The interplay between membrane topology and mechanical forces in regulating T cell receptor activity

Abstract

T cells are critically important for host defense against infections. T cell activation is specific because signal initiation requires T cell receptor (TCR) recognition of foreign antigen peptides presented by major histocompatibility complexes (pMHC) on antigen presenting cells (APCs). Recent advances reveal that the TCR acts as a mechanoreceptor, but it remains unclear how pMHC/TCR engagement generates mechanical forces that are converted to intracellular signals. Here we propose a TCR Bending Mechanosignal (TBM) model, in which local bending of the T cell membrane on the nanometer scale allows sustained contact of relatively small pMHC/TCR complexes interspersed among large surface receptors and adhesion molecules on the opposing surfaces of T cells and APCs. Localized T cell membrane bending is suggested to increase accessibility of TCR signaling domains to phosphorylation, facilitate selective recognition of agonists that form catch bonds, and reduce noise signals associated with slip bonds.

Fig. 1. T cell surface and the immune synapse.

a The T cell surface is populated with receptors displaying different dimensions ranging from one Ig-like domain (3.5 nm) to those with long and bulky extracellular domains such as CD45 with a rigid core of 15.2 nm and variable mucin-line domain that extends the height to 10 (CD45R0), 20 (CD45RA), or 40 (CD45RABC) nm. The repetition of the mucin-like domain may increase the space it occupies in cell interfaces. b Upon APC/T cell engagement, and following TCR triggering, T cell surface proteins sort based on size and F-actin based transport to distinct zones. The mature immune synapse takes 10–30 min to develop, and three zones are arranged in circles, with proteins subjected to strong F-actin and ESCRT based transport in the cSMAC, proteins linked to F-actin/myosin, such as LFA-1 ligated to ICAM-1, in the pSMAC, and proteins with the weakest F-actin coupling in the dSMAC. c In addition, some proteins linked directly to TCR triggering such as Lck kinase accumulate in the pSMAC where TCR signaling is initiated. The CD2–CD58 interaction forms a peripheral corolla of close interactions in the dSMAC when a sufficient number of receptors are engaged. TCR microclusters form at the outer edge, translocate through the corolla and pSMAC to reach the cSMAC.

Fig. 2. TCR triggering by mechanical forces.

a 3T3 cells were engineered to express surface-bound anti-CD3 scFv elongated with a CD43 tether (anti-CD3-CD43) that allows T cell engagement without inducing TCR triggering (illustrated by a heat map of Ca²⁺ flux). b Applying force to T cells bound by anti-CD3-CD43 with a micropipette triggered TCR signaling as measured by Ca²⁺ flux. c–e Similar pulling forces applied to T cells engaged via CD28, CD62L or ICAM-2 did not induce Ca flux. f Shear forces applied to T cells engaged via CD3 induced Ca²⁺ flux. g–i T cells engaged via CD28, CD62L or ICAM-2 did not induce T cell activation.

Fig. 3. Forces exerted from ICAM-1/LFA-1 engagement.

Naïve T cells express LFA-1 in an inactive bent form. Inside out signaling induced upon TCR triggering converts LFA-1 to an open form that can ligate to ICAM-1 on APCs, generating a surface tension of 40 nm intercellular space around engaged pMHC/TCR complexes that span only 15 nm, which provide defined molecular forces around engaged TCRs leading to membrane bending.

Fig. 4. Stochastic membrane displacement.

a The plasma membrane of T cells (and all cells in the body) continuously fluctuates to dissipate thermal energy at physiological temperatures. TCR receptors, therefore, are normally moving up and down on the T cell surface (colored and gray positions A and B). Engagement of TCRs by pMHC on an APC fixes the relative positions of the TCR and pMHC, which opposes the normal membrane fluctuations and provides another source of mechanical force on engaged TCRs. b illustrated displacement of position 0 on the nm scale over a unit of time.

Fig. 5. Protein crowding.

a Proteins associated with biological membranes occupy large surface areas on flat membranes (left), but are crowded in the inner leaflet when the membrane is bent inward (right). This creates steric pressure that opposes membrane bending. b Bending the plasma membrane directly around engaged TCRs may increase steric pressure on the CD3 complex and assist dissociation of buried CD3 ITAMs to the cytosol.

Fig. 6. T cells can be activated by tethered ligands.

a T cells are activated by ligands coated on surfaces such as glass or plastic, activation beads, or APCs. Ligand binding bends the membrane around TCRs to physically accommodate membrane proteins with large ectoplasmic domains. Soluble ligands are poor T cell activators because they bind TCRs without inducing plasma membrane bending: i pMHC-coated glass surface. ii anti-CD3 antibody-coated glass surface. iii soluble anti-CD3 antibodies. iv soluble pMHC. v APC expressing pMHC. vi activation beads coated with anti-CD3 antibodies. b TCR triggering by soluble pMHC can be achieved with pMHC dimers linked via short spacers (left) but not when the pMHC dimers are linked via a long spacer (right). Only short spacers are able to induce membrane bending.

Fig. 7. T cell activation requires ligands with particular dimensions.

a TCR triggering is achieved when the interspatial distance between T cells and APCs is around physiological dimensions which is 4 Ig-like domains (~15 nm). Similar results are obtained using artificial APCs expressing membrane-bound anti-CD3 scFv elongated with different tethers. b High-affinity TCR ligands tethered on artificial APCs can trigger TCR signaling independent of their dimensions (left) while low-affinity TCR ligands lose their potency to activate TCR when elongated.

Fig. 8. Membrane curvature and CD45 exclusion contribute to TCR triggering.

Micropatterned surfaces were prepared in 2D with gold nanoparticles (NPs) at the same level as an SLB (a) or in 3D with gold NPs elevated 10 nm on SiO₂ pillars above the SLB (b). Anti-CD3 Fab was linked to NPs and ICAM-l molecules were present on the SLB for adhesion. Large membrane curvature is generated at engaged TCR clusters in the 2D system to accommodate large ICAM-1/LFA-1 complexes (a) whereas elevation of anti-CD3 Fab in the 3D system generates less membrane curvature for a given ligand spacing (b). However, when the distance between NP pillars is reduced to 40 nm TCR signaling is restored (b, left). This might be explained by a high degree of membrane curvature when ICAM-1/LFA-1 interactions are interspersed with the closely spaced pMHC/TCR (b, left). While ICAM-1 didn’t accumulate in 3D TCR clusters with 40 nm spacing, a relatively small number of interspersed ICAM-1/LFA-1 interactions would be sufficient to enforce curvature; further work is required to test for high curvature in the 3D 40 nm clusters.