Force generation upon T cell receptor engagement

Abstract

T cells are major players of adaptive immune response in mammals. Recognition of an antigenic peptide in association with the major histocompatibility complex at the surface of an antigen presenting cell (APC) is a specific and sensitive process whose mechanism is not fully understood. The potential contribution of mechanical forces in the T cell activation process is increasingly debated, although these forces are scarcely defined and hold only limited experimental evidence. In this work, we have implemented a biomembrane force probe (BFP) setup and a model APC to explore the nature and the characteristics of the mechanical forces potentially generated upon engagement of the T cell receptor (TCR) and/or lymphocyte function-associated antigen-1 (LFA-1). We show that upon contact with a model APC coated with antibodies towards TCR-CD3, after a short latency, the T cell developed a timed sequence of pushing and pulling forces against its target. These processes were defined by their initial constant growth velocity and loading rate (force increase per unit of time). LFA-1 engagement together with TCR-CD3 reduced the growing speed during the pushing phase without triggering the same mechanical behavior when engaged alone. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was monitored simultaneously to verify the cell commitment in the activation process. [Ca(2+)](i) increased a few tens of seconds after the beginning of the pushing phase although no strong correlation appeared between the two events. The pushing phase was driven by actin polymerization. Tuning the BFP mechanical properties, we could show that the loading rate during the pulling phase increased with the target stiffness. This indicated that a mechanosensing mechanism is implemented in the early steps of the activation process. We provide here the first quantified description of force generation sequence upon local bidimensional engagement of TCR-CD3 and discuss its potential role in a T cell mechanically-regulated activation process.

Figure 1. Successive phases during the interaction of a T cell with an anti-CD3 coated bead on the biomembrane force probe.

(A) Schematic view of the experimental setup. A T cell (left) is held by a micropipette, which extremity is at a fixed position x_{cell pipette} along the x-axis, with an aspiration pressure ΔP_{T cell}. The BFP (right) consists in a bead (centered at x_bead(t)) adhering to a red blood cell, which is maintained by a micropipette with an aspiration pressure ΔP_BFP. This aspiration sets the red blood cell stiffness k. The position of the right micropipette edge is located at x_{BFP pipette}(t). After making contact with the bead at time t = 0, the T cell emits a protrusion of length L_protrusion(t) = x_bead(t)-x_bead(t = 0). (B) Representative experiment (Movie S1) with a static BFP-holding micropipette (Left: brightfield images; Right: Fura2 ratio). Bar is 5 µm. (C) Above: Position x_bead(t) during the three consecutive phases. During the “latency phase” the bead is immobile. At t = t_push, the “pushing phase” starts as a protrusion emerges from the T cell, leading to x_bead(t)>x_bead(t = 0). The “pulling phase” starts at t = t_pull, when the protrusion retracts and the T cell pulls on the bead. Below: Fura2 ratio versus time. The ratio increases abruptly at t = t_Calcium, and t_Calcium>t_push. (D) Delay t_Calcium−t_push between the onset of protrusion growth and Fura2 ratio increase.

Figure 2. Characterization of the CD3-induced pushing phase.

(A) The dynamic probe-protocol used to measure T cell protrusion growth consists in stepping back the right pipette along the x-axis as soon as a RBC compression is detected (above), in order to relax this compression (below). The residual force exerted on the T cell protrusion after stepping back the pipette, F_residual , is kept below 25 pN. (B) Representative experiment (Movie S3) using the dynamic-probe protocol (Left: brightfield images; Right: fura2 ratio). Bar is 5 µm. (C) Above: Protrusion length L_protrusion versus time. During the latency phase, L_protrusion is constant and equal to 0. The pushing phase starts at t = t_push, when L_protrusion starts increasing with time. L_protrusion increases initially at a constant velocity v_protrusion. The protrusion reached a maximal length L_max. Below: fura2 ratio versus time. The ratio increases abruptly at t = t_Calcium>t_push, with a maximal amplitude Δr_fura2 relative to the base level. (D) Distribution of L_max over N = 37 cells. L_max = 8.2+/−0.7 µm. (E) Distribution of v_protrusion over N = 37 cells. v_protrusion = 0.12+/−0.01 µm/s.

Figure 3. Characterization of the CD3-induced pulling phase.

(A) During the pulling phase, the T cell protrusion pulls on the bead of the force probe, leading to its elongation over time. Bar is 5 µm. (B) elongation of the BFP ΔL_BFP(t) = L_BFP (t)−L_BFP (0) in a representative experiment. ΔL_BFP increases initially at a constant speed v_pull = dL_BFP (t)/dt. (C) Distribution of the resulting loading rate r = dF/dt (expressed in pN/s), r = 1.6+/−0.2 pN/s for a probe stiffness k = 50 µm/s (N = 15 cells).

Figure 4. Reduced CD3-Dependent Force Generation upon CD18 Engagement.

(A) L_protrusion versus time for anti-CD3 coated beads (open circles), anti-CD3+anti-CD18 coated beads (full circles), and anti-CD18 coated beads (full triangles). The co-engagement of both CD3 and CD18 reduces v_protrusion, while CD18-only engagement abolishes protrusion growth. Inset shows the beginning of the growth. (B) BFP elongation ΔL_BFP versus time, for anti-CD3 coated beads (open circles), anti-CD3+anti-CD18 coated beads (full circles), and anti-CD18 coated beads (full triangles). (C) Comparison of outcome upon CD3-, CD3+CD18- and CD18-engagement when T cells interact with the BFP. We compare times (t = 0 when the bead contacts the T cell) t_push (start of the pushing phase), t_Calcium ([Ca²⁺]_i increase time), t_pull (start of pulling phase), and duration of the pushing phase Δt_push = t_pull−t_push. We also compare the Calcium response amplitude (fura2 ratio Δr_Fura2), pushing speed v_pull and maximal protrusion length L_max (^*p<0.01, ^**p<0.001, ^***p<0.0001).

Figure 5. Loading rates generated by T cells depend on the Probe Stiffness.

Loading rate r = dF/dt versus stiffness k of the force probe, for anti-CD3 coated beads.

Figure 6. Cytoskeleton remodeling in T cells interacting with Abs-coated beads on the force probe.

T cells were transfected with LifeAct-mCherry (left : DIC, right : color code mCherry). (A) Anti-CD3 coated bead. (see Movie S6. (B) Anti-CD18 coated bead. (C) Anti-CD3+anti-CD18 coated bead. (D) L_protrusion versus time for one representative experiment with a latrunculin A treated T cell (open triangles) or a non-treated T cell (open circles).