Summary

从全血人单核细胞来源的树突状细胞的生成

Published: December 24, 2016
doi:

Summary

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Abstract

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity.

The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to naïve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Introduction

树突细胞(DC)是免疫系统中最重要的专门的抗原呈递细胞。未成熟DC(iDC中)驻留在皮肤或粘膜组织,因此是所述第一免疫细胞与入侵的病原体相互作用之间。的DC代表先天和适应性免疫系统1之间的桥梁,因为它们可激活下列病原体检测T细胞和B细胞反应。此外,它们有助于由于大量的细胞因子,如IL-1β,IL-6,和IL-12的分泌的促炎性免疫应答。 DCS还可以激活NK细胞,并吸引其他免疫细胞对感染的趋化网站。

的DCs可分为未成熟树突状细胞(iDC中),并根据它们的形态和功能的成熟树突状细胞(的mDC)2。由(许多模式识别受体之一例如 toll样受体,C型识别外来抗原的后凝集素,或补体受体)在细胞表面上大量表达,将iDC发生重大的变化,并开始成熟。在此成熟过程中,受体抗原捕获被下调,而对于抗原呈递必需分子被上调3。成熟DCs上调主要组织相容性复合物I和II(MHC I和II),共刺激分子象CD80和CD86,这对于T淋巴细胞的抗原呈递和活化所必需的。此外,在细胞表面上的趋化因子受体CCR7的表达被诱导,这使得DC的从外周组织至淋巴结的迁移。迁移由沿趋化因子配体19(CCL19 / MIP-3b)的与趋化因子配体21(CCL21 / SLC)梯度到淋巴结4-6 DC的“滚动”促进。

移植完成之后,MDC在目前的处理抗原天真的CD4 +和CD8 + T细胞,从而INItiating对抗入侵病原体7适应性免疫应答。这与在淋巴结的T细胞相互作用也与病毒8的传播有关。 其他体外研究表明,树突有效地捕捉和HIV一场轰轰烈烈的感染9-12转移到T细胞和这个传输的效果。这些实验强调, 在体内的HIV攻击的DCs作为从外围到淋巴结梭。期间抗原呈递树突分泌塑造效应T辅助细胞的分化,并且因此,对微生物的整个免疫反应的结果,在这个非常相互作用判定键白细胞介素。除了1型(Th1细胞)和2型(Th2细胞)的效应T细胞,已经描述 CD4 + T辅助细胞( 例如,类型17(Th17细胞)和类型22(TH22)T细胞)的其他子集,以及他们的诱导和功能已经被彻底调查。区议会进一步参与产生调节性T细胞(Treg细胞)13,14。这些细胞具有免疫抑制作用,可以停止或下调诱导或效应T细胞的增殖,因而制定免疫力和宽容的关键。

人类传统的DC器(CDC)包括细胞几种亚型与骨髓来源(即朗格汉斯细胞(LCS)和真皮和间质的DC)或淋巴来源(即浆的DC(pDC细胞))。对于体外实验或DC疫苗接种策略,单核细胞衍生的DCs被常规用作用于皮肤的DCs的模型。这些细胞显示在生理,形态和功能与常规骨髓树突状细胞的相似性。它们通过加入白介素-4(IL-4)和粒细胞-巨噬细胞集落刺激因子(GM-CSF)与来自健康供体12,15-18分离的单核细胞产生的。树突细胞也可以直接从皮肤或粘膜活检分离,或可以甚至bE从CD34 +从获得的子宫外脐带血样本中分离出的造血祖细胞的发展。在这里,我们证明单核细胞是如何分离和密度梯度离心的外周血单核细胞(PBMC)富集之后,从抗凝固的人血的刺激。培养5天后,在特定条件下的人单核是分化成的iDC,并准备用于在非临床环境实验程序。

