具有人免疫系统和异体人黑色素瘤生长的人源化小鼠模型的构建和鉴定

doi:10.13481/j.1671-587x.20190344

摘要/Abstract

摘要： 目的：构建同时具有人免疫系统和人肿瘤生长的人源化小鼠模型，为人肿瘤免疫研究和临床肿瘤免疫药物药效评估提供依据。方法：通过给予重度免疫缺陷NOD/SCID IL2rg-/-小鼠亚致死剂量辐照，联合小鼠肾被膜下移植人胚胎胸腺组织和静脉输注CD34⁺胎肝细胞，构建具有人免疫系统重建的人源化小鼠。构建人源化小鼠模型后3周开始，每3周采集外周血监测人免疫细胞重建情况，包括CD45⁺、CD33⁺、CD3⁺、CD3⁺CD4⁺、CD3⁺CD8⁺、CD56⁺细胞比例。构建人源化小鼠模型后，第15周给予小鼠皮下接种人A375黑色素瘤细胞，每5 d测量肿瘤体积。接种6周后处死小鼠，流式细胞术检测人源化小鼠外周血、骨髓、淋巴结、脾脏及肿瘤组织中人免疫细胞的组成和表型。免疫组织化学法检测人源化小鼠肿瘤组织中人免疫细胞的组成和表型。结果：在人源化15周时，人源化小鼠模型外周血中人T细胞比例为（33.53±8.57）%、B细胞比例为（43.33±10.05）%。在A375细胞接种后前3周，人黑色素瘤生长较慢，其后加速生长，6周时肿瘤体积达（1 564±334.3） mm³。免疫组织化学和流式细胞术检测，人黑色素瘤内有大量以CD8⁺ T细胞为主的人T细胞浸润；肿瘤组织中CD8⁺/CD4⁺比值明显高于外周血、骨髓、淋巴结和脾脏（P<0.01）。PD-1分子在肿瘤浸润人CD4⁺ T和CD8⁺ T细胞中的表达水平明显高于其在外周血和免疫器官中的水平。结论：成功建立了具有人类免疫系统和异体人黑色素瘤生长的人源化小鼠模型，并在其外周血、淋巴器官以及人肿瘤组织中均检测到包括人T细胞和人B细胞在内的高水平人CD45⁺免疫细胞。

关键词: 人源化小鼠, 黑色素瘤, 肿瘤微环境, 肿瘤免疫治疗

Abstract: Objective:To construct a humanized mouse models with human immune system and human tumor growth, and to provide the basis for human tumor immunological research and evaluation on the efficacy of tumor immunotherapeutic durgs. Methods:The humanized mice with human immune system reconstruction were established by intravenous injection of human CD34⁺ fetal liver cells combined with implantation of human fetal thymic tissue under the renal capsule of NOD/SCID IL 2rg-/- after sub-lethal total body irradiation. The peripheral blood was collected,and the immune cell reconstitution was monitored every 3 weeks from the 3th week after humanization, including the ratios of CD45⁺, CD33⁺, CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺,and CD56⁺ cells. The human A375 melanoma cells were subcutaneously inoculated at the 15th week after humanization,and the tumor volume was measured every 5 d.. Six weeks after tumor inoculation, the mice were sacrificed and the composition of human immune cells as well as their phenotypes in peripheral blood, bone marrow, lymph node,spleen and tumor tissue were measured by flow cytometry. The composition of human immune cells as well as their phenotypes in tumor tissue were measured by immunohistochemistry. Results:At the 15th week after humanization, the ratio of human T cells was (33.53±8.57)% and the ratio of human B cells was (43.33±10.05)% in the peripheral blood of the humanized mice. The human melanoma grew slowly during the first 3 weeks, and the human tumor sizes expanded rapidly and reached (1 564±334.3) mm³ at the 6th week after tumor inoculation. The immunohistochemical and flow cytometry analysis results showed that a large number of human T cells, which mainly composed by CD8⁺ T subsets, infiltrated in the human melanoma. The ratio of CD8⁺/CD4⁺ in tumor tissue was significantly higher than those in the peripheral blood, bone marrows, lymph nodes and spleen tissue (P<0.01). The levels of PD-1 molecules expressed in tumor-infiltrated human CD4⁺ T and CD8⁺ T cells were higher than those in the peripheral blood and immune organs. Conclusion:A humanized mouse models with human immune system and allogeneic human melanoma is successfully established. The high levels of human immune cells, including human T cells and B cells, can be detected in the peripheral blood, lymph organs, and human tumor tissue in this mouse model.

