Single Molecule Observation of TCR Signaling Complexes in Living T Cells

Abstract

T cell's main function is to detect foreign peptides in a sea of self-peptides displayed on antigen presenting cells via T cell receptors (TCR). Individual TCRs bind to foreign peptides with affinities that differ only slightly from that of self-peptides. As a result, each TCR is likely to bind to a subset of self-peptides whose ever-present signal can overwhelm the signals from genuine foreign peptides. However, previous results demonstrate that T cells can be triggered by a few foreign peptides among thousands of self-peptides. In fact, erroneous triggering of T cells by self-peptides results in autoimmune diseases such as rheumatoid arthritis, Crohn's disease, and some forms of diabetes. This implies that the decision to activate at the cellular level is highly sensitive and selective, and the decision is likely to be based on a combination of factors, such as binding affinity, number of interactions, and spatial and temporal distribution of the signal. While the molecular interaction of TCR and its ligand has been well studied in vivo, any correlations between the binding kinetics and cellular responses are lost within population behaviors. Thus, the precise physical mechanism by which TCR-peptide binding leads to the triggering of T cells remains unclear. Elucidating the molecular mechanism of this process requires simultaneous measurement of the binding kinetics, stoichiometry, and movement of individual signaling molecules in living T cells. In this project, we directly observe molecular interactions within TCR-ligand complexes with single molecule resolution and correlate it with subsequent single cell responses in hybrid junctions between live primary T cells and supported lipid membranes.