Extended Data Fig. 1: TGR CD8⁺ T_E do not sustain mitochondrial metabolism by incorporating more glutamine-derived carbons into the TCA cycle.

WT CD8⁺ T cells isolated from spleens of C7Bl/6 mice were activated with anti-CD3 (5 μg/mL), anti-CD28 (0.5 μg/mL), IL-2 (100 U/mL), expanded for a total of 72 h, and exposed to 10 mM, 3 mM, or 1 mM glucose as indicated in cultures set to 1 million per ml. a, Polar metabolites were extracted from 500 µl media supernatant from 20 h cultures of 10 mM and 1 mM cells. Bar graphs represent glutamine concentration in the indicated conditions compared to complete culture media for n = 3 biological replicates. Significance was calculated using 2-tailed Student’s t tests, no significant changes (ns) were observed. b, T_E were generated as above, but during the final 20 h all groups were cultured in 4 mM heavy labelled (U-¹³C) glutamine (100% U-¹³C glutamine). Polar metabolites were extracted and isotopologue distribution assessed by targeted mass spectrometry. TCA intermediates are plotted as percent label from newly metabolized U-¹³C (open bars) or remaining U-¹²C (black bars) glutamine carbons. Data are from n = 3 biological replicates, representative of 2 independent experiments. Significance was calculated using 2-tailed Student’s t tests, no significant changes were observed. c, T_E were generated as above, but during the final 6 h 2 mM U-¹³C-lactate was added to the culture. Polar metabolites were extracted and isotopologue distribution assessed by targeted mass spectrometry. Significance was calculated using 2-tailed Student’s t tests comparing the predominant m + 2 isotopologue group in both conditions. Data shown for n = 3 biological replicates. Significance was calculated using 2-tailed Student’s t tests, No significant changes were observed. All error bars show SEM.

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