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

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 microm/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.

Fig 1.

Initial polarization and antigen-specific repolarization of CD3ζGFP in D10 T cell clones. T cells transfected with CD3ζGFP were imaged at 15-s intervals during contact with 10 μM CA-134-146-peptide-pulsed APCs. Data show the FURA ratio overlayed with the contrast image and the GFP intensity in a mid z plane at the indicated times relative to first contact. For a movie version of this figure, see Movie 1, which is published as supporting information on the PNAS web site, www.pnas.org.

Fig 2.

3D data segmentation and normalized densities of CD3ζGFP during antigen-specific repolarization. (A) An example of segmentation showing a widefield fluorescence image (Left) and the binary map of captured pixels (Right). (B) A 2D coordinate system for viewing data. Pixel intensities were mapped to polar coordinates (θ, φ), where θ = 0° (front of the T cell) is the vector from the center of the T cell to the center of the B cell, and corresponds to the center of the polar plot. θ = 180° (the rear of the cell) corresponds to the circumference of the polar plot. φ (−180° ≤ φ ≤ 180°) is the azimuthal angle. (C and D) 2D graphical representation of time dependent TCR clustering. Black and red represent low and high intensities. A diffuse TCR distribution before flux (C) becomes a clustered distribution within 10 min of flux (D).

Fig 3.

Instantaneous velocities of CD3 molecules in a developing synapse assessed by 3D quantitation. Calculated temporal and angular dependence of three different velocity measures for three T cells. The “Receptor” velocity describes movement of the receptors, with respect to the membrane motion. The “Membrane” velocity describes only the movement of cell membrane. The “Total” velocity is the sum of the Membrane and Receptor velocities. The data for the three cells show two distinct features: (i) early time velocities can be attributed to a conformational wave that transits the membrane (compare black, blue, and red Membrane and Total velocities); and (ii) preflux Receptor velocities are small, followed by a large increase in receptor motion toward the synapse between 0 and 30 s after flux. Note that negative velocities are toward the synapse.

Fig 4.

Single particle tracking of TCR recruitment to the nascent immunological synapse. Example time-lapse positions of nanosphere-labeled TCRs. Coordinates of each nanosphere in (x, y, z) space were determined from 3D fluorescence images, and the distance from each particle to the interface was calculated by using differential interference contrast images to determine the interface location. Shown are the distances of each particle from the interface at each time point normalized by the length of the cell of individual nanospheres on single T cells responding to APCs pulsed with: •, 10 μM CA 134-146; ○, 1 μM CA 134-146; ▴, 10 μM CA 134-146-E8T. Representative movies of responses to agonist and weak-agonist recruitment are published as Movies 1–3 on the PNAS web site.

Fig 5.

Velocities of TCR as measured by single particle tracking. Instantaneous velocities of nanosphere-labeled TCRs as a function of time were measured by tracking position changes as a function of time. Motion of the tail of T cells was subtracted to correct for translation of the whole cell, as described in Methods. Shown are mean velocities of nanospheres (n ≥ 10) as a function of the time relative to initial intracellular calcium elevation for TCRs on T cells responding to APCs pulsed with 10 μM CA (Top), 1 μM CA (Middle), or 10 μM CA-E8T (Bottom).