对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术
Summary
We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.
Abstract
Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a “pathogen-mimicking” nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.
Introduction
传统疫苗的发展主要采用试错的经验方法。但是，与生物材料和发现免疫激活的分子决定因素的各种各样的最近发展，现在有可能合理设计疫苗制剂与来自病原体1,2-衍生生物物理和生物化学的线索。特别地，各种微粒药物递送平台已经被检查的，因为它们可以被共装载亚基的抗原和免疫刺激剂的疫苗载体，保护疫苗成分免于降解，并提高它们的共同递送至抗原呈递细胞（APC）存在于淋巴节点（逻辑节点），从而最大限度地提高免疫刺激和活化3-5。在这份报告中，我们描述了“病原体模仿”纳米颗粒系统的合成，称为interbilayer交联多层囊泡（ICMVs），先前已经被证明是一种有效的疫苗platfor米为健壮的细胞毒性T淋巴细胞（CTL）和在全身和粘膜组织区室6-9体液免疫应答诱导。特别是，接种ICMVs取得了实质性的提高血清IgG水平对疟疾抗原，与接种常规助剂（ 如明矾和Montanide中）7相比，也引发对肿瘤细胞和小鼠9病毒攻击模式有效的CTL反应。在这里，用ICMVs作为模型疫苗纳米粒子系统，我们描述的方法对疫苗的纳米制剂特性，包括粒径和zeta电位测量和粒子贩卖跟踪，以引流淋巴结（DLNS）利用冰冻切片组织7的共聚焦成像。此外，我们提出了荧光素酶表达的抗原特异性CD8 + T细胞9,10继转移后分析在小鼠中膨胀CTL反应的全动物基于成像的方法。最后，我们去外周血单核细胞（PBMC），用于在接种纳米粒子6,9-小鼠内源性T细胞应答纵向量化的划线四聚体染色。
ICMVs是一个基于脂质的纳米颗粒制剂通过简单的脂质体受控聚变合成为多层结构，然后将其化学地交联的马来酰亚胺官能化的磷脂头部基团稳定化的内脂质层与二硫酚交联剂6。一旦ICMVs合成中，纳米粒子的一小部分可以被用于确定颗粒尺寸和ζ电势（ 即，表面的粒子的电荷）与动态光散射（DLS）系统和ζ电位分析仪。 DLS措施中的光散射颗粒悬浮液的变化，允许确定扩散系数和粒子11的流体动力学大小。获得一致的粒径不同批次的合成是关键由于粒径是通过的APC 12,13影响疫苗颗粒的淋巴引流到DLNS和随后的细胞摄取的主要因素之一。此外，ζ电势可以通过测量当电流被施加在粒子速度，这允许确定颗粒和颗粒表面电荷11的电泳迁移率来获得。确保颗粒一致ζ电位值是重要的，因为颗粒表面电荷确定胶体稳定性，其具有在储存过程中和在体内给药14,15后在颗粒分散体直接影响。为了跟踪的粒子定位到DLNS，ICMVs可以带有所需荧光团包括亲脂性染料和共价标记的抗原。免疫接种后，小鼠可以在不同时间点进行安乐死，DLNS切除，冰冻切片，并用共聚焦显微镜分析。这种技术允许淋巴的DraⅠ可视化宁纳米颗粒疫苗载体和抗原DLNS两者。组织切片可另外被染色用荧光标记的抗体和用于获得详细信息，诸如种类与抗原和形成生发中心有关的细胞，因为我们先前7所示。
一旦粒子合成进行了优化，并贩卖给DLNS一经确认，以验证在体内 CTL扩张启发是非常重要的。为了分析抗原特异性CD8 + T细胞的诱导响应于纳米颗粒疫苗，我们已利用模型抗原，卵白蛋白（OVA），以OVA 257-264肽（SIINFEKL）免疫显性CD8 + T细胞表位，它允许详细的免疫学分析对于最初疫苗开发16,17抗原特异性T细胞应答。特别是，以询问膨胀和抗原特异性CD8 + T细胞的迁移的动力学，我们已经产生了双转基因小鼠模型通过杂交萤火虫荧光素酶表达的转基因小鼠（吕克）与OT-I转基因小鼠具有CD8 + T细胞与T细胞受体（TCR）特异性针对SIINFEKL（在用H-2K B缔合 ）。从这些的OT-I /吕克小鼠，荧光素酶表达，OT-I CD8 + T细胞可以被分离并用于过继转移进入幼稚C57BL / 6小鼠制备。一旦晶种，成功免疫用含有OVA纳米颗粒将导致扩张的转移的T细胞，其可以通过与一个整体动物成像系统9,10监测的生物发光信号来跟踪的。这种非侵入性全身成像技术已用于与其他的病毒或肿瘤抗原，在过去18至20，露出参与T细胞扩增在淋巴组织和传播至外周组织中的纵向的方式处理。
互补继转移抗原特异性CD8 + T细胞的分析，endogeno我们的T细胞应答接种后可以检查与肽-主要组织相容性复合体（MHC）四聚体测定21，其中一个肽-MHC四聚体复合物，由四个荧光团标记的MHC-I类分子装载肽表位，采用结合TCR和标签CD8 + T细胞以抗原特异性方式。肽的MHC四聚体分析可以在终端尸检研究，以确定抗原特异性CD8 + T细胞在淋巴和外周组织或与来自串行抽血获得外周血单核细胞（PBMC）的纵向研究来进行。染色的淋巴细胞与肽-MHC四聚物之后，流式细胞仪分析，执行对CTL的CD8 + T细胞中的频率的表型或定量的详细分析。
Protocol
Representative Results
Discussion
本文中提供的协议说明了合成和一个新的基于脂质的纳米颗粒系统的表征，称为ICMVs，并提供验证的基于纳米颗粒的疫苗制剂的有效性，以诱导抗原特异性CD8 + T细胞应答的过程。 ICMV合成以全含水的条件，这是一个重大的优点与其他常用的聚合物纳米颗粒系统（ 例如 ，聚（丙交酯-共-乙）酸颗粒），通常需要为制备有机溶剂中，经常造成的损失相比，完成在抗原蛋白质抗原29,30。此…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究是支持美国国立卫生研究院授予1K22AI097291-01和国家中心推进美国国立卫生研究院的转化科学奖下数UL1TR000433。我们也承认欧文达雷尔教授在麻省理工学院和斯蒂芬·马蒂亚斯在教授弗雷德·哈钦森癌症研究中心为他们的对疫苗的纳米粒子和OT-I /吕克·转基因小鼠的初步工作所作的贡献。
