Cenk Sumen

Continuous T cell receptor signaling required for synapse maintenance and full effector potential

Although signals through the T cell receptor (TCR) are essential for the initiation of T helper cell activation, it is unclear what function such signals have during the prolonged T cell–antigen-presenting cell contact. Here we simultaneously tracked TCR-CD3 complex and phosphoinositide 3-kinase activity in single T cells using three-dimensional video microscopy. Despite rapid internalization of most of the TCR-CD3, TCR-dependant signaling was still evident up to 10 h after conjugate formation. Blocking this interaction caused dissolution of the synapse and proportional reductions in interleukin 2 production and cellular proliferation. Thus TCR signaling persists for hours, has a cumulative effect and is necessary for the maintenance of the immunological synapse.

Costimulation and endogenous MHC ligands contribute to T cell recognition.

To initiate an immune response, key receptor-ligand pairs must cluster in “immune synapses” at the T cell–antigen-presenting cell (APC) interface. We visualized the accumulation of a major histocompatibility complex (MHC) class II molecule, I-Ek, at a T cell–B cell interface and found it was dependent on both antigen recognition and costimulation. This suggests that costimulation-driven active transport of T cell surface molecules helps to drive immunological synapse formation. Although only agonist peptide–MHC class II (agonist pMHC class II) complexes can initiate T cell activation, endogenous pMHC class II complexes also appeared to accumulate. To test this directly, we labeled a “null” pMHC class II complex and found that, although it lacked major TCR contact residues, it could be driven into the synapse in a TCR-dependant manner. Thus, low-affinity ligands can contribute to synapse formation and T cell signaling.

T cell receptor antagonism interferes with MHC clustering and integrin patterning during immunological synapse formation.

T cell activation by nonself peptide–major histocompatibility complex (MHC) antigenic complexes can be blocked by particular sequence variants in a process termed T cell receptor antagonism. The inhibition mechanism is not understood, although such variants are encountered in viral infections and may aid immune evasion. Here, we study the effect of antagonist peptides on immunological synapse formation by T cells. This cellular communication process features early integrin engagement and T cell motility arrest, referred to as the “stop signal.” We find that synapses formed on membranes presenting antagonist–agonist complexes display reduced MHC density, which leads to reduced T cell proliferation that is not overcome by the costimulatory ligands CD48 and B7-1. Most T cells fail to arrest and crawl slowly with a dense ICAM-1 crescent at the leading edge. Similar aberrant patterns of LFA-1/ICAM-1 engagement in live T–B couples correlate with reduced calcium flux and IL-2 secretion. Hence, antagonist peptides selectively disable MHC clustering and the stop signal, whereas LFA-1 valency up-regulation occurs normally.