| Summary: | <p>Understanding how immune signalling is initiated and regulated spatiotemporally is likely to be helped by investigations of receptor stoichiometry. The pre-TCR expressed by thymocytes shares similarities in structure and signalling components with the mature TCR but, in contrast to the TCR, it has no known ligands. This thesis has therefore analysed the stoichiometry of the pre-TCR using mutagenesis- and imaging-based approaches, and explored how ligand-independent signalling might be initiated by both the TCR and pre-TCR. The mutational analysis, which required considerable optimisation because the pre-TCR is very weakly expressed in transfected cells, suggested that only one contiguous surface on pre-Tα needs to be buried in the pre-TCR complex in order for it to reach the cell surface. This comprised the surface buried at the interface with the constant region of TCRβ and therefore was incompatible with previous suggestions that the pre-TCR assembles into a ‘head-to-tail’ dimer. Unexpectedly, super-resolution imaging experiments combined with cluster analysis, and the method of single-molecule cross-colour coincidence detection were suggestive that, rather than dimers, the pre-TCR forms higher-order oligomers in transfected cells and possibly also in thymocytes. Similar analyses showed that the organisation of the TCR was heterogeneous, depending on the level of expression of the receptor. Overall, technical limitations that emerged in the course of the study highlighted some of the difficulties in studying native receptor stoichiometry on resting cells generally. The final part of the thesis investigated ligand-independent signalling by the TCR, using a novel assay based on the binding of a superagonistic antibody to a non-signalling form of the receptor CD28. Using CRISPR/Cas-mediated gene editing, it was shown that ligand-independent signalling by the mature TCR and pre-TCR was dependent on Lck and Zap70, indicating that it is reliant on the triggering of conventional signalling pathways. Ways in which the pre-TCR might initiate signalling, that could be tolerant of complexes exhibiting a range of stabilities, are also discussed.</p>
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