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Immunologia e infezioni

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

Published: June 22, 2016
doi:
10.3791/54138
, ,
1Department of Biochemistry, College of Life Science & Biotechnology,Yonsei University

Summary

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from naïve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Abstract

调节性T（ 调节性T细胞），其表达的Foxp3作为转录因子，是CD4 + T细胞的亚群。 调节性T细胞通过调节免疫应答发挥免疫耐受和内环境稳定的维护至关重要的作用。 调节性T细胞的主要作用是抑制效应T（T EFF）细胞的增殖和产生细胞因子如IFN-γ，TNF-α和IL-2的。它已被证明持久病原体感染和癌症发展过程中调节性T细胞的抑制T EFF细胞的功能能力得到增强。为了澄清调节性T细胞静息或发炎条件下的功能，已经制订了各种使用鼠标或人类T reg细胞在体外抑制测定的。这项研究的主要目的是建立一个比较表型和休息之间的抑制功能和差异激活调节性T方法细胞。以分离活化的T reg细胞，小鼠感染的淋巴细胞脉络丛脑膜炎病毒（LCMV）克隆13（CL13），LCMV的慢性菌株。与静息从幼稚小鼠分离的T reg细胞比从LCMV CL13感染小鼠的脾分离的T reg细胞表现出两个活化表型和增强的抑制活性。在这里，我们描述了体外的表型分析，从静止T reg细胞区分活化的T reg细胞的基本协议。此外，我们描述了完全活化的T reg细胞的抑制活性的测量的协议。

Introduction

调节性T（ 调节性T细胞）表达叉头框P3（Foxp3的），作为其发育和功能1的转录因子。此外， 调节性T细胞表达的各种其它分子如CD25 2，淋巴细胞激活基因3（LAG-3）3，糖皮质激素诱导的肿瘤坏死因子受体4，和细胞毒性T淋巴细胞相关蛋白4（CTLA-4）5在其表面上或细胞内区域。在慢性感染多种病原体如病毒6,7，细菌8,9和寄生虫10-12，或在癌症发展13,14的过程中，T reg细胞变得分化成活化细胞，显示增强抑制功能靶向效应CD4 +和CD8 + T细胞。若干篇论文表明，扩展和活化的T reg细胞向受损CD8 + T细胞respons朋友逆转录病毒（FV）感染15-17时即FV诱导的T reg细胞抑制IFN-γ或颗粒酶B的表达和CD8 + T细胞15-17的细胞毒活性。此外，在单纯疱疹病毒感染模型，据报道CD4 + CD25 + 调节性T细胞的耗竭导致病毒特异性CD8 + T细胞和由免疫病理学CD4 + T细胞18-20的浸润严重的组织损伤的扩大。

与淋巴细胞脉络丛脑膜炎病毒的克隆13菌株慢性感染的小鼠（LCMV CL13）21-24已被广泛用于慢性病毒感染过程中表征效应T细胞（T EFF）和T reg细胞的表型和功能。期间持续LCMV感染，病毒特异性-T EFF细胞逐渐失去其效应器功能，并成为耗尽T（ŤEXH）细胞。另一方面，Treg细胞增强其抑制病毒特异性T细胞反应能力25。在T EFF细胞的功能能力的降低可以通过几个因素，如对T EFF细胞抑制性受体的上调，抗原呈递细胞的功能改变，生产免疫调节细胞因子，和增加的频率或增强调节性T的功能进行说明细胞26。间参与T细胞抑制因子，细胞程序性死亡蛋白-1（PD-1）-expressingŤEXH细胞和T reg细胞已被广泛认为是抗原持久性和抑制性环境的特点。最近，有人报告说， 调节性T细胞的PD-1途径和消融的封锁导致增强的T细胞功能和LCMV慢性感染27期间降低病毒载量。此外， 调节性T细胞的小鼠慢性感染过程中与LCMV 23,25激活</sUp>和他们的抑制功能得到加强25。 PD-1的高表达对T reg细胞以及为T EXH细胞，和PD-1被T reg细胞表达的水平与他们的抑制功能的强度，以抑制T细胞增殖25相关。

这里，我们描述一个比较来自感染LCMV CL13和从幼稚小鼠分离静止T reg细胞的小鼠中分离的活化T reg细胞的特性的方法。此外，我们解释一系列过程分离活化的T reg细胞，并考察其体外的表型，以及衡量其体外抑制活性。

