| Summary: | T cells have an important role in the resolution of infections and cancer. The mechanisms underlying early contact formation of T cells with antigen-presenting cells, and the contribution of T-cell receptor (TCR) microclusters (MCs) to T-cell activation, are still not well understood, especially given recent evidence demonstrating the importance of membrane topography in T-cell signalling. Furthermore, it is unclear what features of the TCR triggering mechanism are shared with other receptors, such as the B-cell receptor (BCR). For experiments presented in this thesis, cell lines were created that express TCRs with different affinities and surface densities, along with an inhibitable mutant of ZAP-70, which were used to probe the effects of TCR density and signalling in early T-cell contacts and antigen reactivity. TCR density did not affect the spreading or close contact formation of T cells with supported lipid bilayers (SLB) presenting agonist pMHC, suggesting that very few TCRs are needed to trigger these events. MC distribution was uncorrelated with close contacts, implying that these structures are real, and not imaging artefacts. However, T cells with low TCR expression levels signalled robustly, suggesting that MC formation might not
be a pre-requisite of TCR signalling. Nevertheless, signalling triggered under these conditions did not result in full T-cell activation. ZAP-70 inhibited cells displayed reduced cell spreading, formed fewer but larger close contacts, and recruited more ZAP-70 at MCs. This data suggests that ZAP-70 is a strong regulator of T-cell contact formation, and that it also contributes to antigen discrimination, alongside its key role in signal amplification at the TCR. Finally, key requirements of the kinetic segregation (KS) model were confirmed in T and B cells, including the induction of TCR triggering by slowing the receptor’s diffusion within a close contact, and the demonstration that the BCR is unclustered on resting cells, similarly to the TCR, thereby ruling out a popular model of BCR triggering. Work carried out in this thesis provides insight into the mechanisms regulating early contact formation by T
cells, and by demonstrating key predictions of the KS model in both T and B cells, raises the possibility that a unified mechanism for immune receptor triggering could exist.
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