Fig. 5: Chronic TCR stimulation promotes increased βOHB metabolism.
a, Inhibitory receptor expression on in vitro-stimulated T cells. Representative histograms of exhaustion marker expression (PD1, TOX) in CD8⁺ T cells under acute and chronic stimulation conditions. Data represent the means; error bars, s.e.m. (n = 3 mice per group). b, Heatmap showing the relative abundance (z-score) of TCA cycle-derived metabolites from CD8⁺ T cells exposed to acute versus chronic stimulation with anti-CD3 and anti-CD28 antibodies in vitro (n = 3 mice per group). c, Bar graph depicting total abundance of 13C-labelled βOHB in acute versus chronic stimulated CD8⁺ T cells. Data represent the means; error bars, s.e.m. (n = 3 mice per group). Statistical significance was calculated with a two-tailed, unpaired Student’s t-test. d, Bar graph depicting [U-13C6]-glucose labelling into glucose-6-phosphate (G6P), M + 6, in acute and chronic stimulated CD8+ T cells. Statistical significance was calculated with a two-tailed, unpaired Student’s t-test. Data represent the means; error bars, s.e.m. (n = 3 mice per group). e,f, 13C labelling of TCA cycle intermediates in CD8⁺ T cells exposed to chronic antigen stimulation. CD8⁺ T cells exposed to acute versus chronic stimulation were cultured in VIM medium containing 0.85 mM [13C4]-βOHB for 2 h. e, Total abundance of [13C4]-βOHB-derived citrate and malate. f, Ratio of [13C4]-βOHB-labelled malate (M + 2) to citrate (M + 2) for T cells from e. Data represent the means; error bars, s.e.m. (n = 3 mice per group). Statistical significance was calculated with a two-tailed, unpaired Student’s t-test. g, Bar graph showing [13C2]-acetate into UDP-GlcNAc (M + 2) in acute and chronically stimulated CD8+ T cells, as well as [13C4]-βOHB into UDP-GlcNAc (M + 2) acute and chronically stimulated CD8+ T cells (total abundance). Right, bar graph showing [13C2]-acetate into Ac-Met (M + 2) in acute and chronically stimulated CD8+ T cells as well as [13C4]-βOHB into Ac-Met (M + 2) acute and chronically stimulated CD8+ T cells (total abundance). Data represent the means; error bars, s.e.m. (n = 3 mice per group). Statistical significance was calculated via two-way ANOVA with multiple comparisons. h, UMAP of 8,552 human CD8⁺ TILs from GSE98638. Shown are unique clusters for effector (Teff), memory (Tmem), terminally exhausted (TexTerm) and proliferating exhausted (TexEff) CD8⁺ T cell populations. i–k, Violin plots of gene expression across human CD8⁺ TIL subsets. i, Expression of MKI67 human gene between CD8+ T cell subsets. j, Expression of ketolysis signature genes (ketolysis gene set: ACAT1, ACAT2, BDH1, BDH2, HMGCL, HMGCS1, HMGCS2, OXCT1, OXCT2). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple comparisons test and a 5% significance level. k, Violin plots of BDH1 and OXCT1 gene expression between different human CD8+ T cell subsets. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are as follows: c, P = 0.0007; d, P = 0.7405; e, left, P = 0.0120, right, P = 0.0014; f, P = 0.0003; g, left, ns = 0.8633, ***P = 0.002; right, ns = 0.2021, ****P < 0.0001; i–k, P < 0.0001.
