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放射疗法是胸部肿瘤的治疗方法之一,其通常会引起放射性肺损伤(radiation-induced lung injury,RILI),发生率为5%~20%[1]。RILI是一个复杂的动态过程,涉及多种细胞、途径和机制[2-3]。有研究结果显示,胸部在受到照射后的早期,肺组织会出现明显的免疫失衡现象[4-5]。我们的前期研究结果显示,辐射损伤的肺上皮细胞可能会刺激树突状细胞的抗原呈递功能进而激活T细胞;在树突状细胞不存在的情况下,辐射损伤的肺上皮细胞依旧能活化T细胞[6]。外泌体是具有双层膜结构的细胞外囊泡,其可以携带信息物质参与细胞间通信。Ikhlas等[7]的研究结果显示,受损的非专职性抗原呈递细胞分泌的外泌体可以参与免疫反应。而Liu等[8]的研究结果显示,肺组织中的细胞外囊泡主要来源于Ⅱ型肺上皮细胞。我们通过体外模拟肺部微环境中的细胞间通信,以期发现外泌体参与RILI过程的途径,为RILI的临床研究提供新的实验依据。
60Co γ射线照射的小鼠肺上皮MLE-12细胞分泌的外泌体对T淋巴细胞的活化作用
Exosome-mediated T cell activation by mouse lung epithelial MLE-12 cells irradiated with 60Co γ ray
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摘要:
目的 探讨小鼠肺上皮MLE-12细胞(简称MLE-12细胞)受到60Co γ射线照射后分泌的外泌体介导的T细胞活化。 方法 将MLE-12细胞分为对照组和60Co γ射线照射组(2、4、6和8 Gy),采用超速离心法分别从其培养液的上清液中提取外泌体,应用透射电子显微镜和纳米颗粒跟踪分析技术确定外泌体的形态结构和数量特征,采用蛋白质印迹法(WB)检测外泌体中溶酶体相关膜蛋白3(CD63)、四次跨膜蛋白28(CD81)、肿瘤易感基因101蛋白(TSG101)、Ⅰ型内质网膜蛋白(Calnexin)的表达,采用流式细胞术(FCM)检测外泌体表面Ⅰ类主要组织相容性复合体(MHC Ⅰ)、Ⅱ类主要组织相容性复合体(MHC Ⅱ)、免疫调节蛋白B7-1(CD80)和免疫调节蛋白B7-2(CD86)的表达水平。将从小鼠脾脏中分离出来的初始T细胞分别与对照组MLE-12细胞(简称NC MLE-12)分泌的外泌体(简称exo/NC-MLE)、6 Gy γ射线照射组的MLE-12细胞(简称IR MLE-12)分泌的外泌体(简称exo/IR-MLE)共培养,采用FCM检测T细胞亚群CD3+、CD4+和CD8+及其活化指标T细胞特定表面糖蛋白CD28和早期活化抗原1(CD69)的变化;将初始T细胞分别与NC MLE-12、IR MLE-12和外泌体抑制剂GW4869处理组的MLE-12细胞共培养,采用FCM检测T细胞亚群CD3+、CD4+和CD8+及其活化指标CD28和CD69的变化。2组间比较采用两独立样本t检验,多组间比较采用方差分析法,组间两两比较采用Bonferroni调整法。 结果 MLE-12细胞分泌的外泌体显示出典型的一面凹陷的茶托样结构,粒径为30~150 nm;WB结果显示,与MLE-12细胞相比,其外泌体中特异性标志物CD63、CD81和TSG101高表达,而阴性标志物Calnexin低表达。与对照组相比,在6 Gy γ射线照射后不同时间,单个MLE-12细胞分泌的外泌体数量于24、48 h时均增加(t=5.36、6.66,均P<0.05);在不同剂量γ 射线照射后24 h,单个MLE-12细胞分泌的外泌体数量增加的现象具有剂量-效应关系,在照射剂量为 6、8 Gy 时,差异均有统计学意义(t=4.14、5.67,均P<0.05)。与exo/NC-MLE相比,exo/IR-MLE中MHCⅠ、MHC Ⅱ、CD81和TSG101的表达水平均升高。FCM结果显示,与exo/NC-MLE相比,exo/IR-MLE中MHC Ⅰ、MHC Ⅱ、CD80和CD86表达水平均升高(t=4.04~6.47,均P<0.05)。与exo/NC-MLE相比,在与exo/IR-MLE共培养的T细胞中,CD3+、CD4+和CD8+ T细胞均出现增殖现象(t=3.08~5.88,均P<0.05),CD28和CD69表达水平均升高(t=3.02~8.65,均P<0.05);外泌体抑制剂GW4869可以抑制IR MLE-12所诱导的T细胞活化(t=3.64~23.03,均P<0.05)。 结论 60Co γ射线照射后的MLE-12细胞分泌的外泌体可以通过抗原呈递激活T细胞。 Abstract:Objective To evaluate T cell activation driven by exosomes from mouse lung epithelial MLE-12 cells (MLE-12 cells) irradiated with 60Co γ ray. Methods MLE-12 cells were divided into a control group and a 60Co γ irradiation group (2, 4, 6, and 8 Gy), and exosomes were extracted from the supernatant of their culture medium by using ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscope were used to determine the morphological structure and quantity of exosomes. The expression of lysosomal associated membrane protein (CD63), tetraspanin (CD81), tumor susceptibility gene (TSG101), and type Ⅰ endoplasmic reticulum protein (Calnexin) in exosomes were identified by Western blot (WB). Flow cytometry (FCM) was used to detect the expression of major histocompatibility complex class Ⅰ (MHC Ⅰ), major histocompatibility complex class Ⅱ (MHC Ⅱ), immune regulatory protein B7-1 (CD80), and immune regulatory protein B7-2 (CD86) on the surface of exosomes. Naive T cells isolated from mouse spleens were cocultured with exosomes (exo/NC MLE) secreted by MLE-12 cells in the control group (NC MLE-12) and exosomes (exo/IR MLE) secreted by MLE-12 cells in the 6 Gy 60Co γ irradiation group (IR MLE-12), respectively. FCM was used to detect the changes of T cell subsets CD3+, CD4+, and CD8+ and their activated proliferation indicators T cell specific surface glycoprotein CD28 and early activation antigen 1 (CD69). Naive T cells were incubated