猴免疫缺陷病毒特异性CD8分析<sup> +</sup> T细胞猕猴由肽MHC-I四聚体染色
Summary
在这里，我们提出了针对艾滋病病毒枚举和表征猕猴CD8 + T细胞的优化协议。本文不仅对HIV免疫学领域，而且也适用于的 CD8 + T细胞应答已知影响疾病结果的生物医学研究的其它领域是有用的。
Abstract
肽主要组织相容性复合体I类（的pMHC-I）四聚体一直一个宝贵的工具来研究CD8 + T细胞反应。因为这些试剂直接结合到T细胞受体CD8 + T淋巴细胞的表面上，荧光染料标记的pMHC-I四聚体使抗原的精确检测（银）特异性CD8 + T细胞，而无需在体外重新-stimulation。此外，当多色组合流式细胞仪，的pMHC-I四聚体染色可以揭示抗原特异性CD8 + T细胞的重要方面，包括分化阶段，记忆表型，和激活状态。这些类型的分析已经在HIV免疫学领域特别有用的 CD8 + T细胞可影响发展为艾滋病。猴免疫缺陷病毒的猕猴（SIV）的实验感染提供了一个宝贵的工具来研究针对艾滋病病毒的细胞免疫功能。其结果是，相当大的逐行SS已经在该动物模型定义和表征T细胞应答已取得。在这里，我们提出了一个优化的协议用于通过的pMHC-I四聚体染色猕猴列举SIV-特异性CD8 + T细胞。我们的测定法允许每测试二的pMHC-I四聚体+ CD8 + T细胞群，这可能是用于跟踪由疫苗接种或SIV感染产生SIV-特异性CD8 + T细胞应答有用的同时定量和存储器表型。考虑在生物医学研究非人灵长类动物的相关性，这种方法适用于多种疾病设定学习的 CD8 + T细胞应答。
Introduction
CD8 + T细胞包含适应性免疫系统的重要组成部分，因为它们参与肿瘤免疫监视和向细胞内病原体1根除。简单来说，CD8 + T细胞表达特异性识别肽-主要组织相容性复合体I类（的pMHC-I）中的T细胞受体（TCR）的分子存在于宿主细胞的质膜。因为这些肽是从内源性合成的蛋白的水解产生的，细胞表面的pMHC-I复合物提供了一个窗口，进入细胞内环境。在病毒感染，例如，感染的细胞将显示含有病毒蛋白衍生肽，可以作为配体被巡逻的CD8 + T细胞中表达的TCR的MHC-I类分子。在一个特定的病毒特异性CD8 + T细胞遇到感染细胞呈递其的pMHC-1配体的情况下，T细胞受体接合将导致CD8 + T细胞的激活和大招三方共同导致靶细胞裂解。鉴于这些TCR /的pMHC-I相互作用的关键特性，确定了震级，特异性和响应CD8 + T细胞表型往往能揭示人类疾病的重要线索。
直到20世纪90年代初，抗原特异性CD8 + T细胞的量化依靠技术要求有限稀释法（LDA）2,3-。不仅在LDA需要数天才能完成，因此也未能检测缺乏增殖潜能的细胞。其结果是，在LDA大大低估抗原（抗原）特异性的 CD8 +参与免疫应答的T细胞的实际频率。虽然ELISPOT和细胞内细胞因子染色测定的发展极大地提高测量细胞免疫的能力，这些方法仍然需要在体外刺激用于定量抗原特异性T细胞4。但直到1996年的奥特曼，戴维斯和科尔eagues发表了具有里程碑意义的文章报告的pMHC-I技术的四聚体5的发展。临界该技术的成功是的pMHC-I类分子，其延伸的TCR /的pMHC-Ⅰ相互作用的半衰期，从而减少的pMHC-I四聚体中流式细胞测定法的洗涤步骤脱落的概率的多聚化。的pMHC-I四聚体在上述测定法的主要优点是能够精确地直接离体检测抗原特异性CD8 + T细胞，而无需在体外再刺激的能力。此外，具有多色彩的pMHC-I四聚体染色的组合流式细胞仪已经允许分化阶段，记忆表型，和Ag特异性CD8 + T-细胞2-4的激活状态的详细分析。在最近对通过表征的 CD8 + T细胞所有组成成分的技术进步的光的pMHC-Ⅰ多聚体染色6，应用FO广度R此方法很可能会继续扩大。
在生物医学研究领域很少有比艾滋病病毒免疫学7领域受益更多来自的pMHC-I四聚体染色。虽然CD8 + T细胞曾与由奥特曼，戴维斯和同事8,9出版时HIV病毒血症的初步控制了时间相关的，在随后的几年中使用的pMHC-I四聚体的显著扩大了我们的理解的HIV特异性CD8 + T细胞应答。例如，的pMHC-I四聚体染色帮助确认病毒特异性CD8 + T细胞反应在大多数HIV感染者10-12强劲的大小。这种方法还促进艾滋病毒和SIV特异性CD8 + T细胞应答的通过与在不存在的被称为“精英控制”抗逆转录病毒治疗，这种现象的病毒复制的自发控制相关的MHC-I类分子限制的表征<suP> 13-15。此外，的pMHC-I四聚体是在建立程序性死亡-1（PD-1）/ PD配体1（PD-L1）的轴作为可逆通路中的HIV特异性CD8 + T细胞的不受控制的慢性感染的功能失调的表型器乐16,17。总的来说，这些研究强调的pMHC-I四聚体的效用用于监测针对爱滋病病毒的 CD8 + T细胞应答。