Protocol

在这项研究中，将小鼠保持在延世大学的延世实验动物研究中心的一个特定的无病原体的设施。所有的动物实验均按照利用在延世大学延世实验动物研究中心的国际动物护理和使用委员会批准协议的韩国食品和药物管理局的指导方针进行。 1.溶液的制备通过在RPMI稀释牛胎儿血清（FBS），以2％和青霉素 – 链霉素1％制备2％RPMI培养基准备完整的RPMI媒体。以RPMI培养…

Representative Results

我们用2×10 6 PFU LCMV CL13的静脉注射为其生成与持续性病毒感染小鼠。调查在调节性T细胞和慢性病毒感染在t CONV细胞的表型的变化，从幼稚和被感染的小鼠获得脾淋巴细胞用各种抗体染色并通过流式细胞术进行分析。 的 CD4 + T CONV（ 图1A，上图）和Foxp3 – – CD8 + T CONV（ 图1B，…

Discussion

虽然在小鼠和人类只存在调节性T细胞的数量少，必须了解它们的功能，因为它们在调节免疫反应和维持免疫耐受了至关重要的作用是重要的。 T的数目和抑制功能的慢性病毒感染15-20以及癌症进展13,14期间reg细胞增加。这大概是由于持续抗原刺激。为了评估调节性T细胞的抗原下的持久性和疾病的发展发挥作用，其抑制活性需要测量。

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Divulgazioni

The authors have nothing to disclose.

Acknowledgements

This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1A6A3A01020610 to HJP) and a grant from the Korean Health Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (HI15C0493 to SJH).

Materials

FITC Rat Anti-Mouse CD4 RM4-5 BD Biosciences 553047 Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.
Cytofix/Cytoperm BD Biosciences 554714 Use this reagent for cell surface staining.
U-Bottom Tissue Culture Plates BD Biosciences 353077
Fixation buffer BD Biosciences 554655 Use this reagent for cell surface staining.
FITC Rat Anti-Mouse CD25 7D4 BD Biosciences 553072 Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.
Cell strainer, 70mm BD Biosciences 352350 Use this strainer for grinding the whole spleen.
Cell strainer, 40mm BD Biosciences 352340 Use this strainer for filtering the cells before column enrichment.
Brilliant Violet 421 Anti-mouse CD279 (PD-1) 29F.1A12 BioLegend 135217 Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.
Brilliant Violet 605 Anti-Mouse CD4 RM4-5 Biolegend 100547 Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.
APC Anti-Mouse/Rat Foxp3  FJK-16s eBioscience 17-5773 Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.
Foxp3 / Transcription Factor Staining Buffer Set eBioscience 00-5223
PerCP-Cyanine5.5 Anti-Mouse CD8a 53-6.7 eBiosicence 45-0081 Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.
Mouse IFN-gamma Platinum ELISA eBiosicence BMS606
RPMI 1640 GE Life Sciences SH30027
PBS (1X) GE Life Sciences SH30256
ACK Lysing Buffer Gibco A10492-01
L-Glutamine, 200mM solution Gibco  25030
Penicillin-Streptomycin, 10,000U/mL Gibco  10378-016
LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit Life technologies L-34975 Please determine appropriate concentration. In this protocol, this reagent was diluted 500X in FACS buffer.
CD8a+ T Cell Isolation Kit, mouse Miltenyibiotec 130-104-075
CD4+CD25+ Regulatory T Cell Isolation Kit, mouse Miltenyibiotec 130-091-041
MACS Separation Columns, LD columns Miltenyibiotec 130-042-901 Use this column for Treg cell isolation
MACS Separation Columns, LS columns Miltenyibiotec 130-042-401 Use this column for CD8+ T cell and Treg cell isolation
EDTA, 0.5M (pH 8.0) Promega V4231
2-Mercaptoethanol Sigma Life Science M7522
Fetal Bovine Serum Thermo Fisher Scientific SH30919.03
CellTrace Violet Cell Proliferation Kit Thermo Fisher Scientific C34557
BD Canto II flowcytometer BD Biosciences Flow cytometer*
Flowjo TreeStar Flow cytometry software†
Hematocytomer Marienfeld superior
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Tags

Regulatory T CellsChronic Lymphocytic Choriomeningitis VirusPhenotypic AnalysisFunctional AnalysisIn Vitro SuppressionCD4+CD25+ CellsCD8+ Responder T CellsAnti-CD3/CD28 BeadsCytokine Production
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Park, H. J., Oh, J. H., Ha, S. Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice. J. Vis. Exp. (112), e54138, doi:10.3791/54138 (2016).
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