Biophysical Society Thematic Meeting| Aussois 2019

Biology and Physics Confront Cell-Cell Adhesion

Poster Abstracts

15-POS Board 15 SHINING LIGHT ON TOPOLOGY IN EARLY T-CELL ACTIVATION – A NOVEL BILAYER SYSTEM Markus Körbel 1 ; Edward Jenkins 2 ; Kevin Y Chen 1 ; Simon J Davis 2 ; David Klenerman 1 ; 1 University of Cambridge, Department of Chemistry, Cambridge, United Kingdom 2 University of Oxford, MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, Oxford, United Kingdom The very first contacts of a T cell with an antigen presenting cell (APC) are key to understanding antigen discrimination in adaptive immunity. The use of supported lipid bilayers (SLBs) to mimic the APC simplifies imaging analysis and allows controlled changes in contacting-surface composition. In studies of the immune synapse, SLBs are commonly functionalized with peptide-bound MHC (pMHC) and ICAM-1, with pMHC acting as an adhesion molecule and a molecular trigger, and ICAM-1 behaving both as an adhesion protein and a steric barrier to engage pMHC, respectively. By replacing it with CD58, a small adhesion molecule, and the extracellular part of CD45, as glycocalyx, we aimed to produce a more realistic model system allowing new insights into T-cell signalling. On the new surface, the adhesive behaviour of T cells could be fine-tuned by varying the CD45 to CD58 ratio: low densities of CD45 allowed cell attachment and spreading, and higher densities prevented contact formation. At intermediate glycoprotein densities, cells only attached and started to signal if pMHC was present. We utilized the size-based exclusion of CD45 in the SLB together with cell membrane labelling to image contact formation via live TIRF microscopy. Initial small, diffraction-limited contacts that formed and grew were highly dynamic, dominated by centripetal movements at later stages. The small, initial contacts represented the footprint of microvilli, a dominant feature of T-cell topology. T cells treated with agents that reduce microvilli on their cell membrane were unable to detect or respond to pMHC molecules in our model SLB. Our results suggest that T-cell microvilli provide a structure to both penetrate the glycocalyx barrier and detect antigens.

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