CRBN harnesses arginine and glutamine metabolism in activated CD8⁺ T cells. (A-B) Metabolomic LC-MS/MS analysis of 24-hour anti-CD3ε+anti-CD28–activated Crbn^+/+and Crbn^−/− CD8⁺ T cells. (A) Heat map of metabolites that were significantly different and (B) the average fold change in arginine, glutamine, and proline metabolites. Gray indicates downregulated metabolites in the pathway, and pink denotes increased metabolites in the pathway. Fold change in mRNA levels for enzymes and transporters for each reaction are indicated. All enzymes and transporters were upregulated by the fold change indicated in Crbn^−/− CD8⁺ T cells. Enzymes that were not assessed for differential expression are shown in lavender. (C) L-Arg and L-Glu uptake (% of control) by Crbn^+/+ and Crbn^−/− CD8⁺ T cells after 24 hours of anti-CD3ε+anti-CD28 activation. (D) Intracellular putrescine, spermidine, and spermine levels measured by LC-MS/MS of activated Crbn^+/+and Crbn^−/− CD8⁺ T cells (24 hours). (E) Flow cytometric analyses of CD98 expression in Crbn^+/+and Crbn^−/− CD8⁺ T cells after anti-CD3ε+anti-CD28 stimulation for 24 hours. (F-G) Crbn^+/+ and Crbn^−/− CD8⁺ T-cell proliferation after 72 hours of anti-CD3ε+anti-CD28 stimulation with cotreatment of α-difluoromethylornithine (5 mM; DFMO; F) or oligomycin (G). All results are representative of at least 2 independent experiments (excluding metabolomics analysis). n.s., not significant; *P < .05; ** P < .01; ***P < .001; ****P < .0001.