小鼠骨髓基质树突状细胞诱导 CD4 T 细胞活化、增殖和 Th1 分化的体外模型研究
Summary
在这里, 我们提出了一种在体内测定由 GM-脑脊液骨髓 (BM) 衍生树突状细胞 (CD4) 诱导的天真的 t 细胞 (t 细胞) 活化、增殖和 Th1 分化的方法。此外, 该协议还描述了 BM 和 t 细胞分离、dc 生成和 dc 和 t 细胞的移植。
Abstract
量化的天真 CD4 t 细胞活化, 增殖, 并分化为 t 帮助 1 (Th1) 细胞是一个有用的方法来评估 t 细胞在免疫应答中发挥的作用。本协议描述骨髓 (BM) 祖细胞的体外分化, 以获得粒细胞巨噬细胞集落刺激因子 (GM-脑脊液) 衍生树突状干细胞 (DCs)。该协议还介绍了蛋白蛋白肽 (OVAp) 负载的 GM-脑脊液衍生 DCs 和天真的 CD4 T 细胞 OTII 转基因小鼠的移植, 以分析体内活化、增殖和 Th1 分化的转移 CD4 T 细胞。这个协议绕过了纯粹的体内方法的限制, 因为无法专门操作或选择所研究的细胞群体。此外, 本议定书允许在体内环境中进行研究, 从而避免对可能发生在体外的功能性因素的改变, 包括细胞类型和其他仅在完整器官中发现的因素的影响。该协议是一个有用的工具, 以产生变化的 DCs 和 T 细胞, 修改适应性免疫反应, 有可能提供重要的结果, 以了解许多免疫相关疾病的起源或发展。
Introduction
CD4 T 细胞和抗原呈现细胞 (apc), 如 DCs 是需要的调解人免疫的微生物病原体1,2。在外周淋巴器官中, CD4 T 细胞在 apc3、4、5所提出的特异抗原识别后激活。活化 CD4 T 细胞增殖和分化为独特的特定效应细胞, 是必要的发展一个正确的适应性免疫应答6,7。控制这些过程是至关重要的, 以产生适当的适应性防御, 杀死病原体而不产生有害的组织损伤8。Th 细胞是根据表面分子、转录因子和效应细胞因子的表达或产生而定义的, 对病原体的反应执行必要和精确的功能1。细胞 Th1 细胞子集表达转录因子 T 打赌和细胞因子干扰素γ (IFNγ) 和参与宿主防御细胞内病原体1。量化 CD4 t 细胞的活化、增殖和 Th1 分化是评估 t 细胞在免疫应答中发挥作用的有用手段。
该协议能够在体内分析体外生成的 BM 衍生 DCs 的能力, 以调节天真 CD4 T 细胞的活化、增殖和 Th1 分化。该议定书还有助于评估幼稚 CD4 T 细胞被激活、诱导增殖和 Th1 分化的能力 (图 1)。这个多才多艺的协议绕过无法具体地操作或选择被研究的细胞人口在纯粹体内协议。利用基因修饰小鼠5的 BM, 或通过治疗或基因操作分离的 bm 细胞9, 可以研究不同分子和处理对 DCs 的影响。同样, t 细胞反应可以探索通过获得 t 细胞为领养转移从不同的来源或在几操作以后3,8,10。
该协议的主要优点有两个方面。用流式细胞仪分析 T 细胞活化、增殖和 Th1 分化;这与体内研究相结合, 从而避免了在体外可能发生的改变, 包括细胞类型和其他仅在完好器官中发现的因素11。
使用重要染料是一种广泛使用的技术来跟踪细胞增殖, 同时避免使用放射性。用这些试剂进行增殖的测量是基于细胞分裂后的染料稀释。此外, 这些染料可以在多波长检测, 并易于分析的流式细胞术与多种荧光抗体或标记。通过流式细胞术分析 T 细胞的活化、增殖和 Th1 分化, 我们强调了该协议的实用性。
Protocol
Representative Results
Discussion
该协议允许对 BM 衍生 DCs 的能力进行表征, 以调节天真 CD4 T 细胞的活化、增殖和分化。此外, 它还可以用来评估 CD4 T 细胞的易感性, 由 BM 衍生 DCs。通过该协议, 可以在体内测量这些事件的变化。
根据调查的假设, 可以使用 T 细胞和 DCs 的几种组合。例如, 你可以分析敲或敲出特定基因的后果, 或者 CD4 T 细胞活化、增殖或 Th1 分化的特定刺激的效率。这些过程可以在 DCs 或 …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢西蒙博士的英语编辑。这项研究得到了干杯研究所 (ISCIII) 的赠款的支持 (PI14/00526;PI17/01395;CP11/00145;CPII16/00022), 米格尔 Servet 计划和基金会拉蒙 Areces;Fondo Europeo de 收复与发展区域 (菲德) 共同出资。CNIC 得到经济、工业和竞争力部 (经济) 和 CNIC 基金会的支持, 是 Severo 奥乔亚卓越中心 (SEV-2015-0505)。RTF 是由基金会拉蒙 Areces 和 CNIC, VZG 由 ISCIII, BHF 由 Investigación 疗养院医院 12 de Octubre (imas12) 和 JMG G ISCIII 的 Servet 计划和 imas12。
Materials
| Ethanol | VWR Chemicals | 20,824,365 | 5 L |
| Scissors | Fine Science Tools (F.S.T.) | 14001-12 | |
| Forceps | Fine Science Tools (F.S.T.) | 11000-13 | |
| Fine Forceps | Fine Science Tools (F.S.T.) | 11253-20 | |
| Scalpel | Fine Science Tools (F.S.T.) | 10020-00 | Box of 100 blades |
| Fetal Bovine Serum | SIGMA | F7524 | |
| Penicillin/streptomycin | LONZA | DE17-602E | |
| Roswell Park Memorial Institute medium (RPMI) | GIBCO | 21875-034 | |
| Sterile Petri dishes | Falcon | 353003 | |
| 25-gauge needle | BD Microlance 3 | 300600 | |
| 1 ml syringe | Novico | N15663 | |
| 15 ml conical tubes | Falcon | 352096 | |
| 50 ml conical tubes | Falcon | 352098 | |
| 70 μm nylon web filter | BD Falcon | 352350 | |
| Red blood lysis buffer | SIGMA | R7757 | 100 Ml |
| EDTA | SIGMA | ED2SS-250 | |
| Bovine Serum Albumin | SIGMA | A7906 | 100 g |
| Trypan blue | SIGMA | 302643-25G | |
| Culture-plates | Falcon | 353003 | |
| 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) | Cambrex | BE17-737E | |
