Macrophage trained immunity requires a mitochondrial glutamine-restriction checkpoint for antitumor protection in mice.

Trained immunity enables innate immune cells to acquire memory-like responses, offering a strategy to enhance antitumor immunity. However, the metabolic‒epigenetic mechanisms underlying this process remain poorly defined. Here, we show that lipopolysaccharide-induced macrophage training is encoded by a mitochondrial metabolic checkpoint. Integrated transcriptomic, metabolomic, and epigenomic profiling reveals that TLR4-NF-κB signaling represses SLC1A5_var, a mitochondrial glutamine transporter, limiting glutaminolysis and reducing α-ketoglutarate availability. This metabolic restriction limits removal of the activating histone mark histone H3 lysine 4 trimethylation by KDM5B, thereby maintaining inflammatory gene accessibility. Functionally, pharmacological inhibition or myeloid-specific knockdown of SLC1A5_var potentiates macrophage training and improves tumor control in murine cancer models, whereas enforced SLC1A5_var expression or α-ketoglutarate supplementation abrogates these effects. These findings define an SLC1A5_var-α-ketoglutarate-KDM5B metabolic-epigenetic axis that programs macrophage trained immunity and illustrate how targeted metabolic restriction can be leveraged to enhance innate immune responses against cancer.