UC Berkeley

Recognition of antigen by T cells occurs at the junction between a T cell and an antigen-presenting cell. Within this highly constrained interface, the T cell receptor (TCR) interacts with its cognate antigen peptide major histocompatibility complex (pMHC). Additional signaling and adhesion molecules present in this intercellular environment influence its overall topography and geometry, which can significantly impact receptor:ligand binding events. The work presented in this dissertation describes three studies aimed at making direct, in situ measurements of molecular interactions during T cell signal transduction. For all of these studies, I use a hybrid live T cell-supported membrane system to mimic the physiological juxtacrine signaling environment and single molecule fluorescence microscopy to monitor individual receptor:ligand binding events. The supported membrane is functionalized with fluorescently-tagged pMHC and the formation of individual pMHC:TCR complexes is observed via time-resolved single molecule techniques. Using these approaches, I report live cell, two-dimensional pMHC:TCR kinetics and affinity measurements in self-reactive human T cell clones and discuss discrepancies between my results and those determined via mechanical assays. I also describe a novel method for simultaneously monitoring pMHC:TCR binding and downstream cellular response in a single cell. This strategy allows the measurement of all pMHC:TCR interactions leading to T cell activation and reveals a cumulative dwell time threshold required for activation. In order to better understand the molecular interactions of the costimulatory protein CD80, I have developed a novel CD80-SNAP-tag reagent suitable for single molecule receptor:ligand binding studies. I discuss the design, characterization, and application of this reagent, which has been used to demonstrate global environmental changes in the membrane-membrane interface during T cell triggering. Together, these studies emphasize the importance of investigating ligand binding in a physiologically relevant context and will contribute to a better understanding of the early stages of T cell signaling.