Whole-animal imaging and flow cytometric techniques for analysis of antigen-specific CD8+ T cell responses after nanoparticle vaccination.
Whole-animal imaging and flow cytometric techniques for analysis of antigen-specific CD8+ T cell responses after nanoparticle vaccination.
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DOI：
10.3791/52771
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发表时间：
2015-04-29
期刊：
影响因子：
--
通讯作者：
Moon JJ
中科院分区：
文献类型：
--
作者：
Ochyl LJ;Moon JJ
传统的疫苗佐剂，如明矾，会引起次优的CD8+ T细胞反应。为了解决疫苗开发中的这一重大挑战，人们设计了各种纳米颗粒系统来模拟病原体的特征，以改善抗原向引流淋巴结的递送，并增加抗原呈递细胞对抗原的吸收，从而产生了针对抗原特异性CD8+ T细胞反应进行优化的新疫苗配方。在这篇文章中，我们描述了一种“病原体模拟”纳米颗粒系统的合成，称为双层交联多层囊泡（ICMVs），它可以作为一种有效的疫苗载体，共同递送亚基抗原和免疫刺激剂，并引发有效的细胞毒性CD8+ T淋巴细胞（CTL）反应。我们描述了用动态光散射和zeta电位分析仪表征疫苗纳米颗粒的水动力大小和表面电荷的方法，并提出了一种基于共聚焦显微镜的方法来分析纳米颗粒介导的抗原递送到引流淋巴结。此外，我们展示了一种新的生物发光全动物成像技术，利用表达荧光素酶的抗原特异性CD8+ T细胞过继转移到受体小鼠中，然后使用纳米颗粒疫苗接种，可以实时对ctl的扩增和运输模式进行无创询问。我们还描述了四聚体染色和流式细胞术分析外周血单核细胞纵向定量内源性T细胞反应接种纳米颗粒小鼠。
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.