Measuring interaction force between T lymphocytes and their target cells using live microscopy and laminar shear flow chambers.

4区 生物学 Q4 Biochemistry, Genetics and Molecular Biology
Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-08 DOI:10.1016/bs.mcb.2024.09.001
Sophie Goyard, Amandine Schneider, Jerko Ljubetic, Nicolas Inacio, Marie Juzans, Céline Cuche, Pascal Bochet, Vincenzo Di Bartolo, Andrés Alcover, Thierry Rose
{"title":"Measuring interaction force between T lymphocytes and their target cells using live microscopy and laminar shear flow chambers.","authors":"Sophie Goyard, Amandine Schneider, Jerko Ljubetic, Nicolas Inacio, Marie Juzans, Céline Cuche, Pascal Bochet, Vincenzo Di Bartolo, Andrés Alcover, Thierry Rose","doi":"10.1016/bs.mcb.2024.09.001","DOIUrl":null,"url":null,"abstract":"<p><p>Understanding the immunological synapse formation and dynamics can be enriched by measuring cell-cell interaction forces and their kinetics. Microscopy imaging reveals structural organization of the synapse, while physical methods detail its mechanical construction. Various techniques have been reported for measuring forces needed to rupture the interface between a T lymphocyte and its target cell but most of them measure one pair at a time. We describe here a laminar shear flow-based method that exerts dragging forces on T cell-target cells pairs immobilized on the surface of a flow chamber. Increasing flow rate allows us to observe the detachment of hundreds of cell conjugates on the wide field of a light transmission microscope. Monitoring precisely the flow rate gradient exerted on T cells readily yields synapse rupture measurements. Dragging forces are measured at the point of rupture as a linear function of the flow speed in minutes from 10pN to 20nN for each cell pair among a statistically representative cell population in the whole field of view of a single experiment. The output cells can be collected in multi-well plate sorted in the increasing order of rupture forces. We used this approach to unveil the involvement of the cytoskeleton regulator adenomatous polyposis coli (APC) in the stability of immunological synapses formed between human cytotoxic T cell and tumor target cells. APC is a polarity regulator and tumor suppressor associated with familial adenomatous polyposis and colorectal cancer. Reduced APC expression impairs T cell adhesion with tumor target cells suggesting an impact of APC mutation in anti-tumor immune defense.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"175-200"},"PeriodicalIF":0.0000,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in cell biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1016/bs.mcb.2024.09.001","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/11/8 0:00:00","PubModel":"Epub","JCR":"Q4","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 0

Abstract

Understanding the immunological synapse formation and dynamics can be enriched by measuring cell-cell interaction forces and their kinetics. Microscopy imaging reveals structural organization of the synapse, while physical methods detail its mechanical construction. Various techniques have been reported for measuring forces needed to rupture the interface between a T lymphocyte and its target cell but most of them measure one pair at a time. We describe here a laminar shear flow-based method that exerts dragging forces on T cell-target cells pairs immobilized on the surface of a flow chamber. Increasing flow rate allows us to observe the detachment of hundreds of cell conjugates on the wide field of a light transmission microscope. Monitoring precisely the flow rate gradient exerted on T cells readily yields synapse rupture measurements. Dragging forces are measured at the point of rupture as a linear function of the flow speed in minutes from 10pN to 20nN for each cell pair among a statistically representative cell population in the whole field of view of a single experiment. The output cells can be collected in multi-well plate sorted in the increasing order of rupture forces. We used this approach to unveil the involvement of the cytoskeleton regulator adenomatous polyposis coli (APC) in the stability of immunological synapses formed between human cytotoxic T cell and tumor target cells. APC is a polarity regulator and tumor suppressor associated with familial adenomatous polyposis and colorectal cancer. Reduced APC expression impairs T cell adhesion with tumor target cells suggesting an impact of APC mutation in anti-tumor immune defense.

利用活体显微镜和层流剪切室测量T淋巴细胞与靶细胞之间的相互作用力。
通过测量细胞-细胞相互作用力及其动力学,可以丰富对免疫突触形成和动力学的理解。显微镜成像揭示了突触的结构组织,而物理方法详细描述了其机械结构。已有各种技术报道用于测量破坏T淋巴细胞与其靶细胞之间界面所需的力,但大多数技术一次测量一对。我们在这里描述了一种基于层流剪切流的方法,该方法对固定在流室表面的T细胞靶细胞对施加拖曳力。增加流速使我们能够在光透射显微镜的宽视场上观察到数百个细胞共轭物的分离。精确监测施加在T细胞上的流速梯度很容易产生突触断裂的测量结果。在单个实验的整个视场中,在统计上具有代表性的细胞群中,每个细胞对在破裂点处的拖拽力作为流速的线性函数(以分钟为单位,从10pN到20nN)进行测量。输出细胞可以按破裂力的递增顺序收集在多孔板中。我们利用这种方法揭示了细胞骨架调节剂大肠腺瘤性息肉病(APC)在人类细胞毒性T细胞和肿瘤靶细胞之间形成的免疫突触稳定性中的作用。APC是一种极性调节因子和肿瘤抑制因子,与家族性腺瘤性息肉病和结直肠癌有关。APC表达降低会损害T细胞与肿瘤靶细胞的粘附,提示APC突变在抗肿瘤免疫防御中的作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
Methods in cell biology
Methods in cell biology 生物-细胞生物学
CiteScore
3.10
自引率
0.00%
发文量
125
审稿时长
3 months
期刊介绍: For over fifty years, Methods in Cell Biology has helped researchers answer the question "What method should I use to study this cell biology problem?" Edited by leaders in the field, each thematic volume provides proven, state-of-art techniques, along with relevant historical background and theory, to aid researchers in efficient design and effective implementation of experimental methodologies. Over its many years of publication, Methods in Cell Biology has built up a deep library of biological methods to study model developmental organisms, organelles and cell systems, as well as comprehensive coverage of microscopy and other analytical approaches.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信