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Abstract
Introduction
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Inmunología y contagio

在从幼稚的CD4 + T细胞的人CD4 + FOXP3 +诱导调节性T细胞（iTregs的）使用含有TGF-β-协议的体外分化

Published: December 30, 2016
doi:
10.3791/55015
, , ,
1Unit of Computational Medicine, Center for Molecular Medicine, Department of Medicine Solna,Karolinska Institutet, Karolinska University Hospital, & Science for Life Laboratory

Summary

本协议描述从幼稚CD4 + T细胞在体外可重复产生和人为调节性T细胞（iTregs的）的表型。不同的协议FOXP3诱导允许与各自的协议获取特定表型iTreg的研究。

Abstract

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes.

Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human naïve T cells using a range of Treg-inducing factors (TGF-β plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR.

These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained.

A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-β plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

Introduction

CD4 + CD25 + FOXP3 +调节性T细胞（Treg细胞）抑制其他免疫细胞，并外周耐受的关键介质，防止自身免疫和炎症反应过度1。 Treg细胞的重要性是由人类疾病immunodysregulation多内分泌腺病肠病X连锁综合征（IPEX），其中的Treg的损失，由于在`master' Treg细胞转录因子叉头框P3突变（FOXP3）例举导致严重的全身性自身免疫性疾病，致命自幼。然而，调节性T细胞作为免疫系统，因为他们也可以妨碍某些设置2抗肿瘤免疫力一把双刃剑。调节性T细胞的数量和功能的治疗操纵因此受到众多临床研究。在癌症中，调节性T细胞耗竭可能是可取的，临床的方法取得了一些成功鼓励进一步研究3。在自身免疫和炎性疾病，除了在塞弗Treg细胞的治疗效果拉尔小鼠疾病模型，继Treg细胞转移的最近最早在人的试验，以防止移植物抗 -host病（GVHD）4 – 7和评估安全性在治疗1型糖尿病8显示了非常有前途的结果。

天然存在的Tregs（nTregs）包括在保持健康9胸腺衍生tTregs和外周诱导的pTregs，具有非冗余必要功能– 11。然而，nTreg数量有限，鼓励诱导调节性T细胞（iTregs的） 在体外从幼稚T细胞前体12的补充方法。仍然稳定的iTregs的，大概是由于缺乏在FOXP3基因座13的所谓的Treg特异性去甲基化区域（TSDR）去甲基化，仍令人担忧和若干研究表明， 在诱导的Tregs 体内更稳定14。

迄今为止，FOXP3仍然是最好的M蛋白arker对于调节性T细胞，但也并非绝对特定，因为人类的常规的CD4 + CD25-T细胞中瞬时激活后15,16表达FOXP3的中间水平。虽然显著进展已经阐明FOXP3表达的调节而完成的，仍有许多关于特别是在人类细胞中FOXP3的诱导，稳定性和功能被发现。尽管差异nTregs， 在体外诱导FOXP3 + CD4 + T细胞可以作为一个模型系统以研究FOXP3诱导分子机制和作为起点到在将来开发的协议，其允许产生的iTregs的那些更类似于体内产生的Treg，这可能是适用于在将来继转移策略。

没有`金standard'协议以诱导人的iTregs，和当前的协议都基于模仿Treg细胞诱导条件在体内被开发：白细胞介素-2（IL-2）和转化生长因子β（TGF-β）信号对于FOXP3诱导体内 17是至关重要的，和全反式维甲酸（ATRA） -这是在体内通过肠相关树突细胞产生的-被经常用来增强FOXP3感应在体外 18 – 21。我们已经开发出利用丁酸22中 ，最近显示，以增加鼠Treg的感应23,24一个肠道微生物衍生的短链脂肪酸的其他人Treg诱导协议。我们最近还建立用于产生具有体外优异的抑制功能的iTregs的一个新的协议，通过使用TGF-β，ATRA的组合和雷帕霉素22，后者是雷帕霉素（mTOR的）的临床批准哺乳动物靶抑制剂是已知促进人Treg扩张25,26中FOXP3维护。

该方法描述了在再现的体外代人CD4 + FOXP3 +使用一组不同的条件的iTregs，并通过流式细胞术和定量逆转录聚合酶链反应及其后续的表型的（QRT-PCR），以揭示FOXP3的表达等的Treg签名分子如的协议特定的图案如CD25，CTLA-4，EOS，以及IFN-γ和SATB1 表达 22的压制。所产生的细胞群可用于关于抑制活性官能测定或对分子研究，无论是关于一般的FOXP3调节剂或研究特定的某些化合物，如丁酸盐或雷帕霉素的效果。驾驶Treg细胞分化的分子机制进一步的了解是在自身免疫或癌症的未来治疗方法专门针对参与调节性T细胞的产生和功能分子高度相关。

Protocol

人外周血单核细胞（PBMC）中新鲜从卡罗林斯卡大学医院，瑞典购买匿名健康供体的血沉棕黄层分离。从区域伦理审查委员会在斯德哥尔摩（Regionalaetikprövningsnämnden我斯德哥尔摩），得到了该实验的伦理许可证，瑞典（批准文号：2013/1458年至1431年/ 1）。 从外周血1. T细胞分离 PBMC隔离 预先铺设15毫升密度离心培养基在50ml管（每血沉棕黄层5管）（如聚蔗糖）溶液中。?…

Representative Results

图1示出实验装置的方案。 图2示出了用于磁性分离的幼稚的CD4 + T细胞和nTregs的代表性纯度对照染色。 ˚Figure 3A显示仪设门的流动和图3B显示代表FOXP3和CD25流式细胞仪染色的培养6日表示iTreg或控制的条件下。 在体外刺激后，大多数细胞上调CD25，它是在雷帕霉素?…

