(3ah) Integrative Microengraving Process and Immuno-HCR for Ex-Vivo, Highly Sensitive and Multifunctional Characterization of Rare Immune Cells In Periphery

The human immune system comprises a diverse set of cells that contribute to a productive immunological response. Characterizing these dynamic behaviors for individual cells is critical in gaining insights how immune system operates. As yet, current bioanalytical technologies suffer from challenges in resolving the diverse functional activities and identities of individual subsets of cells as well as limits of signal detection and number of output parameters concurrently detectable in the assay. Recent success in nano and micro technologies has brought new insights to improve bioanalytical assays to utilize them in studying single cells. Miniaturization of biological assays using nano and micro technologies has enabled single cell resolution assays overcoming many challenges that traditional biological assays carry on. However, there is still a need to improve the integration of present single-cell analytical tools based on nano and micro technologies that would offer new insights into their operational variability and phenotypic diversity that are difficult to define with stand-alone analytical tools and bulk correlations. This talk will describe a new technology integrating microengraving-a soft lithographic method for printing arrays of secreted proteins from thousands of single cells-with an in situ signal amplification strategy called immuno-HCR, which antibodies are covalently modified with specific oligonucleotides that initiate hybridization chain reaction (HCR) with hairpins labeled with fluorescent molecules that the intensity of the resulting signal doubles with each extension. This new method makes it possible to detect up to 16 different cytokines and chemokines secreted from individual, viable human immune cells with subpicogram sensitivity. In application of this new technology, we will demonstrate measurements of the antigen-dependent, HLA-restricted responses by viable CD4⁺ T cells co-loaded with antigen-pulsed dendritic cells. Specifically, these multiplexed measures allow quantitative measures of the rates of secretion and number of cells responding for eight different secreted proteins concurrently (MIP-1β, IL-2, IL-4, IL-9, IL-10, IL-17, TNF-α, and IFN-γ). This integrated approach for multidimensional single-cell analysis significantly expands the breadth of multifunctional states enumerated for single cells and suggests a new strategy for ex vivo, high-throughput screening of antigen-specific human T cells and may provide new insights in understanding and characterizing the contributions from rare cells in various immune diseases.