with NC MLE-12, IR MLE-12, and MLE-12 cells from exosome inhibitor GW4869-treated groups, respectively. FCM was used to detect the changes of T cell subsets CD3+, CD4+, and CD8+ and their activation indicators CD28 and CD69. Independent samples t-test was used for comparison between two groups. Analysis of variance was used to compare multiple groups. Bonferroni adjustment was applied for pairwise comparison between two groups. Results The exosomes produced from MLE-12 cells showed a typical saucer-like structure, with a particle size of 30–150 nm. WB results showed that the exosomes specific markers CD63, CD81, and TSG101 were highly expressed in exosomes, but the negative marker Calnexin was low in expression, compared with the MLE-12 cells. Compared with the control group, at different times after 6 Gy γ ray irradiation, the number of exosomes secreted by a single MLE-12 cell increased at 24 and 48 hours (t=5.36, 6.66, both P<0.05). The phenomenon of an increase in the number of exosomes secreted by a single MLE-12 cell 24 hours after irradiation with different doses of γ rays has a dose-effect relationship, and the difference is statistically significant at doses of 6 and 8 Gy (t=4.14, 5.67, both P<0.05) after the MLE-12 cells were irradiated with γ ray. The expression levels of MHC Ⅰ, MHC Ⅱ, CD81, and TSG101 increased in exo/IR-MLE compared with exo/NC-MLE. FCM results showed that the expression levels of MHC Ⅰ, MHC Ⅱ, CD80, and CD86 increased in exo/IR-MLE compared with exo/NC-MLE (t=4.04–6.47, all P<0.05). Compared with the exo/NC-MLE, in the T cells cocultured with exo/IR-MLE, the CD3+, CD4+, and CD8+ T cells all proliferated (t=3.08–5.88, all P<0.05), and the expression levels of CD28 and CD69 increased (t=3.02–8.65, all P<0.05). The exosome inhibitor GW4869 can suppress T cell activation induced by IR MLE-12 (t=3.64–23.03, all P<0.05). Conclusion Exosomes from MLE-12 cells irradiated with 60Co γ ray could activate T cells through antigen presentation. -
Key words:
- Radiation /
- Alveolar epithelial cells /
- Exosomes /
- T-lymphocytes /
- Antigen presentation
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[1] Giuranno L, Ient J, De Ruysscher D, et al. Radiation-induced lung injury (RILI)[J/OL]. Front Oncol, 2019, 9: 877[2023-04-02]. https://www.frontiersin.org/articles/10.3389/fonc.2019.00877/full. DOI: 10.3389/fonc.2019.00877. [2] Zhang ZF, Zhou JL, Verma V, et al. Crossed pathways for radiation-induced and immunotherapy-related lung injury[J/OL]. Front Immunol, 2021, 12: 774807[2023-04-02]. https://www.frontiersin.org/articles/10.3389/fimmu.2021.774807/full. DOI: 10.3389/fimmu.2021.774807. [3] Yan YJ, Fu JM, Kowalchuk RO, et al. Exploration of radiation-induced lung injury, from mechanism to treatment: a narrative review[J]. Transl Lung Cancer Res, 2022, 11(2): 307−322. DOI: 10.21037/tlcr-22-108. [4] Guo TT, Zou LQ, Ni JJ, et al. Regulatory T cells: an emerging player in radiation-induced lung injury[J/OL]. Front Immunol, 2020, 11: 1769[2023-04-02]. https://www.frontiersin.org/articles/10.3389/fimmu.2020.01769/full. DOI: 10.3389/fimmu.2020.01769. [5] Cui WC, Zhang P, Hankey KG, et al. AEOL 10150 alleviates radiation-induced innate immune responses in non-human primate lung tissue[J]. Health Phys, 2021, 121(4): 331−344. DOI: 10.1097/hp.0000000000001443. [6] 李倩, 耿爽, 鄢成名, 等. DC细胞在辐射损伤抗原递呈及T细胞活化中的作用[J]. 中国辐射卫生, 2022, 31(6): 657−662, 668. DOI: 10.13491/j.issn.1004-714X.2022.06.003.
