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Spatiotemporal signaling dynamics evoked by single binding interactions between a peptide-bound MHC molecule and a T cell receptor

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Abstract

T cells (TCs) have the crucial immunological task of discriminating foreign peptide fragments from endogenous (self) peptide fragments. This recognition is carried out by the T Cell Receptor (TCR) on the TC surface, which binds to an antigen-derived peptide fragment presented by major histocompatibility complex (pMHC) on the surface of an Antigen Presenting Cells (APC). Despite the centrality of this TCR:pMHC interaction and the myriad techniques used analyze it, few studies exist in which the signaling consequences of individual TCR:pMHC binding events have been determined. Direct examination of the outcomes from single TCR:pMHC encounters provides insight into how the stochastic signaling network of the T cell performs crucial tasks such as signal integration and ligand discrimination. In Chapter 1, individual TCR:pMHC binding interactions (dwell times) are mapped to two proximal events in T cell activation: the assembly of Linker for Activation of T cells (LAT) molecules; and, translocation of Nuclear Factor of Activated T cells (NFAT). After TCR engagement with agonist pMHC, the LAT scaffold protein forms a hub of signaling from which a number of subsequent signaling steps are initiated; hence, LAT assembly is a gatekeeper of downstream signaling. I observe that the probability of LAT assembly formation, localized to ligated TCR, is an increasing function of the dwell time of the TCR:pMHC interaction, suggesting that the formation of LAT assembly from ligated TCR represents a form of kinetic proofreading; however, the size and lifetime of a LAT assembly is independent of the TCR:pMHC dwell time interaction that produced it. This discreteness of LAT clustering from TCR:pMHC dwell time has consequences for signal integration from multiple binding events. In Chapter 2, I explore what distinguishes a productive TCR:pMHC binding event from one that fails to trigger LAT assembly, as an approach to delineate the “kinetic bottlenecks” in antigen discrimination and T cell activation. I found that activation of zeta-chain- associated protein kinase of 70 kDa (ZAP-70), which phosphorylates LAT at multiple sites, is necessary, but insufficient. Aside from ZAP-70 activity, diffusive phospho-LAT requires an additional event to trigger aggregation. Importantly, I found that, at low pMHC densities, PLC-γ1 controls the timing for the initiation of LAT clustering, a dependency not previously identified. One feature I readily observed in my experiments is that LAT assemblies can form without any apparent originating pMHC. The composition and function of these apparent orphan LAT assemblies are investigated preliminarily in Chapter 3. In summary, by measuring the correlation between single receptor (TCR)-ligand (pMHC) binding events and subsequent steps in T cell activation, one obtains unprecedented quantitative insight into the stochastic nature of signal transduction in T cells.

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This item is under embargo until April 28, 2024.