Materials
| 1. Synthesis and characterization of ICMVs co-loaded with protein antigen and adjuvant molecules | |||
| 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (sodium salt) (MPB) | Avanti Polar Lipids, INC. | 870012 | |
| 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) | Avanti Polar Lipids, INC. | 850375 | |
| Monophosphoryl Lipid A (Synthetic) (PHAD™) (MPLA) | Avanti Polar Lipids, INC. | 699800 | |
| 20 mL glass vials | Wheaton | 0334125D | |
| Symphny Vacuum Oven | VWR | 414004-580 | |
| Ovalbumin (OVA) | Worthington | 3054 | |
| Bis-Tris Propane (BTP) | Fisher | BP2943 | |
| Q125 Sonicator (125W/20kHz) | Qsonica | Q125-110 | |
| Dithiothreitol (DTT) | Fisher | BP172 | |
| 2 kDa Thiolated Polyethylene Glycol (PEG-SH) | Laysan Bio | MPEG-SH-2000-1g | |
| Malvern ZetaSizer Nano ZSP | Malvern | ||
| ZetaSizer Cuvettes | Malvern | DTS1070 | |
| 2. Examination of lymph node draining of fluorescence-tagged ICMVs with confocal microscopy | |||
| 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt (DID) | Life Technologies | D-7757 | |
| Alexa Fluor 555-succinimidyl ester (AF555-NHS) | Life Technologies | A37571 | |
| Tissue-Tek OCT freezing medium | VWR | 25608-930 | |
| Tissue Cryomolds | VWR | 25608-922 | |
| 3. Monitoring expansion of antigen-specific, luciferase-expressing CD8+ T cells after nanoparticle vaccination with whole animal imaging | |||
| C57BL/6 mice | Jackson | 000664 | |
| Albino C57BL/6 mice | Jackson | 000058 | |
| OT-1 C57BL/6 mice | Jackson | 003831 | |
| 70 μm nylon strainer | BD | 352350 | |
| EasySep™ Mouse CD8+ T Cell Isolation Kit | StemCell | 19853 | |
| IVIS® whole animal imaging system | Perkin Elmer | ||
| 4. Peptide-MHC tetramer staining of peripheral blood mononuclear cells (PBMCs) for flow cytometric analysis of antigen-specific CD8+ T cells | |||
| K2EDTA tubes | BD | 365974 | |
| ACK lysis buffer | Life Technologies | A10492-01 | |
| Anti-CD16/32 Fc Block | Ebioscience | 14-0161-86 | |
| H-2Kb OVA Tetramer | MBL | TS-5001-1C | |
| Anti-CD8-APC | BD | 553031 | |
| Anti-CD44-FITC | BD | 553133 | |
| Anti-CD62L-PECy7 | Ebioscience | 25-0621-82 | |
| 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) | SIGMA | D8417-10MG | |
| CyAn Flow Cytometer | Beckman Coulter | ||
| FlowJo Software | FlowJo |
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