从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成
Summary
我们描述了从一个单一的人献血的监管，内存和幼稚T细胞产生的一种方法。极化调节性T细胞可以比较多种遗传同质性，这里还详细介绍了包括抑制法与遗传和功能应用中的其他子集。
Abstract
免疫的CD4 +调节性T细胞（Tregs）的开发和维护，促进周边的宽容，需要保持大量对人体的自我和共生抗原在免疫稳态。调节性T细胞和炎症常规T细胞之间的平衡扰动可导致免疫病理或癌症。虽然已被证明是4几个根本的分歧在人类与小鼠的调节性T细胞生物学5 1，I型糖尿病，类风湿关节炎和移植物抗宿主病，结肠炎的小鼠模型中的有效调节性T细胞治疗注射迄今排除了临床使用。由于缺乏足够数量，纯度，稳定性和特异性治疗调节性T细胞归巢的需要Treg的人类发展的动力平台上，以优化条件下体外扩增 6。
在这里，我们倒是描述一个从一个单一的人外周血献血，其中可分为四个阶段的分化诱导调节性T细胞（iTregs）分离外周血单个核细胞，CD4 + T细胞磁性选择的方法： 在体外细胞培养和荧光激活T细胞亚群细胞分选（FACS）。由于Treg的签名转录因子叉头框P3（FOXP3）是人类7激活诱导的转录因子，并没有其他存在独特的标记，标记的组合面板必须用于识别与抑制活性的T细胞。 6天培养后，细胞在我们的系统可以被划分成幼稚T细胞，记忆T细胞或基于其相对表达CD25，CD45RA的iTregs。由于内存和幼稚T细胞有不同的极化要求和可塑性8，牛逼成CD45RA +和CD45RO细胞人口的初始预分拣+亚群可以E中检查这些差异。与他人一致，CD25表达喜 CD45RA的– iTregs快递高水平的FOXP3 9，GITR和CTLA-4的第11和CD127 12的水平低。按照每个人口的流式细胞仪，由此产生的细胞可用于在评估相对延缓羧基琥珀酰亚胺酯（CFSE）标记的自体T细胞增殖能力的抑制实验。
Protocol
1。分离人外周血单个核细胞（单核细胞从白膜） 从医院或附近的血液中心位置，获得一个单位白膜。我们的血液中心提供约40-60毫升单位正常献血者中获得的棕黄色大衣。 倒入蒸压500毫升玻璃瓶含无菌PBS稀释白膜血。最终体积的PBS +白膜应该是250毫升。 装满十50 ml锥形管各20毫升Lymphoprep的解决方案。 轻轻覆盖25毫升稀释白膜Lymphoprep解决方案，小心不扰乱Lymphoprep?…
Discussion
虽然拥有巨大的调节性T细胞的转移在打击自身免疫排斥反应和其他免疫或炎症介导的疾病，其高效发电和维护稳定的方法治疗的承诺尚未开发。由于只有1-5％的循环人类T细胞是调节性T细胞，他们控制的扩张和分化克服这种缺乏作为实施调节性T细胞进入临床的主要威慑。另一方面，我们从审判13 TGN1412了解到，这是科学和伦理的必要深入了解分子事件，编排实施任何治疗方案之前，人类T?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
<p class="jove_content"作者希望感谢他们协助流式细胞仪分析和排序詹妮弗怪和格雷格·鲍曼。这项工作得到了NIH的资助从NCRR 2P20 RR020171和大学由肯塔基启动资金调频; GIE位承认总统的研究生奖学金和肯塔基机遇奖学金的支持。</p
Materials
| Name of the reagent | Company | Catalogue Number | Comments |
| Lymphoprep | Axis-Shield | 1114547 | Keep at room temperature |
| Refrigerated Bench-Top Centrifuge | Eppendorf | 5810R | |
| Bright-Line Hemocytometer | Hausser Scientific | 3110 | |
| EasySep Human CD4+ T Cell Enrichment Kit | Stem Cell Technologies | 19052 | |
| EasySep Human Memory CD4+ T Cell Enrichment Kit | Stem Cell Technologies | 19157 | |
| EasySep Human Naïve CD4+ T Cell Enrichment Kit | Stem Cell Technologies | 19155 | |
| The Big Easy EasySep Magnet | Stem Cell Technologies | 18001 | Silver |
| 5 mL Round Bottom Polystyrene Tubes | BD Falcon | 352008 | |
| 15 mL Polypropylene Centrifuge Tubes | VWR | 89004-368 | |
| 14 mL Polypropylene Round-Bottom Tubes | BD Falcon | 352059 | |
| 50 mL Polypropylene Centrifuge Tubes | VWR | 89004-364 | |
| Human anti-CD3 Antibody | Bio X Cell | BE0001-2 | Clone: OKT3 |
| 24 Well Cell Polystyrene Culture Plate | BD Falcon | 353047 | |
| 96 Well Round Bottom Tissue Culture Plate | Grenier Bio-One | 650180 | |
| RPMI 1640 with Glutamax-I and HEPES Buffer | Gibco | 72400 | |
| Fetal Bovine Serum (FBS) | Gibco | 16000 | |
| β-Mercaptoethanol | Sigma | M7522 | |
| Recombinant Human TGF-β1 | eBioscience | 14-8348-62 | |
| Recombinant Human IL-2 | eBioscience | 14-8029-63 | |
| Bovine Serum Albumin (BSA) | MP Biomedicals Inc. | 810531 | |
| 0.5 M EDTA | Amresco | E177 | |
| Mouse Anti-Human CD25-PE | Miltenyi Biotec | 130-091-024 | Clone: 4E3 |
| Mouse Anti-Human CD45RA-PE-Cy5 | eBioscience | 15-0458-42 | Clone: HI100 |
| Mouse Anti-Human CD127-APC | Miltenyi Biotec | 130-094-890 | Clone: MB15-18C9 |
| DNase II | MP Biomedicals Inc. | 190370 | |
| MoFlo Flow Cytometer | Beckman Coulter | ||
| FlowJo Software | Tree Star, Inc. | ||
| Cell Quest Pro Software | BD Biosciences | ||
| Newborn Calf Serum | Gibco | 16010 | |
| L-glutamine | Gibco | 25030 | |
| AIM-V | Gibco | 0870112 | |
| CellTrace CFSE Cell Proliferation Kit | Invitrogen | C34554 | |
| Treg Suppression Inspector Beads | Miltenyi Biotec | 130-092-909 | |
| Penicillin-Streptomycin | Gibco | 15140 | |
| 40 μM Nylon Cell Strainer | BD Falcon | 352340 |
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Tags
Induced Regulatory T CellsPrimary Human Naive T CellsMemory T CellsImmunosuppressive CD4+ Regulatory T CellsPeripheral ToleranceImmunologic HomeostasisSelf AntigensCommensal AntigensTreg BiologyTherapeutic Injection Of TregsMurine ModelsColitisType I DiabetesRheumatoid ArthritisGraft Versus Host DiseaseEx Vivo ExpansionPeripheral Blood Mononuclear CellsCD4+ T CellsIn Vitro Cell CultureFluorescence Activated Cell Sorting (FACS)Forkhead Box P3 (FoxP3)Activation-induced Transcription Factor
Citer Cet Article
Ellis, G. I., Reneer, M. C., Vélez-Ortega, A. C., McCool, A., Martí, F. Generation of Induced Regulatory T Cells from Primary Human Naïve and Memory T Cells. J. Vis. Exp. (62), e3738, doi:10.3791/3738 (2012).