Imaging of cell-cell communication in a vertical orientation reveals high-resolution structure of immunological synapse and novel PD-1 dynamics

The immunological synapse (IS) is one of the most pivotal communication strategies in immune cells. Understanding the molecular basis of the IS provides critical information regarding how immune cells mount an effective immune response. Fluorescence microscopy provides a fundamental tool to study the IS. However, current imaging techniques for studying the IS cannot sufficiently achieve high resolution in real cell-cell conjugates. In this study, we present a new device that allows for high-resolution imaging of the IS with conventional confocal microscopy in a high-throughput manner. Combining micropits and single-cell trap arrays, we have developed a new microfluidic platform that allows visualization of the IS in vertically "stacked" cells. Using this vertical cell pairing (VCP) system, we investigated the dynamics of the inhibitory synapse mediated by an inhibitory receptor, programed death protein-1, and the cytotoxic synapse at the single-cell level. In addition to the technique innovation, we have demonstrated novel biological findings by this VCP device, including novel distribution of F-Actin and cytolytic granules at the IS, programed death protein-1 microclusters at the NK IS, and kinetics of cytotoxicity.We propose that this high-throughput, cost-effective, easyto-use VCP system, along with conventional imaging techniques, can be used to address a number of significant biological questions in a variety of disciplines.