Shifted substrate preference toward FA oxidation in NFATc1-dysfunctional T cells. (A) Summary dot plot showing the lipid uptake of patient and HC T lymphoblasts after 24 hours of stimulation with soluble anti-CD3/CD28 antibodies using Bodipy FL C16 staining. (B) Bar graph showing the measured levels of total lipid species in HCs and patients with and without stimulation. (C) Volcano plot showing the log₂ fold changes (x-axis) in lipid species upon stimulation in HCs vs patients. The y-axis represents the P values for −log₁₀ of the measurements. Red full circles represent the TAG species, each significantly changed TAG species is indicated (statistics performed using t tests). (D) Heatmap showing fold changes in concentrations of different lipid species in patient and HC T lymphoblasts measured by mass spectrometry–based lipidomics after 24 hours of stimulation with soluble anti-CD3/CD28 antibodies. (E) Scheme depicting the TCA cycle and the TCA cycle intermediates that are detected in the current analysis. Summary graphs show the relative ¹³C enrichment of TCA cycle intermediates in patient T lymphoblasts incubated in [U-¹³C₁₆]palmitate, normalized to the HCs. (F) SCENITH analysis showing the metabolic alterations of patients (P1 and P2) and HCs (experiment 1: n = 4; experiment 2: n = 3) CD8⁺ TEM population after CD3/CD28 bead stimulation. The experiment was performed twice and the plot shows the averaged results from each experimental replicate. The negative percentage values shown in the graph are inherent to the analysis of the assay. The calculation formulas are indicated in the supplemental Methods. Statistical analysis was performed using 2-way ANOVA with Bonferroni post hoc test to correct for multiple comparison. Sorted naïve T cells from HCs (n = 4) were treated with vehicle, CsA (300 nM), or 2-DG (1 μM), and stimulated them with Dynabeads coated with CD3 and CD28 antibodies. (G) Summary graphs showing the percentage of CD8⁺ T cells with upregulated CD25 surface expression after stimulation with CD3/CD28 Dynabeads and percentage of proliferation by measuring dilution of the VPD450 in CD8⁺ T cells at basal state (day 0), and on days 2, 3, 4, and 8 after stimulation in vehicle-, CsA-, or 2-DG–treated cells. Summary graphs showing the gMFI of 2-NBDG and Bodipy FL16 in CD8⁺ T cells after stimulation with CD3/CD28 beads measured via flow cytometry at basal state (unstimulated), and on days 2, 3, 4, and 8 after stimulation. (H) (Left) Immunoblot showing the protein expression levels of HK2, CPT1a, and GAPDH. (Right) Graphs showing the quantification of the western blots averaged from 3 independent experiments displaying the expressions of HK2 and CPT1a on HC naïve T cells harvested on day 4 after stimulation with Dynabeads. (I) Summary graphs show the relative ¹³C enrichment of TCA cycle intermediates (citrate, malate, and aspartate) in HC T cells stimulated with Dynabeads (for 4 days) and treated with CsA, 2-DG, or vehicle, incubated in [U-¹³C₁₆] palmitate and normalized to the vehicle treatment. All data are mean ± SEM. Statistics performed using one-way ANOVA with post hoc Bonferroni correction if not stated otherwise.