this new in vitro model system for the study of immunosupressive mechanism of regulatory T cells.

Ⅲ. Results
Firstly, we developed new cell culture system based on microfabrication and microfluidics. A new system that can precisely control the size of multicellular cluster sizes of T cells, thus enable the modulation of contact-mediated cell-cell interactions and the size of multicellular clusters, was successfully developed. Using this system, we demonstrated that proliferation and apoptosis can be differentially regulated depending on group-decision behavior of T cells. Furthermore, we developed a tool that can quantitatively assess the amount of cytokines secreted by a single T cell so that we can figure out the role of directional-secretion of cytokines and their diffusion on T cell fate decision. Antibodies capturing the secreted cytokines were attached along with anti-CD44, and using multiple-fluorescent-labeled antibodies, we could amply signals from cytokines to have great sensitivity.
In order to mimic complex cytokine gradient formed around the imflammation sites along with lymphatic flow, we integrated microfluidics with microwell systems. Utilizing the fact that material transfer predominantly occurs by diffusion in microfluidic systems, we could successfully develop microfluidic system where spatial distribution of cytokines can be stably controlled. By using microscope projection photolithography, microwells could be successfully fabricated inside microfluidic channels.
In order to use these novel microfabrication/microfluidics based systems for the study of immunosuppressive mechanisms of regulatory T cells, we established protocols for nTreg isolation/expansion and iTreg differentiation to obatin sufficient numbers of T cells for the experiments. Also, T cells from FoxP3 knock-in transgenic mice were used for in situ monitoring of regulatory T cell phenotype change by many environmental factors.
In this way, we could study the immunosuppressive functions of re gulatory T cells more systemically than conventional in vitro co-culture system. We demonstrated that contact-mediated interactions, spatial distributions of cells, and imflammatory cytokines play very important role on the immunosuppressive capability of regulatory T cells.

Ⅳ. Plans for application of the results
The results of this research may provide new insights into the stratigy for the therapeutics against various immune-related diseases. Also, in vitro model system developed in this study can be used for the study of cell-cell communication study as well as tools for drug screening.

목차 Contents

• 겉표지 (앞면) ...1
• 제출문 ...3
• 보고서 요약서 ...4
• 요약문 (한글) ...5
• 요약문 (영문) ...7
• 연구 성과 실적 및 향후 계획 ...9
• 참여연구원 현황표 ...13
• II. 총괄연구과제 연구결과 ...14
• 1. 연구개발과제의 배경 및 필요성 ...15
• 2. 국내외 기술개발 현황 ...18
• 3. 연구개발과제의 추진체계 ...19
• 4. 연구개발수행 내용 및 결과 ...21
• 5. 목표달성도 및 관련분야 기여도 ...36
• 6. 향후 연구성과 추진 계획 ...37
• 7. 연구개발결과의 파급효과 ...37
• 8. 연구개발결과의 활용계획 ...38
• 9. 연구개발과정에서 수집한 해외과학기술정보 ...38
• 10. 참고문헌 ...39
• III. 첨부서류 ...41
• ○. 실적증빙자료 및 첨부서류 목차 ...42
• 1. 자체평가의견서 ...43
• 2. 실적 증빙자료 ...47
• 3. 기타 첨부서류 ...52