(19e) Elucidating T-Cell Activation Threshold Using Precisely Defined Three-Dimensional Artificial Antigen Presentation

Mason Smith, Fei Wen Adaptive immune response is shaped in large part by T cells recognizing and binding cognate antigen on the surface of antigen presenting cells (APCs). While much is known about the behavior of T cells during an immune response, the exact molecular mechanism that translates peptide-major histocompatibility complex (pMHC)- T-cell receptor (TCR) binding to T cell activation remains a controversial topic in immunology. Several studies using antigen presenting fluid lipid bilayers have attempted to shine a light on this mechanism by looking at the minimum number of pMHC required to initiate T cell activation. These studies have found that the early steps of T cell activation (TCR triggering) can be initiatedby as few as three agonist pMHCs in a membrane, and that there exists a global pMHC density threshold below which T cell activation does not occur. While these studies have provided unique insights into TCR triggering, their predicted T-cell activation thresholds vary in planar intermolecular pMHC spacing from 34 nm to greater than 150 nm. Further, conflicting results regarding the importance of local pMHC density and total pMHC encountered have been reported. Planar fluid lipid bilayers provide robust and scalable experimental systems with incredibly fine control over molecular density; however, it is not immediately clear if their results can be extrapolated to describe T cell response to pMHC on a three-dimensional cell surface. Additionally, planar pMHC display systems assume sustained interfacial contact between a T cell and a flat antigen-presenting surface and are typically characterized by some uncertainty in the number of pMHC molecules immobilized in each defined nanoclusters.

We have engineered a novel artificial antigen presentation system that provides precise control over the local and global arrangement of pMHC molecules on the surface of a living cell using recombinant scaffold protein. Using this artificial antigen presentation system, we are able to display thousands of clusters of up to six individual pMHCs with single-molecule precision on a cell surface. Further, cells presenting unique local pMHC densities can be sorted using fluorescence activated cell sorting (FACS) to isolate cells presenting different global pMHC display levels. By using FACS to isolate cells with the same global pMHC display level but discretely different pMHC cluster size, we can study T cell response to a wide range of local and global pMHC densities in a three-dimensional cell-culture environment. Preliminary T-cell activation results using sorted artificial antigen presenting cells support the existence of a minimum global pMHC display threshold for T cell activation, which is different from that reported using planar lipid bilayers. More importantly, they suggest that local pMHC valency can compensate for a reduction in the global pMHC display level. These results suggest that while two-dimensional systems for in vitro T cell activation present an important, scalable research platform, quantitative thresholds for T cell activation derived from these systems may underestimate the activation threshold in physiologically relevant systems.