[보고서]CD137-CD137L 양방신호 네트워크에 의한 종양 면역관용조절

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[국가R&D연구보고서] CD137-CD137L 양방신호 네트워크에 의한 종양 면역관용조절
Regulation of tumor immunological tolerance via the CD137-CD137L bi-directional signaling network 원문보기

보고서 정보
주관연구기관	울산대학교 University of Ulsan
연구책임자	권병석
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2017-05
과제시작연도	2016
주관부처	미래창조과학부 Ministry of Science, ICT and Future Planning
연구관리전문기관	한국연구재단 National Research Foundation of Korea
등록번호	TRKO201900027114
과제고유번호	1711036944
사업명	개인연구지원
DB 구축일자	2020-09-19
키워드	CD13L 역신호.수지상세포.대식세포.종양 관용.면역감시.종양미세포환경.면역치료.항체치료.T 세포.CD13L reverse signal.dendritic cell.macrophage.tumor tolerance.immune surveillance.tumor microenvironment.immunotherapy.antibody therapy.T cell.

초록 ▼

□ 연구의 목적 및 내용
CD137 KO 생쥐와 항-CD137 항체를 주사한 생쥐에서 공통적으로 항암반응이 증가함.
본 과제에서는 이 두 현상에 관여하는 공통 항암기작을 규명하고자 하였음.

□ 연구결과
1. CD137 KO 생쥐와 항-CD137 항체를 주사한 생쥐의 종양에서 공히 IFN-γ를 생산하는 CD8+ T 세포가 증가함.
2. CD137 KO 생쥐와 항-CD137 항체를 주사한 생쥐의 종양에서 공히 IL-12를 생산하는 CD103+ DC와 M1 대식세포가 증가함.
3. 종양에서 IL-12는 IFN-γ를 생산하는 CD8+ T 세포의 생성에 필수적임.
4. 종양에서 IFN-γ는 CD103+ DC와 M1 대식세포의 생성에 필수적임.
5. Treg 세포의 CD137은 종양에서 M2 대식세포의 분화에 필수적임.
6. CD137L 신호는 IFN-γ에 의한 CD103+ DC 분화를 억제함.
7. CD137L 신호는 GM-CSF 존재 하에서 M2 대식세포의 분화를 촉진함.
이상의 결과를 요약하면 CD137L 신호는 종양에서 CD11b+ DC와 M2 대식세포의 분화를 촉진하여 종양의 성장을 촉진하는 역할을 함. 그러므로 CD137L 신호를 차단되면 CD103+ DC와 M1 대식세포와 IFN-γ를 분비하는 CD8+ T 세포가 서로 분화를 촉진하는 면역감시 주기가 고착됨.

□ 연구결과의 활용계획
1. CD137L 표적을 대상으로 항암제 개발에 대한 이론적 근거 마련.
2. 종양의 미세환경에서 골수성 세포의 분화를 조절하는 인자가 항암제의 치료표적이 될 수 있는 이론적 근거 제공.
3. 위의 원리는 이용한 항암제는 다른 원리에 근거한 항암제와 병용요법으로 사용할 수 있다는 근거 제공

(출처 : 연구결과 요약문 4p)

Abstract ▼

□ Purpose& contents
CD137-/- and anti-CD137-injected mice commonly higher anticancer immunity. In this project, we identified the role of CD137L reverse signaling in tumor growth.

□ Result
1. There were increased percentages of IFN-γ-producing CD8+ T cells within tumors of CD137-/- and anti-CD137-injected mice.
2. There were increased percentages of IL-12-producing DCs and M1 macrophages within tumors of CD137-/- and anti-CD137-injected mice.
3. IL-12 was required for the generation IFN-γ-producing CD8+ T cells within tumors.
4. IFN-γ was required for the generation of IL-12-producing CD103+ DC and M1 macrophages within tumors.
5. CD137 expression on Treg cells was required for the differentiation of M2 macrophages within tumors.
6. CD137L signaling inhibited the differentiation of CD103+ DCs induced by IFN-γ.
7. CD137L signaling promoted the differentiation of M2 macrophages in the presence of GM-CSF.
Taken together, these results suggest that CD137L signaling is a differentiation factor of intratumoral CD11b+ DC and M2 macrophages, subsequently promoting tumor growth. Thus, blockade of CD137L signaling results in perpetuation of immune surveillance by driving mutual differentiation between IL-12-producing CD103+ DCs and M1 macrophages and IFN-γ-producing CD8+ T cells in tumors.

□ Expected Contribution
1. CD137L is a good target for anticancer drug.
2. Regulation of intratumoral differentiation of myeloid cells can be used for cancer therapy.
3. Blockers of CD137L signaling can be used for combination therapy with other cancer drugs.