Li Q, Geng S, Yan CM, et al. Antigen presentation and T cell activation by dendritic cells in radiation damage[J]. Chin J Radiol Health, 2022, 31(6): 657−662, 668. DOI: 10.13491/j.issn.1004-714X.2022.06.003.[7] Ikhlas S, Usman A, Kim D, et al. Exosomes/microvesicles target SARS-CoV-2 via innate and RNA-induced immunity with PIWI-piRNA system[J/OL]. Life Sci Alliance, 2022, 5(3): e202101240[2023-04-02]. https://www.life-science-alliance.org/content/5/3/e202101240. DOI: 10.26508/lsa.202101240. [8] Liu BW, Jin Y, Yang JY, et al. Extracellular vesicles from lung tissue drive bone marrow neutrophil recruitment in inflammation[J/OL]. J Extracell Vesicles, 2022, 11(5): e12223[2023-04-02]. https://onlinelibrary.wiley.com/doi/10.1002/jev2.12223. DOI: 10.1002/jev2.12223. [9] Théry C, Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines[J/OL]. J Extracell Vesicles, 2018, 7(1): 1535750[2023-04-02]. https://www.tandfonline.com/doi/full/10.1080/20013078.2018.1535750. DOI: 10.1080/20013078.2018.1535750. [10] Hill GR, Koyama M. Cytokines and costimulation in acute graft-versus-host disease[J]. Blood, 2020, 136(4): 418−428. DOI: 10.1182/blood.2019000952. [11] Reddy M, Eirikis E, Davis C, et al. Comparative analysis of lymphocyte activation marker expression and cytokine secretion profile in stimulated human peripheral blood mononuclear cell cultures: an in vitro model to monitor cellular immune function[J]. J Immunol Methods, 2004, 293(1/2): 127−142. DOI: 10.1016/j.jim.2004.07.006. [12] Chattopadhyay PK, Roederer M. Good cell, bad cell: flow cytometry reveals T-cell subsets important in HIV disease[J]. Cytometry A, 2010, 77A(7): 614−622. DOI: 10.1002/cyto.a.20905. [13] Berg J, Halvorsen AR, Bengtson MB, et al. Circulating T cell activation and exhaustion markers are associated with radiation pneumonitis and poor survival in non-small-cell lung cancer[J/OL]. Front Immunol, 2022, 13: 875152[2023-04-02]. https://www.frontiersin.org/articles/10.3389/fimmu.2022.875152/full. DOI: 10.3389/fimmu.2022.875152. [14] 郗停停, 耿爽, 孙泽文, 等. γ射线胸部照射小鼠早期肺组织的免疫细胞反应[J]. 国际放射医学核医学杂志, 2020, 44(5): 286−290. DOI: 10.3760/cma.j.cn121381-202003038-00025.
Xi TT, Geng S, Sun ZW, et al. Early response of immune-related T cells in the lung tissue of mice exposed to gamma rays in the chest[J]. Int J Radiat Med Nucl Med, 2020, 44(5): 286−290. DOI: 10.3760/cma.j.cn121381-202003038-00025.[15] Kulshreshtha A, Ahmad T, Agrawal A, et al. Proinflammatory role of epithelial cell-derived exosomes in allergic airway inflammation[J]. J Allergy Clin Immunol, 2013, 131(4): 1194−1203. DOI: 10.1016/j.jaci.2012.12.1565. [16] Admyre C, Grunewald J, Thyberg J, et al. Exosomes with major histocompatibility complex class Ⅱ and co-stimulatory molecules are present in human BAL fluid[J]. Eur Respir J, 2003, 22(4): 578−583. DOI: 10.1183/09031936.03.00041703. [17] Shahbaz S, Okoye I, Blevins G, et al. Elevated ATP via enhanced miRNA-30b, 30c, and 30e downregulates the expression of CD73 in CD8+ T cells of HIV-infected individuals[J]. PLoS Pathog, 2022, 18(3): e1010378. DOI: 10.1371/journal.ppat.1010378. [18] Xu YY, Liu ZC, Lv LX, et al. MiRNA-340-5p mediates the functional and infiltrative promotion of tumor-infiltrating CD8+ T lymphocytes in human diffuse large B cell lymphoma[J]. J Exp Clin Cancer Res, 2020, 39(1): 238. DOI: 10.1186/s13046-020-01752-2.