Key words: humanized mice, melanoma, tumor microenvironment, cancer immunotherapy

中图分类号:

R739.5

邹俊, 陈冰, 方铭慧, 胡正. 具有人免疫系统和异体人黑色素瘤生长的人源化小鼠模型的构建和鉴定[J]. 吉林大学学报(医学版), 2019, 45(03): 718-724.

ZOU Jun, CHEN Bing, FANG Minghui, HU Zheng. Establishment and identification of humanized mouse models with human immune system and allogeneic human melanoma growth[J]. Journal of Jilin University(Medicine Edition), 2019, 45(03): 718-724.

参考文献

[1] BRAY F, FERLAY J, SOERJOMATARAM I, et al. Global cancer statistics 2018:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J].CA Cancer J Clin, 2018, 68(6):394-424.
[2] LAN P, TONOMURA N, SHIMIZU A, et al. Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34⁺ cell transplantation[J]. Blood, 2006, 108(2):487-492.
[3] SHULTZ L D, BREHM M A, GARCIA-MARTINEZ J V,et al. Humanized mice for immune system investigation:progress, promise and challenges[J]. Nat Rev Immunol, 2012, 12(11):786-798.
[4] COVASSIN L, JANGALWE S, JOUVET N, et al. Human immune system development and survival of non-obese diabetic (NOD)-scid IL2γ(null) (NSG) mice engrafted with human thymus and autologous haematopoietic stem cells[J]. Clin Exp Immunol, 2013, 174(3):372-388.
[5] DE LA ROCHERE P, GUIL-LUNA S, DECAUDIN D, et al. Humanized mice for the study of immuno-oncology[J]. Trends Immunol, 2018, 39(9):748-763.
[6] HU Z, XIA J X, FAN W, et al. Human melanoma immunotherapy using tumor antigen-specific T cells generated in humanized mice[J]. Oncotarget, 2016, 7(6):6448-6459.
[7] HU Z, VAN ROOIJEN N, YANG Y G. Macrophages prevent human red blood cell reconstitution in immunodeficient mice[J]. Blood, 2011, 118(22):5938-5946.
[8] HU Z and YANG Y G. Full reconstitution of human platelets in humanized mice after macrophage depletion[J]. Blood, 2012, 120(8):1713-1716.
[9] SAMAL J, KELLY S, NA-SHATAL A, et al. Human immunodeficiency virus infection induces lymphoid fibrosis in the BM-liver-thymus-spleen humanized mouse model[J]. JCI Insight, 2018, 3(18):e120430.
[10] BUROVA E, HERMANN A, WAITE J, et al. Characterization of the anti-PD-1 antibody REGN2810 and its antitumor activity in human PD-1 knock-in mice[J]. Mol Cancer Ther, 2017, 16(5):861-870.
[11] WANG M, YAO L C, CHENG M, et al. Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy[J]. FASEB J, 2018, 32(3):1537-1549.
[12] ROSATO R R, DAVILA-GONZALEZ D, CHOI D S, et al. Evaluation of anti-PD-1-based therapy against triple-negative breast cancer patient-derived xenograft tumors engrafted in humanized mouse models[J]. Breast Cancer Res, 2018, 20(1):7-16.
[13] MOSIER D E, GULIZIA R J, BAIRD S M, et al. Transfer of a functional human immune system to mice with severe combined immunodeficiency[J]. Nature, 1988, 335(6187):256-259.
[14] KING M A, COVASSIN L, BREHM M A, et al. Human peripheral blood leukocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex[J]. Clin Exp Immunol, 2009, 157(1):104-118.
[15] TRAGGIAI E, CHICHA L, MAZZUCCHELLI L, et al. Development of a human adaptive immune system in cord blood cell-transplanted mice[J]. Science, 2004, 304(5667):104-107.
[16] SHULTZ L D, LYONS B L, BURZENSKI L M, et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells[J]. J Immunol, 2005, 174(10):6477-6489.
[17] WATANABE Y, TAKAHASHI T, OKAJIMA A, et al. The analysis of the functions of human B and T cells in humanized NOD/shi-scid/gammac(null) (NOG) mice (hu-HSC NOG mice)[J]. Int Immunol, 2009, 21(7):843-858.
[18] LI H J, VAN DER LEUN A M, YOFE I, et al. Dysfunctional CD8 T cells form a proliferative, dynamically regulated compartment within human melanoma[J]. Cell, 2019, 176(4):775-789.
[19] XIA J X, HU Z, YOSHIHARA S, et al. Modeling human leukemia immunotherapy in humanized mice[J]. EBio Med, 2016, 10(1):101-108.
[20] JIN C H, XIA J X, RAFIQ S, et al. Modeling anti-CD19 CAR T cell therapy in humanized mice with human immunity and autologous leukemia[J]. EBio Medicine, 2019, 39:173-181.