Effects of Intracellular Calcium and Actin Cytoskeleton on TCR Mobility Measured by Fluorescence Recovery

Background: The activation of T lymphocytes by specific antigen is accompanied by the formation of a specialized signaling region termed the immunological synapse, characterized by the clustering and segregation of surface molecules and, in particular, by T cell receptor (TCR) clustering. Methodology/Principal Findings: To better understand TCR motion during cellular activation, we used confocal microscopy and photo-bleaching recovery techniques to investigate the lateral mobility of TCR on the surface of human T lymphocytes under various pharmacological treatments. Using drugs that cause an increase in intracellular calcium, we observed a decrease in TCR mobility that was dependent on a functional actin cytoskeleton. In parallel experiments measurement of filamentous actin by FACS analysis showed that raising intracellular calcium also causes increased polymerization of the actin cytoskeleton. These in vitro results were analyzed using a mathematical model that revealed effective binding parameters between TCR and the actin cytoskeleton. Conclusion/Significance: We propose, based on our results, that increase in intracellular calcium levels leads to actin polymerization and increases TCR/cytoskeleton interactions that reduce the overall mobility of the TCR. In a physiological setting, this may contribute to TCR re-positioning at the immunological synapse

Systems Imaging of the Immune Synapse

Three-dimensional live cell imaging of the interaction of T cells with antigen presenting cells (APC) visualizes the subcellular distributions of signaling intermediates during T cell activation at thousands of resolved positions within a cell. These information-rich maps of local protein concentrations are a valuable resource in understanding T cell signaling. Here, we describe a protocol for the efficient acquisition of such imaging data and their computational processing to create four-dimensional maps of local concentrations. This protocol allows quantitative analysis of T cell signaling as it occurs inside live cells with resolution in time and space across thousands of cells

T cell receptor (TCR) clustering in the immunological synapse integrates TCR and costimulatory signaling in selected T cells

During T cell activation, T cell receptors (TCR) cluster at the center of the T cell/antigen-presenting cell interface forming a key component of the immunological synapse. The function of this TCR clustering is still unresolved. A comprehensive search for such a function yielded a very limited and specific result. A micrometer-scale receptor clustering integrated the TCR and CD28 signals required for IL-2 secretion in primary 5C.C7 T cells, a low-affinity/avidity TCR system. 5C.C7 TCR signaling itself was not affected. In addition, central TCR accumulation was not required for any T cell effector function tested in three other TCR transgenic models. Central TCR accumulation thus had a specific role in signaling integration in low-affinity T cells