Optimization of T Cell Activation Protocols to Enrich Stem Cell Memory Phenotypes: Metabolic and Health Assays as Predictive Tools

The generation of T cells with a stem cell memory phenotype (T_SCM) holds significant promise for immunotherapy due to their longevity and potent antitumor activity. This study aimed to optimize T cell activation and expansion conditions to selectively enrich for the T_SCM population. Utilizing a suite of cell health and metabolism assays, we monitored T cell cultures to identify predictive markers of T_SCM enrichment. We observed that early activation steps correlated with a surge in ATP production and alterations in cell reducing potential, indicative of metabolic reprogramming. Subsequent activation phases were marked by an upregulation in both catabolic and anabolic pathways, as evidenced by the increased turnover of key metabolites including glucose, lactate, glutamine, glutamate, branched-chain amino acids (BCAA), and pyruvate. Notably, the rate of T cell activation influenced the composition of the resultant T cell populations, with the media composition further affecting these dynamics. Our findings suggest that metabolic assays, particularly those measuring changes in ATP levels and primary metabolite flux, can serve as effective early predictors of T_SCM enrichment. These insights may guide the development of more efficacious T cell-based immunotherapies by facilitating the targeted generation of T cells with a stem cell-like memory phenotype.