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

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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.