猕猴（ 猕猴 ）的实验SIV感染仍然是防治艾滋病毒/艾滋病18,19评估免疫干预的最佳动物模型。在过去的25年中，实质性的进展已在SIV-特异性CD8 + T细胞在此猴类的鉴定和表征制成，包括MHC-I等位基因的发现和肽结合基序13,20-27的定义。其结果是，的pMHC-I四聚体已经开发了用于SIV-特异性CD8 + T细胞的分析在这种动物模型28的反应。大多数这些试剂是由四猕猴MHC-I等位基因的基因产物： 马目-A1 * 001，马目-A1 * 002：01，马目-B * 008：01，和马目-B * 017：01。值得注意的是，猕猴不发表MHC-C座29。绝大多数在本实验中使用的的pMHC-I四聚体均在埃默里大学美国国立卫生研究院四聚体核心工厂生产。然而，一些这些试剂，包括结合到免疫的Gag CM9表位马目-A1 * 001四聚体，只能从由于许可协议商业来源获得。四个猕猴等位基因使用的pMHC-I四聚体以上列出的，我们已经成功地枚举CD8 + T细胞对共21 SIV抗原表位（ 表1），这是诱导疫苗接种或主SIV感染30,31（马丁等人 ，未发表意见）。
本稿件提供优化的pMHC-I四聚体染色方案，用于确定在恒河猴SIV-特异性CD8 + T细胞的频率和存储器的表型。为了降低TCR内化，从而改善的pMHC-I四聚体染色32;该测定用择孵育30分钟与蛋白激酶抑制剂（这里，达沙替尼使用PKI）的开始。 01四聚体：如下所述，使用马目-B * 017时这种处理是非常有用的。还提供了关于如何标记与荧光标记的pMHC-I四聚体和单克隆抗体（mAb）的细胞的指示。该协议还包括用于将细胞溶解相关分子颗粒酶B（GZM B）的细胞内检测一个细胞透步骤。抗CD3的mAb在此步骤中加入，以及改进这种TCR信号分子的检测。作为参考，在这种染色面板使用的所有荧光被列出。
Protocol
Representative Results
Discussion
在此过程中值得讨论的有几个步骤，因为它们是产生最佳效果的关键。首先，由于生物样本的质量是任何流式细胞测定法38的成功的一个强有力的预测，都必须小心，以确保细胞是染色过程中存活，并在悬浮液中。与冷冻保存的样品时，因为它们更容易结块和通常含有死细胞的更高数量，这是特别相关的。在这些情况下，通过将细胞悬浮液通过的pMHC-I四聚体标记过程之前的70微米的细胞滤?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们要感谢大卫·沃特金斯支持，使该方法存在的优化实验。研究本出版物中报道了由迈阿密中心的美国国立卫生研究院艾滋病研究下奖号P30AI073961提供一个试点补助的部分资助。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。
Materials
| Dasatinib | Axon medchem | Axon 1392 | Must be resuspended in DMSO and immediately stored at -20°C |
| RPMI w/ Glutamax | gibco/ Life Technologies | 61870-036 | Must be stored at 4 °C |
| Heat Inactivated FBS | gibco/ Life Technologies | 10082-147 | Must be stored at 4 °C |
| Penicillin-Streptomycinp-Amphotericin B | Lonza | 17-745E | Must be stored at 4 °C |
| DMSO, Anhydrous | Life Technologies | D12345 | Store at room temperature. |
| 5-mL Round-Bottom Polypropylene Tubes | VWR | 60819-728 | |
| Fluorochrome-conjugated pMHC-I tetramers | NIH Tetramer Core or MBL, Inc. | Must be stored and maintained at 4 °C. Centrifuge at 20,000 x g for 15 min before use. Do not freeze. | |
