Characterizing T cell activation using bioluminescent metabolic and cytokine assays: insights into molecular pathways and immune modulation 3380

Abstract Description Advancing our understanding of T cell activation and its regulatory pathways is pivotal for immunology research and therapeutic development. This study evaluates T cell health and functional status under diverse activation conditions, illustrating key molecular mechanisms that govern immune responses. We focus on critical pathways, including mTOR, AMPK, and the metabolic interplay between glycolysis and oxidative phosphorylation. Using selective inhibitors, we identify critical immune regulatory modulators within these pathways. Our findings reveal a strong correlation between T cell metabolic state and cytokine secretion, with media composition identified as a significant factor influencing T cell function. Our methodology employs a panel of bioluminescent Lumit and Metabolite-Glo assays, enabling sensitive quantification of cytokines and metabolites. This method enables monitoring with minimal sample handling, preserving cell integrity and improving reproducibility. Its scalability and adaptability support analysis across diverse activation conditions and high-throughput screening applications. Furthermore, we show correlation to cell surface markers using traditional methods. Our findings and methods provide a robust platform for detailed exploration of signaling, metabolic, and immune dynamics during T cell activation, offering new insights for targeted immunomodulation strategies. Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)