Protocol

伦理学声明:书面知情同意从所有参与献血中央研究所输血与免疫学系,奥地利因斯布鲁克获得。使用匿名剩余标本用于科学目的的批准因斯布鲁克医科大学伦理委员会。 1.富集外周血单个核细胞(PBMC中) 通过离心外周血单个核细胞的浓度。 根据血液中收到的金额开血包和分裂的血液在无菌的50ml离心管中。 如果需要的话,调节音量至50ml对于每个管,用?…

Representative Results

的使用蔗糖缓冲抗凝血离心分离后,外周血单核细胞(PBMC)富集在密度梯度介质( 图1)的顶部的相间。在PBMC中被抽出后,被执行以使用谱系标志物的PBMC中( 例如,CD3对T淋巴细胞,CD14为单核细胞和CD19为B淋巴细胞)中表征不同细胞群体的FACS分析。 图2A示出了收集和密度梯度离心后染色的PBMCs的代表性FACS分析的结果。出选通人口( ?…

Discussion

这个协议描述通过使用磁性纳米微粒测定从抗凝固的血人单核细胞的分离单核细胞来源的树突细胞(MDDCs)的产生。在这个协议中,在离心步骤上游的细胞分离方法,这导致PBMC部分的富集进行。虽然细胞离心过程中丢失,叠加密度梯度介质上的全血包的内容将需要200-300密度梯度培养基中,因此不能被认为是成本有效的。此外,的PBMC通过在清洁的细胞群,在分离过程产生积极影响的抗人CD14磁性颗?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

We would like to thank our technician Karolin Thurnes, Divison of Hygiene and Medical Microbiology, and Dr. Annelies Mühlbacher and Dr. Paul Hörtnagl, Central Institute for Blood Transfusion and Immunological Department, for their valuable help and support regarding this manuscript. We thank the Austrian Science Fund for supporting this work (P24598 to DW, P25389 to WP).

Materials

APC Mouse Anti-Human CD19  Clone  HIB19 BD Biosciences 555415
APC Mouse Anti-Human CD83  Clone  HB15e BD Biosciences 551073
BD Imag Anti-Human CD14 Magnetic Particles  BD Biosciences 557769
BD Imagnet BD Biosciences 552311
BSA (Albumin Fraction V) Carl Roth EG-Nr 2923225
Costar 6 Well Clear TC-Treated Multiple Well Plates Costar 3506
Density gradient media: Ficoll-Paque Premium GE Healthcare Bio-Sciences 17-5442-03
Dulbecco’s Phosphate Buffered Saline (D-PBS) Sigma-Aldrich D8537
Falcon 10mL Serological Pipet Corning 357551
Falcon 25mL Serological Pipet Corning 357525
Falcon 50mL High Clarity PP Centrifuge Tube Corning 352070
Falcon Round-Bottom Tubes Corning 352054
FITC Mouse Anti-Human CD3  Clone  HIT3a BD Biosciences 555339
Ghost Dye Violet 510 (Cell Viability Reagent) Tonbo biosciences 13-0870
GM-CSF MACS Miltenyi Biotec 130-093-862
Heat Inactivated FBS (Fetal Bovine Serum), EU Approved Origin (South America) Gibco 10500-064
Hettich Rotanta 460R Hettich
IL-4 CC PromoKine C-61401
Isolation buffer: BD IMag Buffer (10X)  BD Biosciences 552362
L-Glutamine solution Sigma-Aldrich G7513
Microcentrifuge tubes, 1,5 ml, SuperSpin VWR 211-0015
PE Mouse Anti-Human CD14  Clone  M5E2 BD Biosciences 555398
PE Mouse Anti-Human CD209  Clone  DCN46 BD Biosciences 551265
RPMI-1640 medium Sigma-Aldrich R0883
UltraPure 0.5M EDTA, pH 8.0 Invitrogen 15575020

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Posch, W., Lass-Flörl, C., Wilflingseder, D. Generation of Human Monocyte-derived Dendritic Cells from Whole Blood. J. Vis. Exp. (118), e54968, doi:10.3791/54968 (2016).

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