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组成部分，在调节一系列 身体机能--特别是免疫力。尽管它们意义重大，但对单一细胞分泌物的研究- 由于缺乏可访问的技术，细胞水平一直受到限制。理想的化验方法应该是单细胞 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。不是 一种化验方法就能满足所有这些要求。缺乏识别和审问细胞的方法 高水平的分泌能力有效地阻碍了对这些潜在的高潜能细胞的研究 用于细胞治疗。在单细胞水平上表征细胞分泌的最常用技术是 然而，几十年前的ELISpot技术，这项技术可以产生的分泌物数量有限 并且不能根据检测结果对细胞进行分选。因此，迫切需要发展 可访问的技术，提供在高度多路复用的情况下对信元进行功能性筛选和恢复的能力 分泌形式。为了解决这一差距，我们将设计纳米小瓶，即含有空腔的水凝胶颗粒，以创造 一种强大的5-plex分析模式，能够测量单个细胞中的五种分泌细胞因子，支持几种 不同的工作流程，如稀有多功能T细胞分选，或按功能筛选其他免疫细胞。在 这个项目的首要目标是，我们将通过以下方式开发与5-复合体分泌物检测相兼容的纳米小瓶制剂 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，并 干扰素-γ、肿瘤坏死因子-α、粒-巨噬细胞集落刺激因子、白介素2和颗粒酶B的修饰化学计量法。 将通过比较人类白细胞抗原相合的PBMC的单细胞分泌物来验证纳米瓶单细胞分析 通过使用ELISpot和开发的纳米小瓶检测的多肽呈现而激活。成功完成 我们的目标将解决传统的基于分泌物的单细胞图谱的关键空白，如ELISpot或 细胞内细胞因子染色。这种多重分析将使研究人员能够高度区分和恢复 功能细胞，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.