A metabolic-oxidative axis contributes to NK cell cryo-vulnerability.

While immunotherapy has revolutionized cancer and other disease treatment, its clinical application requires effective cryopreservation for scalable manufacturing and global distribution. However, many immune cells exhibit substantial freeze-thaw sensitivity, limiting their scalable production and distribution. Using metabolic flux analysis, ROS quantification, lipidomics, and xenograft models, we identified metabolic state as a determinant of immune cell cryo-sensitivity. Natural killer (NK) cell activation induced a metabolic shift with elevated glucose utilization and excessive ROS, causing profound post-thaw viability and functional loss (∼25% survival). Targeted pretreatments-glucose metabolism inhibitors, antioxidants, and lipid peroxidation inhibitors-restored post-thaw recovery to ∼90% while preserving effector activity and antitumor efficacy in immunodeficient mice. Similar protection in αβ T cells, γδ T cells, and macrophages indicates that metabolic and oxidative features broadly emerge as key determinants of immune cell cryopreservation sensitivity. These findings support a metabolic-oxidative axis as an important feature associated with NK cell cryo-vulnerability.