人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具
Summary
调节性T细胞是免疫系统的强有力的抑制器。有一个独特的表面标志物来定义它们的缺乏，因此，调节性T细胞的定义主要功能。在这里，我们描述了一个优化的<em>在体外</em>检测能够识别风险科目的免疫失衡发展T1D。
Abstract
调节性T细胞（Treg细胞）的自身抗原免疫耐受的关键调解员。此外，他们被感染后的免疫反应的关键调节因子。尽管努力找出独特的调节性T细胞表面标记，唯一的功能是抑制效应T细胞的增殖和功能的能力。虽然很明显，只有在体外试验 ，可用于评估人Treg功能，这将成为问题时，评估的结果，从横断面研究健康细胞和细胞与自身免疫性疾病（如1型糖尿病的T1D）需要的科目中分离进行比较。有一个响应者的T细胞，刺激的性质和强度，Treg细胞的数量和类型的实验室之间的变化很大：应答者比率和使用的数量和类型的抗原提呈细胞（APC）在人类体外抑制实验。这种差异使得比较难以测量Treg细胞功能的研究。 Treg的领域，需要一个标准化的抑制与正常人和自身免疫性疾病的检测工作。我们已经开发出在体外抑制实验表明很少批内变异在健康志愿者相比，与底层的胰腺β细胞的自身免疫性破坏的科目分离的T细胞的刺激。这件作品的主要目的是描述一个在人类体外抑制实验，允许不同的学科组之间的比较。此外，该法有可能划定nTreg功能的一个小的损失，并预计在未来的进一步丧失，从而确定谁可以受益于预防免疫疗法1的科目。下面，我们提供在此过程中所涉及的步骤的详细描述。我们希望用来衡量Treg细胞功能的体外抑制实验的标准化做出贡献。此外，我们提供了这个实验作为一种工具，认识到早期免疫失衡状态和T1D功能的一个潜在的生物标志物。
Protocol
1。设立抑制法检测之前，需要对细胞的刺激和事后大衣tosylactivated珠抗人CD3（克隆UCHT1，终浓度为1μg/ml）检查是否有效地通过设立在体外增殖实验中使用人类的涂层珠T细胞以从原来的小瓶，在磁场的立场地点的M – 450 tosylactivated珠1毫升并按住，直到所有的珠子都坚持管方。删除缓冲区，而管仍然是在磁场的立场。以管的磁立场，并添加了缓存1毫升，放置在磁场的立场管，并删?…
Discussion
作为唯一的独特功能，调节性T细胞，抑制功能应可靠，统一的测试在同一疾病发展的不同阶段和不同的研究主体之间。我们提供我们这个实验的标准化贡献在我们的实验室开发的抑制实验的细节。在我们广泛的优化研究，我们有决心，刺激T细胞与抗人CD3涂层珠（UCHT1克隆，在1μg/ml浓度组合（相对于市售5μg/ml）作为APC PBMC提供了天然的刺激激活测试科目1都响应者的T细胞和调节性T细胞的能…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了最大麦吉少年Diabetesat医学院的威斯康星州和威斯康星儿童研究所国家工程研究中心。出资者已经没有任何作用，在研究设计，数据收集和分析，或准备的手稿。
Materials
| Name of the reagent or instrument | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| Ficoll-Paque PLUS | Amersham Pharmacia Biotech | 17-1440-03 | |
| DPBS-1X | Gibco | 14190-144 | |
| Trypan Blue | Invitrogen | 15250-061 | |
| anti-CD4 microbeads | Miltenyi | 130-045-101 | |
| Pre-separation filters | Miltenyi | 130-041-407 | |
| LS column | Miltenyi | 130-042-401 | |
| EDTA | Invitrogen | 15575-020 | |
| BSA | Sigma-Aldrich | B4287 | |
| Anti-human CD4-APCCy7 (clone RPA-T4) | BD Pharmingen | 557852 | |
| Anti-human CD25-PE (clone M-A251; IL-2Rα) | BD Pharmingen | 555432 | |
| Anti-human CD8-FITC (clone RPA-T8) | BD Pharmingen | 555366 | |
| Anti-human CD14-FITC (clone M5E2; LPS receptor) | BD Pharmingen | 555397 | |
| Anti-human CD32-FITC (clone FLI8.26; FcγR-type II) | BD Pharmingen | 555448 | |
| Anti-human CD116-FITC (clone M5D12; GM-CSFRα chain) | BD Pharmingen | 554532 | |
| Dynalbeads M-450 tosylactivated | Invitrogen | 140-13 | |
| Anti-human CD3 | Ancell | 144-024 | |
| Buffer1 | Homemade | 0.1M Na2B4O7 pH7.6 | |
| Buffer2 | Homemade | PBS/2mM EDTA/ 0.1% BSA pH7.4 | |
| Buffer3 | Homemade | 0.2M Tris/0.1% BSA pH8.5 | |
| Complete RPMI media | Homemade | RPMI 1640 media 2 mM L-glutamine 5 mM HEPES 100 U/μg/ml peni/strept 0.5 mM sodium pyruvate | |
| [3H] thymidine | Perkin Elmer | NET027Z005MC | |
| human pooled AB serum | Atlanta Biologicals | S40110 | |
| Multiscreen harvest plate | Millipore | MAHFC1H60 | |
| Microscint 20 | Perkin Elmer | 6013621 |
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Tags
Human In Vitro SuppressionScreening ToolRecognitionEarly StateImmune ImbalanceRegulatory T CellsTregsImmune ToleranceSelf-antigensInfectionSurface MarkerProliferationFunctionEffector T CellsIn Vitro AssaysCross-sectional StudiesAutoimmune DiseasesType 1 Diabetes-T1DLaboratoriesResponder T CellsStimulationTreg:responder RatiosAntigen-presenting Cells (APC)VariabilityComparison Between StudiesStandardized Suppression AssayHealthy SubjectsAutoimmune Destruction Of Pancreatic β-cells
Cite This Article
Waukau, J., Woodliff, J., Glisic, S. Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance. J. Vis. Exp. (53), e3071, doi:10.3791/3071 (2011).