Discussion

所描述的协议使人类CD4 + FOXP3 +来自人幼稚CD4 + T细胞的iTregs的鲁棒诱导。它包括我们最近描述的，使用TGF-β，全反式维甲酸和雷帕霉素的组合，对于具有优异的体外抑制功能22的iTregs的感应一个新的协议。相比其他发表的方案，另一个优点是不同iTreg种群的并联感应由不同的协议，从而使的某些iTreg诱导因子效应的直接比较，与在存在单独的IL-2活化的对照细胞沿着。所描述的…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

Nina Nagel is gratefully acknowledged for technical assistance during the video shoot and experimental preparation. We thank Eva-Maria Weiss for help with the intracellular FOXP3 staining protocol and Elisabeth Suri-Payer and Nina Oberle for establishing the nTreg isolation protocol. Matilda Eriksson and Peri Noori are acknowledged for laboratory management.

Funding: A.S. was supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme, the Dr. Åke Olsson Foundation and KI research foundations; A.S. and J.T. were supported by a CERIC (Center of Excellence for Research on Inflammation and Cardiovascular disease) grant, J.T. was supported by Vetenskapsrådet Medicine and Health (Dnr 2011-3264), Torsten Söderberg Foundation, FP7 STATegra, AFA Insurance and Stockholm County Council.

Materials

All-trans retinoic acid Sigma Aldrich R2625-50MG  
anit-human Foxp3-APC clone 236A/E7 eBioscience 17-4777-42
anti-human CD25 microbeads Miltenyi Biotec 130-092-983
anti-human CD25-PE Miltenyi Biotec 130-091-024
anti-human CD28 antibody, LEAF Purified  Biolegend 302914
anti-human CD3 Antibody, LEAF Purified  Biolegend 317315
anti-human CD45RA , FITC Miltenyi Biotec 130-092-247
anti-human CD45RO PE clone UCHL1 BD Biosciences 555493
anti-human CD4-PerCP clone SK3; mIgG1 BD Biosciences 345770
anti-human CD8-eFluor 450 (clone OKT8), mIgG2a eBioscience 48-0086-42 
anti-human CTLA-4 (CD152), clone BNI3, mIgG2ak, Brilliant violet 421 BD Biosciences 562743
anti-human IFN-g FITC clone 4S.B3; mIgG1k eBioscience 11-7319-81 
Brefeldin A-containing solution: GolgiPlug BD Biosciences 555029
cDNA synthesis kit: SuperScript VILO(Reverse transcriptase) cDNA Synthesis Kit Invitrogen 11754-250
Density centrifugation medium: Ficoll-Paque GE healthcare 17-1440-03
DMSO 99,7% Sigma Aldrich D2650-5X5ML
FBS, heat inactivated Invitrogen 10082-147
Fixable Viability Dye, eFluor 780   eBioscience 65-0865-14 or 65-0865-18 
Foxp3 Staining Buffer Set eBioscience 00-5523-00  Caution, contains Paraformaldehyde
Can be also bought in combined kit with antibody; 77-5774-40 Anti-Human Foxp3 Staining Set APC Clone: 236A/E7 Set 
GlutaMAX (200 mM L-alanyl-L-glutamine) Invitrogen 35050-061
human naive CD4 T cell isolation kit II Miltenyi Biotec 130-094-131
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human naive CD4 T cell isolation kit II
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Miltenyi Biotec
130-094-131
Human serum albumin 50 g/l Baxter 1501057
Ionomycin from Streptomyces conglobatus >98% Sigma Aldrich I9657-1MG
MACS LS-columns Miltenyi Biotec 130-042-401
mouse IgG1 K Isotype Control APC Clone: P3.6.2.8.1  eBioscience 17-4714-42 
mouse IgG1 K Isotype Control FITC 50 ug  eBioscience 11-4714-81 
mouse IgG2a isotype control, Brilliant violet 421, clone MOPC-173 BD Biosciences 563464
Pasteur pipet plastic, individually packed Sarstedt 86.1172.001  
PMA PHORBOL 12-MYRISTATE 13-ACETATE Sigma Aldrich P1585-1MG 
Rapamycin EMD (Merck) 553210-100UG
Recombinant Human IL-2, CF R&D 202-IL-050/CF
Recombinant Human TGF-beta 1, CF RnD 240-B-010/CF
RNA isolation kit: RNAqueous-Micro Kit Ambion AM1931  
RPMI 1640 Medium  Invitrogen 72400-054 
Sodium butyrate Sigma Aldrich B5887-250MG 
T cell culture medium: X-Vivo 15 medium, with gentamicin+phenolred Lonza 04-418Q
TaqMan Gene Expression Assay, FOXP3 (Best Coverage)  Applied Biosystems 4331182; assay ID: Hs01085834_m1 Caution, contains Paraformaldehyde
TaqMan Gene Expression Assay, RPL13A (Best Coverage) Applied Biosystems 4351370; assay ID: Hs04194366_g1   Caution, contains Paraformaldehyde
TaqMan Gene Expression Master mix Applied Biosystems 4369514
DOWNLOAD MATERIALS LIST

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Tags

In VitroDifferentiationHumanCD4+FOXP3+Induced Regulatory T CellsITregsNaive CD4+ T CellsTGF-βPhenotypeT Cell ImmunologyPBMCMagnetic Bead IsolationBiotin AntibodyAnti-biotin MicrobeadsLS Column

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Schmidt, A., Éliás, S., Joshi, R. N., Tegnér, J. In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Naïve CD4+ T Cells Using a TGF-β-containing Protocol. J. Vis. Exp. (118), e55015, doi:10.3791/55015 (2016).
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