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

摘要 细胞分泌物构成蛋白质组的重要组成部分，在控制一系列蛋白质方面发挥着至关重要的作用 身体机能——特别是免疫力。尽管具有重要意义，但对单细胞分泌物的研究 由于缺乏可用的技术，细胞水平受到限制。理想的检测方法是单细胞 分辨率，测量几种分泌产物，并允许回收细胞以进行进一步的下游研究。否 一种测定可以满足所有这些要求。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用技术是 已有数十年历史的 ELISpot 技术，然而，该技术可检测的分泌物数量有限。 测量并且不能根据测定结果对细胞进行分选。因此，迫切需要开发 易于使用的技术，能够在高度多重的条件下对细胞进行功能性筛选和回收 分泌形式。为了解决这一差距，我们将设计纳米瓶，即含有空腔的水凝胶颗粒，以创造 强大的 5 重检测格式，能够测量单个细胞的五种分泌细胞因子，支持多种 不同的工作流程，例如罕见的多功能 T 细胞分选，或按功能筛选其他免疫细胞。在 该项目的第一个目标是，我们将开发与 5 重分泌测定兼容的纳米瓶制剂 增加分泌物捕获的结合位点的数量。我们还将确定合适的抗体对和 IFN-γ、TNF-α、GM-CSF、IL-2 和粒酶 B 的化学计量修改。在第二个目标中，我们 将通过比较 HLA 匹配的 PBMC 的单细胞分泌来验证纳米瓶单细胞测定 使用 ELISpot 和开发的纳米瓶测定法通过肽呈递激活。顺利完成 我们的目标将解决传统基于分泌的单细胞分析（例如 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高度区分和恢复 功能细胞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.