流感病毒感染人扁桃体类器官的免疫效应  

Immunological effects of human tonsil organoids after influenza virus infection

作　　者：李思琪 徐赟 李红 费蕾[1] 黄小勇[1] 陈永文[1] LI Siqi;XU Yun;LI Hong;FEI Lei;HUANG Xiaoyong;CHEN Yongwen(Institute of Immunology of PLA,College of Basic Medical Sciences,Army Medical University(Third Military Medical University),Chongqing,400038;Department of Pharynx and Larynx,Chongqing Renpin Ear Nose Throat Hospital,Chongqing,400042,China)

机构地区：[1]陆军军医大学(第三军医大学)全军免疫学研究所,重庆400038 [2]重庆仁品耳鼻喉医院咽喉科,重庆400042

出　　处：《陆军军医大学学报》2024年第9期978-987,共10页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(82371735);国家自然科学基金青年科学基金(82001693);国家重点研发计划(2023YFC2308604)。

摘　　要：目的 建立人扁桃体免疫类器官模型并探讨流感病毒感染后产生的免疫学效应。方法 经扁桃体腺样体肥大手术摘除后的人扁桃体组织在经过单个核细胞梯度离心后,利用2.5D-Transwell进行体外培养并添加IL-2及Baff等成细胞因子促扁桃体类器官(tonsil organoids, TO)的形成。通过单细胞转录组测序(single cell RNA sequencing, scRNA-seq)技术,分析流感病毒细胞受体在类器官各细胞亚群的分布;体外感染流感病毒,使用免疫荧光法(IF)和流式细胞术(FCM)检测扁桃体类器官感染病毒后的免疫学效应。结果 于体外成功诱导扁桃体类器官;scRNA-seq分析结果显示流感病毒细胞受体广泛表达于所有的细胞类型中。流感病毒感染48 h后,刺激扁桃体类器官产生大量可分泌特异性IgG抗体的浆细胞和记忆性B细胞。病毒直接感染还促进CD8^(+)T细胞分泌细胞因子TNF-α与IL-2及CD4^(+)T细胞分泌细胞因子TNF-α、IFN-γ和IL-2。结论 人扁桃体类器官在体外可以被成功诱导且在培养第6天左右达到成熟;流感病毒受体广泛分布于人扁桃体类器官的各细胞亚型;流感病毒可通过受体直接感染T细胞,刺激T细胞活化并分泌细胞因子;同时流感病毒直接感染也可促B细胞分化为浆细胞分泌IgG抗体,并在后期诱导记忆性B细胞的形成。Objective To establish a human tonsil organoid model and investigate the immunological effects of this tonsil organoid infected by influenza virus.Methods Human tonsil tissue removed by adenoid hypertrophy surgery were subjected to gradient centrifugation,and then the obtain cells were cultured in vitro with using a 2.5D-Transwell chamber in combination with IL-2 and B cell activating factor(Baff)to construct a human tonsil organoid.Single cell transcriptome sequencing(scRNA-seq)was used to analyze the distribution of influenza virus receptors in different cell subsets of the tonsil organoid.Immunofluorescence assay and flow cytometry were applied to detect the immunological effects of the tonsil organoid in 48 h after influenza virus infection.Results The tonsil organoids were established successfully using this 2.5D-Transwell culture technique.scRNA-seq analysis showed that the receptors for influenza virus were extensively distributed in nearly all of cell subsets of tonsil organoids.In 48 h after influenza virus infection,the tonsil organoids could generate a large amount of plasma cells and memory B cells to produce specific IgG antibodies.In addition,direct infection of the virus promoted the production of TNF-αand IL-2 from CD8^(+)T cells and the secretion of TNF-α,IFN-γand IL-2 by CD4^(+)T cells.Conclusion The tonsil organoids can be established successfully and reach maturity at about the 6th day after culture.The receptors for influenza virus are widely distributed in human tonsil organoids.Influenza virus directly infects human T cells,leading to T cell activation and cytokine releasing.Moreover,influenza virus infection also promotes B cells differentiate into plasma cells and induce the secretion of IgG as well as the formation of memory B cells.

关 键 词：扁桃体类器官 流感病毒 免疫记忆 炎症因子 浆细胞 

分 类 号：R373.13[医药卫生—病原生物学] R392.11[医药卫生—基础医学] R392.32