(52g) Functional Single-Cell Analysis of T-Cell Activation by Supported Lipid Bilayer Tethered Ligands On Arrays of Nanowells

The use of ligands immobilized on supported lipid bilayers (SLBs) has been an invaluable tool for characterizing T cell receptor (TCR)-mediated activation and re-distribution of associated membrane-expressed proteins during the formation of the immunological synapse. The use of bilayers on planar supports and fluorescent visualization of protein dynamics is limited in its ability to determine other activation-dependent cellular responses such as cytokine secretion that occur on timescales longer than the typical periods of observation for molecular dynamics. We have developed a method to measure the functional responses of individual T cells when engaged by lipid bilayers supported on an elastomeric array of subnanoliter wells (nanowells). In this approach, each nanowell artificially mimics an antigen-presenting cell (APC), thus allowing the formation of a T cell synapse upon contact with the surface. After cell activation by membrane-bound ligands, secretion of cytokines from single T cells were captured and quantified by microengraving in both antigen-independent and dependent manners. This method thus allows a flexible system for relating molecular events in TCR signaling to complex functional responses such as cytokine secretion and cell proliferation.