T cell activation requires force generation

Abstract

Triggering of the T cell receptor (TCR) integrates both binding kinetics and mechanical forces. To understand the contribution of the T cell cytoskeleton to these forces, we triggered T cells using a novel application of atomic force microscopy (AFM). We presented antigenic stimulation using the AFM cantilever while simultaneously imaging with optical microscopy and measuring forces on the cantilever. T cells respond forcefully to antigen after calcium flux. All forces and calcium responses were abrogated upon treatment with an F-actin inhibitor. When we emulated the forces of the T cell using the AFM cantilever, even these actin-inhibited T cells became activated. Purely mechanical stimulation was not sufficient; the exogenous forces had to couple through the TCR. These studies suggest a mechanical-chemical feedback loop in which TCR-triggered T cells generate forceful contacts with antigen-presenting cells to improve access to antigen.

Figure 1.

AFM delivery of antigenic stimulation. (A) Schematic showing AFM cantilever for stimulation of T cells and for monitoring mechanical responses. (B, left) Bright-field image of cantilever showing the dark silicon pad with the tip and the silicon nitride body of the cantilever. (right) Fluorescence image of fluorescent phycoerythrin-conjugated streptavidin assembled from a projection of multiple slices of spinning-disk confocal images. The cantilever tip is bright with labeled streptavidin (arrow). Bars, 50 µm. (C–I) Ca²⁺ responses to AFM-delivered stimulation. The AFM cantilever was brought into continuous contact with Fluo-4–labeled T cells for 180 s and imaged every 1 s. t = 0 is when the AFM force trigger was reached upon initial contact. Fluo-4 intensity was normalized to the Fluo-4 intensity at t = 0. Fluo-4 intensity in T cells contacted with a cantilever coated with anti-CD3 (C), pMHC (D), or irrelevant monoclonal antibody (hCD25 mAb; E). Shaded area under each curve represents the time-integrated calcium flux. (F–H) Fluorescent micrographs of the cells in C–E being touched at the start, peak flux, and end of experiment at the times shown. The cells are false colored according to the bar underneath. Bars, 5 µm. (I) Time-integrated Ca²⁺ flux shown for cantilevers coated with anti-CD3, pMHC, and control mAb. Data were pooled for pMHC (n = 15 across three independent experiments), anti-CD3 (n = 32 across nine independent experiments), and control antibody (n = 15 across four independent experiments). Each dot is a contact on one T cell. Box shows the bootstrapped mean and 95% CI. ns, not significant.

Figure 2.

Mechanical forces generated by AFM-delivered stimulation. Time course of force exerted on the cantilever during contact with the T cell. Graphs begin with the trigger point at t = 0. The 0-force baseline is set to the deflection at the trigger point. All cells are the same as in Fig. 1. Cantilever was coated with anti-CD3 (A) or irrelevant mAb (B). The lightly shaded area represents the push phase. The magnitude of the push force is the height of the peak from the base, calculated from the inflection. The darker shaded region represents the pull phase, with the magnitude of the pull calculated from the minimum of the region to the 0-force baseline. The time lags in G are calculated as shown by the dashed lines. Magnitudes of pushing (C) forces and pulling (D) forces for cells stimulated with anti-CD3–coated cantilevers versus control antibody–coated cantilevers. These data are from the same cells used in Fig. 1. Boxes show the bootstrapped mean and 95% CI. ns, not significant. Time-integrated calcium flux versus magnitude of pushing (E) and pulling (F) forces. Density contours are shown in gray. (G) Histograms show the time lag between the onset of calcium flux to the start of the pulling force for anti-CD3–coated and pMHC-coated cantilevers.

Figure 3.

Inhibitor treatments weaken force generation and Ca²⁺ flux through several mechanisms. Time courses of both force on the cantilever and normalized Fluo-4 intensity. The AFM cantilever was brought into continuous contact with Fluo-4–labeled T cells for 180 s and imaged every 1 s. Flux was normalized to the Fluo-4 intensity at t = 0, when the AFM force trigger was reached upon initial contact. Cells were treated with 1 µM LatA (A), 10 µM ML-7 (B), and 40 µM 2-APB (C). Data were pooled for LatA (n = 14 cells from four independent experiments), ML-7 (n = 10 cells from two independent experiments), and 2-APB (n = 10 cells from two independent experiments). Comparison of integrated calcium flux (D), pushing forces (E), and pulling forces (F) for cells with or without inhibitor treatment. The pooled anti-CD3 results are the same points as in Fig 2. ns, not significant.

Figure 4.

Application of cyclical force rescues Ca²⁺ flux in LatA-treated cells. (A) Z-position of cantilever during application of cyclical force. The ramp-up indicates lowering of the cantilever until the contact. The cantilever is held in place for 15 s and then begins a sinusoidal movement with amplitude 0.5 µm and period 2 s. After 180 s, the tip is again held constant for 15 s, then retracted. (B) Time courses for force on the cantilever and normalized Fluo-4 intensity for a cell treated with LatA subjected to the cyclical force in A. (C) Z-position of cantilever during the spaced cyclic force. Sinusoidal pulses from A were spaced out with constant dwells lasting 8 s, resulting in a waveform with a 10-s period. (D) Time-integrated calcium flux for LatA-treated cells with no external force, continuous cyclical force, with a spaced cyclical force or cyclical force with an irrelevant antibody. Data were pooled for LatA continuous contact (same points as Fig. 3 D), LatA cyclic contact (n = 20 cells from five independent experiments), LatA cyclic contact with control antibody (n = 8 cells from two independent experiments), and spaced cyclic contacts (n = 11 from two independent experiments). Box shows the mean and 95% CI.

Figure 5.

Cyclical force in LatA-treated cells does not increase the number of engagements. Amplitude of force oscillations over time during application of cyclical forces for LatA-treated cells with anti-CD3–treated (A) and control-treated (B) cells with pMHC-coated tips. For clarity, every fourth cycle is plotted, showing amplitude from maxima to minima. (C) Comparison of force amplitudes for the first cycle (1) and the final cycle (80), showing that treatment with LatA blocks an increase in force oscillations over the experiment. Data were pooled for cyclic contacts with pMHC without LatA (n = 5 cells from two independent experiments), cyclic contacts with anti-CD3 with LatA (same cells as Fig. 4), cyclic contacts with anti-CD43 (n = 5 cells), and cyclic contacts with anti-hCD25 (same cells as Fig. 4). ns, not significant.