Extended Data Fig. 3: Active aldehydes, but not hydrogen peroxide (H2O2), enhance glycolysis and proliferation of CD8+ T cells.
(A) Acrolein concentration (μM) in MC38 tumor tissue on day 14 (n = 6 mice). An experimental control group was not included, as the experiment aimed to determine the physiological acrolein levels in tumor tissues. (B) Frequencies of D1–D4 populations among CD8+ T cells 21 hours after stimulation with anti-CD3/CD28 beads in the presence or absence of 2 μM acrolein (ACR) (n = 5 technical replicates). (C) MFI of pAkt, pmTOR and pS6 in naïve-derived CD8+ T cells on day 8 of repetitive stimulation in the presence or absence of ACR (n = 5 technical replicates). (D) Frequencies of PD-1− Tim-3− and PD-1+ Tim-3+ cells among naïve-derived CD8+ T cells on day 8 of repetitive stimulation in the presence or absence of ACR (n = 5 technical replicates). (E) Western blotting images of enzymes associated with glycolysis and FAO in CD8+ T cells stimulated with anti-CD3/CD28 beads for 3 hours in the presence or absence of 5 μM ACR. (F) Seahorse measurement of real-time ECAR of naïve CD8+ T cells 3 hours after stimulation with anti-CD3/CD28 beads in the presence or absence of 0.5 μM 4-hydroxynonenal (4-HNE) (n = 4 technical replicates). (G) Thymidine incorporation of CD8+ T cells 7 hours after stimulation with anti-CD3/CD28 beads in the presence or absence of 0.5 μM 4-HNE (n = 4 technical replicates). The control data are shared with those in Fig. 3f to compare results under the same conditions in the same experiment, as supportive data. (H) Seahorse measurement of real-time ECAR of CD8+ T cells 3 hours after stimulation with anti-CD3/CD28 beads in the presence or absence of 120 μM H2O2 (n = 4 technical replicates). (I) Frequencies of D1–D4 populations among CD8+ T cells 7 hours after stimulation with anti-CD3/CD28 beads in the presence or absence of 120 μM H2O2 (n = 6 technical replicates). (J) Frequencies of D1–D4 populations among CD8+ T cells 14 hours after stimulation with anti-CD3/CD28 beads in the presence or absence of 60 μM H2O2 (n = 5 technical replicates). Data are means ± SEMs. P values were determined using unpaired two-tailed Student’s t tests. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. All data are representative of two or more independent experiments with similar results.
