SNAP-tag Fusion Proteins as a Platform for Studying T Cell Costimulation

Abstract

The costimulatory molecules CD80 and CD86 regulate T cell signaling through their interactions with CD28 on the T cell surface. Reports indicate that costimulation increases T cell activation and lowers the TCR triggering threshold. The regulatory role of these proteins is implicated in autoimmune disease and has been exploited in clinical therapies. Despite the biomedical importance of this process, the physical mechanism by which CD28 modulates T cell signaling is unknown. We have developed a platform to study various aspects of T cell costimulation using SNAP-tag fusions of CD80 and CD86. This approach provides a means for selective functionalization of the ligands through the SNAP-tag domain. The costimulatory molecules are displayed on a supported lipid bilayer (SLB) mimicking the antigen-presenting cell. By labeling each SNAP-tag with a single fluorophore, we can characterize protein mobility and dimerization via fluorescence correlation spectroscopy and photon counting. The SLB can also be chemically functionalized with peptide-MHC and ICAM-1 to trigger the activation of primary T cells interacting with the artificial membrane. Using this live T cell-supported membrane system and high-resolution imaging, we can quantify the effects of CD28- mediated costimulation on early T cell signaling.