(출처 : SUMMARY 5p)

목차 Contents

표지 ... 1
목차 ... 2
연구계획 요약문 ... 3
연구결과 요약문 ... 4
한글요약문 ... 4
SUMMARY ... 5
연구내용 및 결과 ... 6
1. 연구개발과제의 개요 ... 6
2. 국내외 기술개발 현황 ... 9
3. 연구수행 내용 및 결과 ... 11
4. 목표달성도 및 관련분야에의 기여도 ... 17
5. 연구결과의 활용계획 ... 18
6. 연구과정에서 수집한 해외 과학기술정보 ... 18
7. 주관연구책임자 대표적 연구실적 ... 19
8. 참고문헌 ... 19
9. 연구성과 ... 21
10. 국가과학기술지식정보서비스에 등록한 연구시설‧장비 현황 ... 23
11. 연구개발과제 수행에 따른 연구실 등의 안전조치 이행실적 ... 23
12. 기타사항 ... 23
[별첨1] 대 표 연 구 실 적 ... 24
[별첨2] 세부 목표 관련 증빙 ... 34
끝페이지 ... 34

표/그림 (16)

표 종양-면역 사이클에서 암면역치료법의 표적인 자극 및 억제 인자 (출처: Immunity 2013)
표 종양 미세환경에서 국지적인 면역억제 기전에 대한 모식도 (출처: Immunity 2013)
표 CD137/CD137L 신호의 기능을 보여주는 모식도 (출처: Immune Netw 2012)
표 허혈-재관류에 의한 신손상 발병축을 보여주는 모식도 (출처: PNAS 2012; J Immol 2013)
표 이상적인 항암치료법 표적을 나타낸 모식도 (출처: Nature 2013)
표 3상 임상시험 중인 암면역치료법의 건수 (출처: Nature 2013)
표 입양세포전달 치료법의 과정을 나타내는 모식도 (출처: Nature 2013)
표 CD137-/-and anti-CD137-injected mice show systemic antitumor immunity CD8+ T cells. Mice were inoculated with CT26 cells on day 0 and anti-CD137 antibody was injected on day 5 or 6. Anti-CD8 antibody was injected for CD8+T-cell depletion every 5 day starting from day 0. Mice were sacrificed for analysis on day 14 or 16. A and D. Tumor growth curves (n = 56/group). Tumor volumes are shown as mean ± SEM. ***P < 0.001 between the two groups. The result is representative of at least independent experiments. B and E. Percentages of activated splenic CD4+ and CD8+ T cell (n = 56/group). Data are presented as mean ± SEM. *P < 0.05 **P < 0.01 between the two groups. C and F. CTL assays. Purified splenic CD8+ T cells were incubated with gp70 peptide-pulsed CT26 cells for 5 h and CD107a-expressing CD8+ T cells were counted using FACS. *P < 0.05 ***P < 0.001 between the groups. G. Kinetics of tumor growth mice that received anti-CD8 or control antibody (n = 56/group). ***P < 0.001 between the indicated groups
표 Increase in intratumoral CD103+DCsandM1-likeTAMsinCD137-/-andanti-CD137-injectedmice. Tumors were analyzed on day 9, 10 or 16. A and E. FACS dot plots for intratumoral CD103+ DCs and CD11b+ DCs isolated on day 9 or 10 (left panel) and the ratio of CD103+DCs to CD11b+ DCs (right panel). Tumors were pooled from 4 mice/group. CD103+ DCs and CD11b+ DCs were gated from CD45+ MHCIIhiF4/80-CD11c+ cells. B and F. FACS dot plots for intracellular staining of IL-12 in CD45+F4/80-CD11c+ DCs on day 9 or 10. Data are representative of independent experiments (n =4/group). C and G. FACS analysis TAMs isolated on day 16. CD206+/- macrophages were gated from CD45+Gr-1-CD11b+F4/80+ cells (left panel). The ratio of CD206- TAMs to CD206+ TAMs (right panel). ** P < 0.01 *** P < 0.001. D and H. qRT-PCR was performed using total RNA extracted from purified F4/80+TAMs(n=4/group)onday16.*P<0.05 ** P < 0.01 *** P < 0.001. Data are representative of independent experiments
표 Increase in intratumoral Tc1 cells in CD137-/-and anti-CD137-injected mice. Tumors were analyzed on day 14 or 16. A and D. Intratumoral leukocytes were isolated, stimulated in vitro with PMA and ionomycin, and stained for intracellular IFN-g. FACS dot plots for intracellular IFN-g staining of CD3+CD8+ T cells (left panel) and percentages of IFN-g+ cells in CD3+CD8+ T cells (right panel) (n = 4/group). **P < 0.01. B and E. FACS dot plots for activated intratumoral CD62LloCD8+ T cells (left panel) and percentages of CD62Llo cells in CD8+ T cells (right panel). (n = 4/group). *P < 0.05 **P < 0.01. C and F. Levels of IFN-g in tumor lysates (n = 4/group). *P < 0.05 ***P < 0.001