| Beta-mercaptoethanol | Merck | 8-05740-0250 | |
| Sodium pyruvate | LONZA | BE13-115E | |
| L-glutamine | LONZA | BE17-605E | |
| Recombinant murine Granulocyte Macrophage colony-stimulating factor (GM-CSF) | Prepotech | 315-03 | |
| lipopolysaccharide (LPS) | SIGMA-ALDRICH | L2018 | |
| 96-well-plate | Costar | 3799 | |
| v450 anti-mouse CD11b antibody | BD Biosciences | 560455 | |
| PE anti-mouse CD64 antibody | BioLegend | 139303 | |
| PE anti-mouse CD115 antibody | BioLegend | 135505 | |
| FITC anti-mouse CD11c antibody | BioLegend | 117305 | |
| FITC anti-mouse MHCII antibody | BioLegend | 125507 | |
| APC anti-mouse CD86 antibody | BioLegend | 105011 | |
| APC anti-mouse CD80 antibody | BioLegend | 104713 | |
| Flow Cytometry tubes | Zelian | 300800-1 | PS 12X75 5mL |
| OTII Ovoalbumine peptide | InvivoGen | 323-339 | |
| anti-mouse biotinylated CD8α antibody | Tonbo Biosciences | 30-0081-U500 | |
| anti-mouse biotinylated IgM antibody | BioLegend | 406503 | |
| anti-mouse biotinylated B220 antibody | Tonbo Biosciences | 30-0452-U500 | |
| anti-mouse biotinylated CD19 antibody | Tonbo Biosciences | 30-0193-U500 | |
| anti-mouse biotinylated MHCII (I-Ab) antibody | BioLegend | 115302 | |
| anti-mouse biotinylated CD11b antibody | Tonbo Biosciences | 30-0112-U500 | |
| anti-mouse biotinylated CD11c antibody | BioLegend | 117303 | |
| anti-mouse biotinylated CD44 antibody | BioLegend | 103003 | |
| anti-mouse biotinylated CD25 antibody | Tonbo Biosciences | 30-0251-U100 | |
| anti-mouse biotinylated DX5 antibody | BioLegend | 108903 | |
| streptavidin-coated magnetic microbeads | MACS Miltenyi Biotec | 130-048-101 | |
| Magnetic cell separator | MACS Miltenyi Biotec | 130-090-976 | QuadroMACS Separator |
| Separation columns | MACS Miltenyi Biotec | 130-042-401 | |
| PE anti-mouse CD4 antibody | BioLegend | 100408 | |
| APC anti-mouse CD3 antibody | BioLegend | 100235 | |
| FITC anti-mouse CD8 antibody | Tonbo Biosciences | 35-0081-U025 | |
| Cell Violet Tracer | Thermofisher | C34557 | |
| APC anti-mouse CD25 antibody | Tonbo Biosciences | 20-0251-U100 | |
| Alexa647 anti-mouse CD69 antibody | BioLegend | 104518 | |
| PerCPCY5.5 anti-mouse CD45.1 antibody | Tonbo Biosciences | 65-0453 | |
| APC anti-mouse CD45.1 antibody | Tonbo Biosciences | 20-0453 | |
| PECY7 anti-mouse CD45.1 antibody | Tonbo Biosciences | 60-0453 | |
| FITC anti-mouse CD45.2 antibody | Tonbo Biosciences | 35-0454 | |
| Ionomycin | SIGMA-ALDRICH | I0634 | |
| Phorbol 12 Myristate 13 Acetate (PMA) | SIGMA | P8139 | |
| Brefeldin A (BD GolgiPlug) | BD | 555029 | |
| Paraformaldehyde | Millipore | 8-18715-02100 | |
| Intracellular permeabilization buffer | eBioscience | 00-8333 | |
| APC anti-mouse IFNg antibody | Tonbo Biosciences | 20-7311-U100 | |
| Fc-block (anti-mouse CD16/CD32) | Tonbo Biosciences | 70-0161-U100 | |
| B6.SJL CD45.1 mice | The Jackson Laboratory | 002014 | |
| BD LSRFortessa™ Cell Analyzer | BD Biosciences | 649225 | |
| DAPI Solution | Thermofisher | 62248 |
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