从全血人单核细胞来源的树突状细胞的生成
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，已经描述的 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
Representative Results
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?…
Divulgazioni
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 |
Riferimenti
- Banchereau, J., Steinman, R. M. Dendritic cells and the control of immunity. Nature. 392, 245-252 (1998).
- Steinman, R. M., Hemmi, H. Dendritic cells: translating innate to adaptive immunity. Curr Top Microbiol Immunol. 311, 17-58 (2006).
- Steinman, R. M., Inaba, K., Turley, S., Pierre, P., Mellman, I. Antigen capture, processing, and presentation by dendritic cells: recent cell biological studies. Hum Immunol. 60, 562-567 (1999).
- Schwarz, J., Sixt, M. Quantitative Analysis of Dendritic Cell Haptotaxis. Methods Enzymol. 570, 567-581 (2016).
- Weber, M., Sixt, M. Live cell imaging of chemotactic dendritic cell migration in explanted mouse ear preparations. Methods Mol Biol. 1013, 215-226 (2013).
- Sixt, M., Lammermann, T. In vitro analysis of chemotactic leukocyte migration in 3D environments. Methods Mol Biol. 769, 149-165 (2011).
- Randolph, G. J., Angeli, V., Swartz, M. A. Dendritic-cell trafficking to lymph nodes through lymphatic vessels. Nat Rev Immunol. 5, 617-628 (2005).
- Wilflingseder, D., Banki, Z., Dierich, M. P., Stoiber, H. Mechanisms promoting dendritic cell-mediated transmission of HIV. Mol Immunol. 42, 229-237 (2005).
- Pope, M., Gezelter, S., Gallo, N., Hoffman, L., Steinman, R. M. Low levels of HIV-1 infection in cutaneous dendritic cells promote extensive viral replication upon binding to memory CD4+ T cells. J Exp Med. 182, 2045-2056 (1995).
- McDonald, D., et al. Recruitment of HIV and its receptors to dendritic cell-T cell junctions. Science. 300, 1295-1297 (2003).
- Pruenster, M., et al. C-type lectin-independent interaction of complement opsonized HIV with monocyte-derived dendritic cells. Eur J Immunol. 35, 2691-2698 (2005).
- Wilflingseder, D., et al. IgG opsonization of HIV impedes provirus formation in and infection of dendritic cells and subsequent long-term transfer to T cells. J Immunol. 178, 7840-7848 (2007).
- Kapsenberg, M. L. Dendritic-cell control of pathogen-driven T-cell polarization. Nat Rev Immunol. 3, 984-993 (2003).
- Mills, K. H. TLR-dependent T cell activation in autoimmunity. Nat Rev Immunol. 11, 807-822 (2011).
- Banki, Z., et al. Complement as an endogenous adjuvant for dendritic cell-mediated induction of retrovirus-specific CTLs. PLoS Pathog. 6, e1000891 (2010).
- Posch, W., et al. Antibodies attenuate the capacity of dendritic cells to stimulate HIV-specific cytotoxic T lymphocytes. J Allergy Clin Immunol. 130, 1368-1374 (2012).
- Posch, W., et al. Complement-Opsonized HIV-1 Overcomes Restriction in Dendritic Cells. PLoS Pathog. 11, e1005005 (2015).
- Wilflingseder, D., et al. Immediate T-Helper 17 Polarization Upon Triggering CD11b/c on HIV-Exposed Dendritic Cells. J Infect Dis. 212, 44-56 (2015).
- Grassi, F., et al. Monocyte-derived dendritic cells have a phenotype comparable to that of dermal dendritic cells and display ultrastructural granules distinct from Birbeck granules. J Leukoc Biol. 64, 484-493 (1998).