표 IL-12 is responsible for changes the tumor microenvironment induced by CD137 gene deletion or anti-CD137 antibody. Mice were injected with control IgG or antiIL-12 antibody on days 0, 5, and 10. Tumors were analyzed on day 9, 10, or 16.A and E. Percentages of IFN-g+ cells CD3+CD8+ T cells on day16.Tumors were pooled from 4 mice (WT and control Ig-treated CD137-/-mice) or individually analyzed for anti-IL-12-injected CD137-/- mice (n=4). Data are representative of experiments. B and F. Tumor growth curves (n = 46/group). *P < 0.05 **P < 0.01. C and G. The ratio of intratumoral CD103+ DCs to CD11b+ DCs on day 9 or 10. Data were pooled from experiments (n = 4/group/experiment). *P < 0.05 **P < 0.01. D and H. The ratio of CD206- TAMs to CD206+ TAMs on day16. **P<0.01 ***P < 0.001 (n = 4/group). Data are representative of independent experiments
표 IFN-g drives the differentiation of intratumoral CD103+ DC and M1-like TAMs in CD137-/- and anti-CD137-injected mice. Mice were injected with control IgG or anti-IFN-g antibody on days 0, 5, and 10. Tumors were analyzed on day 9, 10, or 16. A and E. Percentages of IFN-g+ cells CD3+CD8+ T cells on day 16.Tumors werepooled from 4 mice (WT and control Ig-treated CD137-/- mice) or individually analyzed for anti-IL-12-injected CD137-/- mice (n=4). Data are pooled from experiments. B and F. The ratio of intratumoral CD103+ DCs to CD11b+ DCs on day 9 or 10. Tumors were pooled from 4 mice/group or individually analyzed. Data are representative of experiments. C and G. The ratio of CD206- TAMs to CD206+ TAMs on day16. *P<0.05 **P < 0.01 (n = 4/group). D and H. Tumor masses at day 10 or day 13 (n = 46/group). *P < 0.05 ***P < 0.001. Data are representative of two independent experiments
표 CD137 expression on Treg cells is required for inhibition of intratumoral M1-like TAMs and tumor growth. Tumors were analyzed on day 14. A. FACS analysis CD137 expression in intratumoral leukocytes. CD45+ leukocytes were gated and CD137 expression was analyzed in CD11b+Gr-1+/- myeloid cells and CD11bcells. B. CD137 expression on T cells. CD137 expression was analyzed CD4+ Foxp3+/- and CD4- T cells. C. CD137L expression on intratumoral leukocytes. D and E. CD4+CD25+ Treg cells of WT mice were isolated by FACSAria and adoptively transferred to WT and CD137-/- mice 1 day before tumor cell inoculation. As negative control, CD137-/- mice received CD137-/- Treg cells. D. The ratio of CD206- TAMs to CD206+ TAMs on day 16. **P < 0.01 (n = 4/group). E. Tumor growth curves (n = 4-6/group). **P < 0.01. Data are representative of independent experiments
표 CD137L signaling inhibits in vitro differentiation of CD103+ DCs and M1 macrophages. A. Bone marrow cells of WT or CD137-/- mice were cultured in human IgG1 or CD137-Fcin the presence of murine Flt3L for 9 days. IFN-g and GM-CSF were added and cells were cultured for 2 days to polarize differentiated DCs toward CD103+ subsets. CD103+ DCs (middle panel) were gated from CD11c+F4/80- cells (upper panel). Macrophages are F4/80+CD11b+CD11c+ or F4/80+CD11b+CD11c- (upper and lower panels). B-F. MDSCs were isolated from the spleen of CT26 tumor-bearing mice on day 14. Cells were cultured in the presence of GM-CSF coated CD137-Fc or human IgG1 for 25 days. B. FACS dot plots for splenic MDSCs before and after purification. C-E. FACS dot plots for neutrophils and macrophages (C) and percentages of neutrophils and macrophage (D) or CD206+macrophages (E) after 4day culture. F. qRT-PCR was performed using RNA purified from cells cultured for 35 days. G and H. Percentages of neutrophils in tumor-infiltrating leukocytes (TIL). Data are presented as mean ± SEM. *P < 0.05 *** P < 0.001. Data are representative of two independent experiments
표 CD137L 신호차단에 의한 면역감시 기능을 증가하는 사실을 보여주는 모식도
표 염증과 종양에서 CD137L 신호의 역할을 보여주는 모식도

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[국가R&D연구보고서] CD137-CD137L 양방신호 네트워크에 의한 종양 면역관용조절
Regulation of tumor immunological tolerance via the CD137-CD137L bi-directional signaling network 원문보기