Glutamine synthetase deficiency enhances CD8 T cell survival and stress resilience in the tumor microenvironment.

Cellular immunotherapy has revolutionized the treatment of hematologic malignancies yet has had limited success in the solid tumor microenvironment (TME). While insufficient nutrients can lead to T cell metabolic stress in the TME, the glutamine antagonist DON can paradoxically enhance antitumor immunity. Because DON inhibits both essential and nonessential enzymes whose impairment may contribute to dose-limiting toxicities, mechanisms underlying DON-induced antitumor activity have remained unclear. Here, we aimed to identify specific DON targets that increase T cell antitumor activity and test if more selective inhibition of glutamine metabolism could replicate the effects of DON with reduced toxicity. CRISPR screening in the TME of DON-relevant glutamine metabolizing enzymes identified some targets that were essential in tumor-infiltrating CD8 T cells, but that tumor-infiltrating CD8 T cells lacking the DON target glutamine synthetase (GS) were enriched. Upon adoptive T cell transfers, GS-deficient CD8+ T cells displayed improved survival, a higher proportion TCF-1+ Tox- stem-like cells, and greater antitumor and memory function. GS converts glutamate to glutamine and GS-deficient cells exhibited increased intracellular glutamate and reduced glutathione levels, which correlated with enhanced mitochondrial respiration and resistance to reactive oxygen species. Pharmacological inhibition of GS reduced tumor burden in multiple orthotopic murine tumor models in a manner dependent on adaptive immunity. Our findings establish GS as a key metabolic regulator of CD8+ T cells stress resilience in the TME. By preserving intracellular glutamate, GS inhibition reprograms T cells for improved survival and function, offering a promising therapeutic strategy to enhance immune-based cancer treatments.