Enhancing Virus-Specific T Cell Persistence: L-Arginine Supplementation Improves the Durability of CD8 + T Cells for Immunotherapy

Abstract Virus-specific T cell (VST) therapy offers a promising treatment for life-threatening viral infections following allogeneic hematopoietic stem cell transplantation (HSCT). However, the efficacy of VST therapy remains limited by apoptosis, exhaustion, and loss of stem-like properties with advancing differentiation. In this study, we demonstrate that supplementation of basic culture medium with excess L-arginine significantly enhances the durability and function of VSTs targeting persistent viruses. L-arginine treatment preserved a less differentiated, less exhausted phenotype and conferred resistance to activation-induced cell death (AICD) and freeze-thaw-induced damage. Mechanistically, these effects were associated with increased mitochondrial membrane potential and MCL-1 expression, suggesting enhanced mitochondrial biogenesis and metabolic fitness. L-arginine-treated VSTs initially exhibited reduced cytotoxic activity upon primary stimulation, likely due to the suppression of exhaustion and differentiation; however, they acquired superior polyfunctionality and cytotoxic potential following secondary stimulation. These benefits were achieved without detectable activation of mTORC1 signaling, indicating a favorable metabolic reprogramming independent of effector-skewing pathways. Our findings position L-arginine supplementation as a clinically applicable strategy to improve the persistence and efficacy of human VST therapy and other adoptive T cell therapies requiring in vitro expansion and cryopreservation.