The importance of membrane order in T cell signalling and viral infection

Abstract

The plasma membrane is a dynamic and heterogeneous structure, whose lateral organization into lipid nanodomains critically regulates cellular processes such as signaling, adhesion, and pathogen entry. In T cells, membrane order, which reflects the degree of lipid packing, plays a pivotal role in organizing signaling platforms and modulating immune responses. In this thesis, membrane order was investigated using the environment-sensitive fluorescence probes Laurdan and C-laurdan, with generalized polarization (GP) used to quantify lipid packing. Paper I presents an optimized GP imaging approach for C-laurdan-labeled T cells by introducing a systematic deconvolution protocol. Deconvolution improved the spatial resolution of fluorescence images, enabling more accurate delineation of plasma membrane regions of interest (ROIs) and enhancing the sensitivity for detecting differences in membrane order between plasma and intracellular membranes. A detailed protocol for optimizing deconvolution parameters was established, allowing for reproducible and higher-precision GP measurements. Paper II investigates how different substrate types and environmental conditions influence T cell plasma membrane order at the equatorial plane and examines how these factors relate to early activation events. Laurdan-based imaging revealed that membrane order at the equatorial plane did not differ significantly across conditions, including CD3- and CD45-functionalized supported lipid bilayers, PLL-coated surfaces, hydrogels, and microfluidic suspension. Although CD3-functionalized SLBs showed a non-significant trend toward increased membrane order, changes were subtle overall. Cells in suspension exhibited a wider range of GP values, while those in hydrogels showed no significant increase in order compared to TESPA. Calcium imaging demonstrated that CD3-functionalized SLBs triggered significantly faster responses than CD45-functionalized SLBs, despite both supporting similar activation levels. Additionally, PLL-coated surfaces induced a higher fraction of activated cells compared to TESPA, though with similar timing. These findings indicate that while substrate composition and receptor engagement strongly influence early signalling dynamics, they do not induce major changes in global membrane order at the equator. Paper III examines the effects of simvastatin on plasma membrane organization and SARS-CoV-2 infection. Treatment with simvastatin reduced plasma membrane order, as assessed by C-laurdan GP imaging, and led to a significant decrease in both intracellular viral load and extracellular virus release in lung epithelial cells. These effects were not solely explained by alterations in ACE2 expression, suggesting that modulation of membrane order contributed directly to limiting viral infection and propagation. Overall, this thesis demonstrates that plasma membrane order is a key determinant in T cell signaling and viral infection. By advancing imaging methodologies and elucidating the interplay between membrane organization and cellular function, this work provides new insights into the biophysical regulation of immune responses and identifies membrane order as a potential target for therapeutic intervention.