Molecular Mechanisms of Lymphocyte Activation - Please Mind the Gap
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Lippert, Anna Helena
University of Cambridge
Doctor of Philosophy (PhD)
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Lippert, A. H. (2020). Molecular Mechanisms of Lymphocyte Activation - Please Mind the Gap (Doctoral thesis). https://doi.org/10.17863/CAM.52527
The underlying mechanism of lymphocyte triggering is still not fully understood, but the kinetic-segregation (KS) model of receptor triggering in T cells has been supported by several recent studies. This model proposes that the small gap between an antigen-presenting cell and an immune cell excludes large, inhibitory phosphatases, causing cell signalling. This work examines lymphocyte signalling phenomena in the light of the KS model. Single molecule and bulk experiments explore how the gap size between immune cells and a surface affects the behaviour of important signalling proteins. The first part of this thesis investigates whether the KS model explains the signalling-related epi-phenomena of superagonism. Experiments show that contacts mediated by highly potent superagonistic antibodies increase the exclusion of phosphatases. These changes are gap-size dependent and, complemented by T cell stimulation assays, support the role of phosphatase exclusion in superagonistic T cell activation predicted by the KS model. This signalling principle is likely to be generalisable to other tyrosinekinase-dependent receptors,including immune checkpoints. The second partof the thesis asks whether the KS mechanism might activate other immunoreceptors. This work focuses on the B cell receptor (BCR), which as a large,bivalent immunoreceptor poses its own challenges to the KS model. Functional activation assays and three-color imaging reveal a link between size-dependent phosphatase exclusion and B cell activation, providing evidence for KS as a general activation mechanism in lymphocytes. Finally, stiffness-dependent T cell activation is explored. While substrate stiffness is known to influence T cell activation,studies are typically performed on glass, which is orders of magnitude stiffer than any substrate in vivo. This work introduces a new platform for single-molecule imaging on bilayers supported by substrates with variable stiffness. Early T cell activation markers show a correlation between stiffness and sustained signalling. This platform enables future experiments to study the underlying mechanism of stiffness-dependent lymphocyte activation. As a whole, this thesis shows how physical parameters like gap-size and substrate stiffness can influence lymphocyte activation. Thus, it enhances our general understanding of lymphocyte activation and motivates antibody design for immunotherapy as well as close-to-physiological signalling studies.
Single-molecule Imaging, Immunology, Biophysics, Lymphocytes
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This record's DOI: https://doi.org/10.17863/CAM.52527
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