Trapping or slowing the diffusion of T cell receptors at close contacts initiates T cell signaling

Abstract

T cell activation is initiated by T cell receptor (TCR) phosphorylation. This requires the local depletion of large receptor-type phosphatases from “close contacts” formed when T cells interact with surfaces presenting agonistic TCR ligands, but exactly how the ligands potentiate signaling is unclear. It has been proposed that TCR ligands could enhance receptor phosphorylation and signaling just by holding TCRs in phosphatase-depleted close contacts, but this has not been directly tested. We devised simple methods to move the TCR in and out of close contacts formed by T cells interacting with supported lipid bilayers (SLBs) and to slow the receptor’s diffusion in the contacts, using a series of anti-CD3ε Fab- and ligand-based adducts of the receptor. TCRs engaging a Fab extended with the large extracellular region of CD45 were excluded from contacts and produced no signaling. Conversely, allowing the extended Fab to become tethered to the SLB trapped the TCR in the close contacts, leading to very strong signaling. Importantly, attaching untethered anti-CD3ε Fab or peptide/MHC ligands, each of which were largely inactive in solution but both of which reduced TCR diffusion in close contacts approximately fivefold, also initiated signaling during cell/SLB contact. Our findings indicate that holding TCRs in close contacts or simply slowing their diffusion in phosphatase-depleted regions of the cell surface suffices to initiate signaling, effects we could reproduce in single-particle stochastic simulations. Our study shows that the TCR is preconfigured for signaling in a way that allows it to be triggered by ligands acting simply as receptor “traps.”

Influence of the extracellular domain size on the dynamic behavior of membrane proteins

“…There are currently multiple mechanisms proposed for T cell signaling (and most are applicable to other immune cell types). Size-dependent, lipid-dependent, or diffusiondependent exclusion of inhibitory molecules (such as CD45) from the immune synapse are three of the main and seemingly distant mechanisms explaining T-cell signaling (3)(4)(5)(6)(7)(8). However, our results suggest that all three phenomena can be intertwined.…”

“…Multiple mechanisms are proposed to explain the initial protein reorganization that leads to the activation (phosphorylation) of the receptors. Partitioning of signaling proteins into membrane domains (2), their ECD size (3)(4)(5), their allosteric interactions with lipids (6), and their mobility altered by the ligands (7,8) are some examples of these mechanisms. Although there is compelling evidence for each of these mechanisms for immune signaling, the dominating mechanism is still unclear.…”

Understanding immune signaling using advanced imaging techniques

“…The triggering of the T-cell receptor (TCR) occurs within a few seconds of cell–cell contact [ 1 ], which involves reorganization of membrane proteins over 20–100 nm [ 3 ] and the fast motion of finger-like microvilli structures [ 4 , 5 ] that continuously change in shape [ 6 ]. These conditions all together present highly technical imaging challenges.…”

“…Tall molecules are excluded from the tight contact between the immune and target cells, which can be a biophysical regulation mechanism for activation of various immune cells [ 3 , 116 , 117 ]. Finally, diffusion and aggregation of the molecules at the IS have been suggested as a crucial biophysical mechanism to fine-tune signaling [ 4 , 118 ]. The IS has been an important target for therapies such as immunotherapy and the accumulating evidence on the biophysical aspects of the IS shows that such collective biophysical properties should be considered for therapies targeting the IS [ 119 ].…”

T cells use distinct topological and membrane receptor scanning strategies that individually coalesce during receptor recognition

“…Upon pMHC recognition, TCR-enriched microvilli (6), visible as ‘microclusters’ (7, 8), are ‘stabilized’ with half-lives that are 2-5x that of unengaged ‘scanning’ microvilli (6). Microvillar tips are presumed to facilitate TCR-pMHC binding both because they approach APCs to within 15 nm (6), which is the approximate transmembrane-transmembrane dimensions of TCR-pMHC complexes (9, 10), and because TCRs are observed to accumulate in these zones of close contact in response to normal ligand recognition (6, 11).…”