| Fluorochrome-conjugated mAbs | Various companies | Must be stored and maintained at 4 °C. Do not freeze. | |
| LIVE/DEAD Fixable Aqua Dead Cell Stain Kit | Life Technologies | l34957 | Must be stored at -20 °C. Resuspend each aliquot in 50 μL of DMSO prior to use. |
| Brilliant Stain Buffer | BD Biosciences | 563794 | Must be stored at 4 °C |
| Phosphate Buffered Saline | VWR | 97064-158 | Store at room temperature |
| Albumine Bovine | VWR | 700011-230 | Must be stored at 4 °C |
| Sodium Azide | VWR | 97064-646 | Store at room temperature. Toxic substance. Do not mix with bleach. |
| Bleach | VWR | 89501-620 | Corrosive chemical, cannot be mixed with sodium azide. Handle with care |
| Paraformaldehyde | Electron Microscopy Sciences | 15714-S | Flammable, corrosive, and toxic reagent. Handle with care |
| Polysorbate 20 (Tween-20) | Alfa Aesar | L15029 | Store at room temperature |
| Permeabilization Solution 2 | BD Biosciences | 340973 | Toxic and corrosive reagent. Handle with care |
| Sarsdet Tubes 1.5mL screw top | VWR | 72.692.005 | |
| 2.0ml DNA/RNA Low bind Tubes | Eppendorf | 22431048 | The use of Sterile microtubes is preffered |
| Vortex mixer | To discretion of scientist | ||
| Biosafety Cabinet | To discretion of scientist | ||
| Milli Q Intergral Water Purification system | EMD Millipore | ZRXQ010WW | Molecular Biology grade water from any provider may be used |
| Microcentrifuge | To discretion of scientist | ||
| Centrifuge | To discretion of scientist | ||
| 4 °C refrigerator | To discretion of scientist | ||
| BD LSR II | BD Biosciences | Flow cytometer must contain lasers and filters that are compatible with the staining panel used. | |
| Deionized water | |||
| Aluminum Foil | VWR SCIENTIFIC INC. | 89068-738 | |
| Incubator | Must be able to maintain 37 °C internal temperature | ||
| FACS Diva software | BD Biosciences | ||
| Flowjo software version 9.6 | Flowjo | Used to analyze FCS files generated by FACS Diva software | |
| Micropippette tips |
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