A microfluidic platform for high performance sorting of immune cells based on their distinct secretion patterns - Project Summary Cellular heterogeneity limits the identification of regulatory networks that control specific cellular functions such as cell secretion. This is particularly challenging for deciphering cellular immunity as immune responses often involve intermediate extracellular signalling acting as autocrine or paracrine modulators. Characterizing such dynamic stimulus-response relationships by analyzing the genes and respective regulatory networks responsible for immune cell secretion at single-cell resolution would be valuable for better understanding and controlling immune responses and would significantly aid drug discovery. However, functional genetic screening required for the identification of genetic regulators of cell secretion dictates sorting large number of cells based on their secretion patterns, which is challenging with existing cell secretion approaches. In addition, performing single- cell analysis of immune cells within a population is critical to accurately decouple autocrine and paracrine secretion cascades, which is not possible via current cell segregation approaches. This proposal will leverage a new microfluidic platform for sorting cells based on their secretion with demonstrated robust performance for the identification of druggable regulators of cell secretion. Our technology – referred to as SECRE (Secretion- Enabled Cell Ranking and Enrichment) – can be used in conjunction with next-generation sequencing to identify the genetic regulators of cell secretion. In this proposal, we will apply the SECRE platform technology for studying multiple types of immune cells and their cell secretion products using a genome-wide CRISPR activation and/or knockout screening. The SECRE assay will be optimized for each cell type and deployed to sort immune cells based on the enhancement of cell secretion. This approach will be used to identify the druggable regulators of IL-2 and CXCL13 secretion by T cells, thus providing an innovative tool for controlling their proliferation and potency. In addition, this approach will be used to explore the druggable regulators of small lytic proteins by NK cells and immunosuppressive cytokine IL-10 by regulatory B cells to enhance their respective potencies, which would provide means to discover new therapeutics for multiple autoimmune diseases. Top screen hits will be selected using well established bioinformatic algorithms and validated through genetic perturbation-based gain-of-function and/or loss-of-function analyses. Using in-vitro functional assays, the cell-type specific secretory networks will be identified and evaluated for potential clinical utility and nominated for further in vivo investigations. The project deliverables will include a first-in-class system for high-performance cell sorting based on their secretory products at single-cell resolution that can be combined with functional genomics to accelerate the development of effective therapeutics for immune disease. This will have immense utility for bioanalysis and/or immunoengineering communities and is expected to enhance drug discovery toolkits. Lastly, this approach will impact the American population by accelerating the development of durable adoptive cell therapies.
