Summary

人外周血和淋巴组织中原发性 T 细胞突触界面的评价

Published: July 30, 2018
doi:

Summary

该协议描述了一种研究原发性多克隆人 T 细胞通过平面脂质双层形成突触界面的能力的技术。利用该技术显示了从淋巴结和外周血中提取的人原发性 T 细胞的差异突触形成能力。

Abstract

目前对 t 细胞突触界面的动力学和结构特征的理解, 主要是通过使用玻璃支持的平面双层和体外衍生的 t 细胞克隆或12 线来确定的. ,3,4。这些发现如何适用于从血液或淋巴组织中分离出来的主要人类 T 细胞是不知道的, 部分原因是在获得足够数量的细胞进行分析5方面存在重大困难。在这里, 我们通过开发一种利用多通道流幻灯片来构建含有活化和粘附分子的平面脂双层来解决这一问题。低高度的流动幻灯片促进快速细胞沉淀, 以同步细胞: 双层附着, 从而允许研究人员的动态的突触界面形成和动力学的颗粒释放。应用这种方法分析了 104至 105只从淋巴结 (LN) 和外周血 (PB) 中分离出的低温保存 T 细胞的突触界面。结果表明, 新的平面脂质双层技术能够研究健康和疾病背景下从血液和组织中提取的人 T 细胞的生物物理特性。

Introduction

对 t 细胞免疫突触的结构特征及其与 t 细胞功能活动的联系的科学知识, 主要来源于从 PB 中提取的细胞系和克隆的研究。这些发现与从血液或人类淋巴组织获得的初级 t 细胞的程度仍不清楚, 因为在淋巴和其他组织中的 t 细胞的突触界面迄今尚未分析。重要的是, 新出现的数据表明, 与 PB67相比, 组织居民和淋巴器官衍生的 T 细胞在表型和功能活性上可能有显著差异。这进一步巩固了需要更好地了解 t 细胞突触界面的特点在原发性人类 T 细胞。

为此, 我们开发了一种新的小尺度方法, 利用多通道流幻灯片中的脂质双层, 使我们能够进行 t 细胞/双层界面的成像, 其分离出的人 PB 和 LN 不到 105个初级 t 细胞。这项新技术允许研究人类 T 细胞突触界面的生物物理特性, 以便更好地模型和了解体内细胞间的相互作用。

Protocol

这项研究是按照《赫尔辛基宣言》进行的。从所有参与者获得书面知情同意, 在宾夕法尼亚大学的机构审查委员会 (IRB#809316, IRB 815056 号) 的批准下获得了血液和淋巴结样本。所有的人类臣民都是成年人。在托马斯杰斐逊大学产科 & 妇科的分娩和分娩中, 提供脐带血样本。所有样品均被取消鉴定。 1. 为图像分析分离 CD4+ T 细胞 从采集的样品中解冻1毫升整除…

Representative Results

首先, 我们比较了在传统的大型流动细胞系统中建立的由活化脐带-血源 CD8+ T 细胞形成的双层的突触界面结构 (见材料表详细资料)1 、2、3、4或多通道流幻灯片。双层含有荧光标记的 anti-CD3 和 ICAM-1, 分别为50分子/µm2和300分子/µm2。从人脐血…

Discussion

此处描述的新技术利用了在常规流单元5中构建平面双层所需的类似试剂, 并可成功地应用于 cell–bilayer4 的初级人类 T 型接口的成像. ,15。该技术提供了显着减少荧光分子的使用, 并要求10–20x 较少的 T 细胞相比, 流细胞系统5, 创造机会, 以分析主要人类 T 细胞从血液和其他组织。

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Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作得到了 R01AI118694 NIH 的资助, 包括566950分奖给尤里 Sykulev。我们感谢悉尼 Bioimaging 癌症中心共享资源, 他们的出色支持。

Materials

CD4 T cell isolation kit, human Miltenyl Biotec 130-096-533
CD8 T cells Isolation Kit, human Miltenyl Biotec 130-096-495
DOPC Avanti Polar Lipids 850375C
DOGS NTA Avanti Polar Lipids 790528C
Biotinyl Cap PE Avanti Polar Lipids 870273C
Human Serum Albumin Octapharma USA NDC 68982-643-01
sticky-Slide VI 0.4 ibidi 80608
Coverslips for sticky-Slides ibidi 10812
Bioptech FCS2 Chamber Bioptech 060319-2-03
anti-CD3 antibody Thermo Fisher Scientific 16-0037-81 OKT3 clone, hybridoma cells are available from ATCC
anti- CD28 antibody Genetex GTX14664 9.3 clone
Casein Sigma C5890
Biotin-PEO4-NHS Thermo Fisher Scientific 21329
DMSO Sigma D2650-5
Alexa Fluor 488 protein labeling kit with column for labeled protein purification Thermo Fisher Scientific A10235
Alexa Fluor 568 protein labeling kit with column for labeled protein purification Thermo Fisher Scientific A10238
Amersham Cy5 NHS Ester GE Life Science PA15101
pMT/V5-His A, B, C Drosophila Expression Vectors Thermo Fisher Scientific V412020
pcopneo, G418 Drosophila expression vector for positive selection ATCC 37409
Serum free Drosophial media Insect-XPRESS Lonza 12-730Q
Hybridoma YN1/1.7.4 ATCC CRL1878 The hybridoma secrets antibody against ICAM-1.
Cyanogen bromide-activated-Sepharose 4B Sigma-Aldrich C9142 Utilized for preparation of Sepharose with covelently bound anti-ICAM antibody.
MasterFlex tangential flow concentrator Cole-Parmer 77601-60 7592-40 Used for ICAM-1 containing supernatant concentration and dialysis of ICAM-1 containing supernant
Centramate Lab Tangential Flow Systems Pall Laboratory FS002K10 OS010T12 FS005K10 Used for ICAM-1 containing supernatant concentration and dialysis of ICAM-1 containing supernant
Ni-NTA Agarose QIAGEN 30210
Dialysis tubing Spectra/Por 131384
Papain from papaya latex Sigma P3125
mouse anti-human antibody against CD107a BD Bioscences 555798 Clone H4A3
Ansell Natural Blue Gloves Fisher Scientific 19-014-539
Nalgene Polypropylene Scissor-Type Forceps Thermo Fisher Scientific 6320-0010
Streptavidin ProZyme SA10
Confocal microscope Nikon Nikon TiE inverted microscope equipped with PFS for long-term image stability control, 60x oil objectives, 4 lasers with excitation lines at 405, 458, 488, 514, 561, and 640 nm, 2 GaAsP detectors and 2 high sensitivity PMTs, DIC transmitted light, Programmable X,Y,Z stage for multiple positions and stitching of large areas, time lapse functions, Tokai-Hit temperature and CO2-controlled chamber for live imaging, and anti-vibration isolation table
TIRF microscope Andor Andor Revolution XD system equipped with Nikon TIRF-E illuminator, Lasers with 405,488,561 and 640 lines, DIC transmitted light, Yokogawa CSU-X1 spinning disk head for confocal imaging, 100/1.49 NA objective, Andor iXon X3 EM-CCD camera, objective heater, and a piezoelectric motorized stage with Perfect Focus System (PFS)
MetaMorph Premier Image Analysis Software Molecular devices

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Steblyanko, M., Anikeeva, N., Buggert, M., Betts, M. R., Sykulev, Y. Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue. J. Vis. Exp. (137), e58143, doi:10.3791/58143 (2018).

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