Validation of a fluorescence spectroscopy method for the determination of T cell membrane order, towards a high-throughput approach 2612

Abstract Description Immune cells are characterized by unique lipidomes. Lipids play critical roles in cell metabolism, signaling, and as structural components of membranes. In T cells, biochemical and biophysical cues facilitate the formation of the immunological synapse, enabling proper signaling and activation. With conflicting reports on the association between cholesterol and CD8 T cell function, clarifying the relationship between membrane biophysical properties and T cell state offers an innovative perspective. As such, we developed a fluorescence spectroscopy-based high-throughput method to assess membrane biophysical properties using molecular probe Di-4-ANEPPDHQ. We evaluated changes in membrane order as a function of activation with a-CD3/28 antibodies in Jurkat and primary naïve CD8 T cells from PBMC. Cells were collected at specific time points post-activation and imaged live. A cholesterol depletion dose curve was also performed in Jurkat cells. Measurements, obtained through ratiometric confocal microscopy and fluorescent plate reader, showed significant fluctuations in membrane order in Jurkat and primary human CD8 T cells post-activation (p < 0.05). Similar directional changes were observed between the two methods. These findings suggest a link between plasma membrane order and T cell state, introducing a novel high-throughput method for assessing membrane biophysical properties. Future studies will explore the functional implications of these changes in cell-mediated immunity. Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)