Quantifying signaling-induced reorientation of T cell receptors during immunological synapse formation

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Abstract
Productive T cell recognition of antigen-presenting cells (APCs) is normally accompanied by the formation of a cell–cell contact called the “immunological synapse.” Our understanding of the steps leading up to this formation has been limited by the absence of tools for analyzing 3D surfaces and surface distributions as they change over time. Here we use a 3D fluorescence quantitation method to show that T cell receptors are recruited in bulk within the first minute after the onset of activation and with velocities ranging from 0.04 to 0.1 μm/s; a speed significantly greater than unrestricted diffusion. Our method reveals a second feature of this reorientation: a conformational change as the T cell pushes more total membrane into the interface creating a larger contact area for additional receptors. Analysis of individual T cell receptor velocities using a single-particle tracking method confirms our velocity measurement. This method should permit the quantitation of other dynamic membrane events and the associated movement of cell-surface molecules.