34 Suiting up T cells: Strengthening membrane “armor” to improve anti-cancer immunity

Objectives/Goals: The plasma membrane (PM) is the nexus for cell signaling, where lipid composition governs receptor organization and T cell function. We aim to define PM dynamics in T cell states and develop PM-based strategies to preserve anti-tumor activity. Methods/Study Population: As such, we induced a dysfunctional T cell state, known as T cell exhaustion, through chronic stimulation or TGF-β1, hypoxia, and tumor cell co-culture and measured membrane biophysical properties through ratiometric confocal imaging of molecular probe Di-4-ANEPPDHQ. Complementary biochemical data from shotgun lipidomic analysis, immunofluorescence staining, and biochemical fluorescent assays paint a more complex picture of membrane fluidity changes during T cell exhaustion. Results/Anticipated Results: In both cases, we observed decreased PM rigidity compared to acute or normoxic T cell activation, specifically in T cells with upregulated PD1, LAG-3, and Tim-3. Dysfunctional T cells also exhibit changes in specific lipid species. This includes increased free cholesterol and decreased esterified cholesterol among other species. Discussion/Significance of Impact: Adoptive cell therapy is limited in solid tumors due to a hypoxic, suppressive TME that drives T cell exhaustion. Our data suggest exhausted T cells have fluid plasma membranes that impair synapse formation. Future work will validate this in patient-derived xenograft/TCR-T models that will pave the way for clinical translation.