During an immune response, dendritic cells (DCs) capture and process antigen, upregulate co-stimulatory molecules and migrate to lymphoid organs to maximise antigen recognition by rare T cell clones. A crucial step for successful T cell activation is cell-cell interaction at the immunological synapse (IS), the organised contact interface which allows optimal communication. IS formation requires the dynamic remodelling of the actin cytoskeleton to spatially distribute membrane areas with distinct protein compositions. While evidence exists for a role of the T cell cytoskeleton in this process, the driving force behind the specific organisation on the DC side is unknown. One important actin regulator is Wiskott-Aldrich Syndrome protein (WASp), expressed exclusively in immune cells. Using a murine model, we investigated the role of WASp in DC-mediated actin-dependent synapse formation. Utilising both confocal and electron microscopy (EM), we observed a disorganised, asymmetric interface between T cells and WASp-deficient DCs. Immunofluorescent staining shows reduced polarisation of certain synapse markers in WAS knock-out conjugates; while high resolution 3D EM reconstructions show severely impaired cell spreading and a reduced contact area. We have used fluorescence recovery after photobleaching (FRAP) to show reduced stability of the actin network in WASp DCs. Our experiments on supported planar bilayers have shown an unexpected interface organisation and highlight a novel role of the DC cytoskeleton in this process. Crucially, we have demonstrated reduced T cell activating capacity of WASp-deficient DCs, as measured by T cell proliferation, cytokine production and transcription factor expression. The results highlight the critical role of cytoskeletal regulation in DC-mediated IS formation. Further, a second murine immunodeficiency model, Dock8 deficiency, and two novel human primary immunodeficiencies were briefly investigated to examine the role of other actin regulators in normal DC function.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:631935 |
| Date | January 2014 |
| Creators | Malinova, D. S. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1435421/ |
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