Multiplexed analysis of secreted proteins from single-cells using high dynamic range nanovials

摘要 细胞分泌物构成了蛋白质组的重要部分，在控制一系列蛋白质代谢中起着至关重要的作用。 身体功能-特别是免疫力。尽管它们的意义，在细胞分泌物的研究，在单一的- 由于缺乏可获得的技术，细胞水平受到限制。理想的测定将具有单细胞 分离，测量几种分泌产物，并允许回收细胞用于进一步的下游研究。没有 一种测定法满足所有这些要求。缺乏识别和询问细胞的方法， 显示出高水平的分泌能力，有效地阻碍了对这些潜在的高效细胞的研究。 用于细胞疗法。在单细胞水平上表征细胞分泌的最常见技术是 然而，几十年前的ELISpot技术，这种技术在可以被检测的分泌物的数量上是有限的。 细胞不能被测量并且不能基于测定的结果被分选。因此，迫切需要发展 这些技术提供了在高度多路复用的环境中功能性地筛选和回收细胞的能力。 分泌形式。为了解决这一差距，我们将设计纳米管，含有空腔的水凝胶颗粒， 一个强大的5重测定格式，能够测量五个分泌的细胞因子从单细胞，支持几个 不同的工作流程，如罕见的多功能T细胞分选，或其他免疫细胞功能筛选。在 该项目的第一个目标是，我们将开发与5重分泌测定相容的纳米管制剂， 增加了用于分泌捕获的结合位点的数量。我们还将确定合适的抗体对， IFN-γ、TNF-α、GM-CSF、IL-2和粒酶B的修饰化学计量。在第二个目标中， 将通过比较HLA匹配的PBMC的单细胞分泌物来验证纳米管单细胞测定法 使用ELISpot和开发的纳米管测定法通过肽呈递活化。成功完成 我们的目标将解决传统的基于分泌的单细胞分析中的关键差距，如ELISpot或 细胞内细胞因子染色。这种多重检测将使研究人员能够区分和恢复高度 这将刺激基础研究和治疗开发的创新。

ABSTRACT Cellular secretions make up a significant portion of the proteome, playing a crucial role in governing a range of bodily functions – in particular immunity. Despite their significance, the study at cellular secretions at the single- cell level has been limited due to a lack of accessible technologies. An ideal assay would have single-cell resolution, measure several secreted products, and allow for recovery of cells for further downstream study. No one assay fulfills all of these requirements. The lack of methodologies to identify and interrogate cells which display high levels of secretory capacity has effectively stalled research into these potentially highly potent cells for cell therapies. The most common technique to characterize cell secretion at the single-cell level is the decades old ELISpot technology, however, this technology is limited in the number of secretions that can be measured and cells cannot be sorted based on the results of the assay. Thus, there is a critical need to develop accessible technologies which offer the capability to functionally screen and recover cells in a highly multiplexed secretion format. To address this gap, we will engineer nanovials, cavity-containing hydrogel particles, to create a robust 5-plex assay format capable of measuring five secreted cytokines from single cells, supporting several different workflows such as rare polyfunctional T cell sorting, or other immune cell screening by function. In the first aim of this project, we will develop nanovial formulations compatible with a 5-plex secretion assay by increasing the number of binding sites for secretion capture. We will also identify suitable antibody pairs and modification stoichiometry for IFN-gamma, TNF-alpha, GM-CSF, IL-2, and Granzyme B. In the second aim we will validate the nanovial single-cell assays by comparing the single-cell secretions of HLA-matched PBMCs activated by peptide presentation using both ELISpot and the developed nanovial assays. Successful completion of our aims will address critical gaps in traditional secretion-based profiling of single cells such as ELISpot or intracellular cytokine staining. This multiplexed assay will enable researchers to discriminate and recover highly functional cells which will spur innovation in both fundamental research and therapeutic development.