Extended Data Fig. 5: PGE₂ affects mitochondrial fitness, T cell oxidative response and lipid metabolism.

a, Relative mitochondrial DNA copy number in unstimulated, repeatedly activated T cells and TILs after 24 h PGE₂ (Fold change to CTRL) (n = 6). b, Representative Electron Microscopy images and c, representative quantitative plot of mitochondrion number/cell in unstimulated and repeatedly activated T cells upon 24 h PGE₂ (n = 3). d, Representative histogram of mitochondrial potential (TMRM) staining in CTRL versus PGE₂ treated TILs. e, Fold change (relative to CTRL) of mitochondrial potential (TMRM) in repeatedly activated CD8⁺ T cells after 24 h PGE₂ (n = 3). f, Quantification of basal respiration, spare respiratory capacity (SRC) and ATP production in repeatedly activated T cells treated with PGE₂ for 24 h (n = 5). g, Fold change (relative to CTRL) of protein synthesis (OPP) in CD39⁻ and CD39⁺ CD8⁺ TILs treated with PGE₂ for 24 h (n = 5). h, Oxidized (GSSG)/reduced (GSH) glutathione ratio quantified by mass-spectromety in CD8⁺ repeatedly activated T cells treated with PGE₂ for 24 h (n = 5). i, Western Blot (top) and quantification (bottom) of PGC1α in TILs treated with IL-2 and PGE₂ for 48 h (representative of 2 biological replicates). j, PGC1A mRNA relative expression in repeatedly activated T cells treated with PGE₂ or mTOR inhibitor Everolimus for 12 h and then stimulated with IL-2 for 15 min (n = 6). k, PD1, TOX protein expression and EP2 and EP4 gene expression in unstimulated or repeatedly activated murine OT1 T cells (n = 3). l, PGC1A mRNA relative expression in repeatedly activated OTI murine T cells transduced with a PGC1α overexpressing vector (n = 4). m, Oxidized (GSSG)/reduced (GSH) glutathione ratio in response to 72 h PGE₂ in repeatedly activated OTI murine T cells transduced with a PGC1α -overexpressing vector (n = 4). n, Heatmap of free fatty acids relative abundance (%CTRL) measured by mass spectrometry in repeatedly activated CD8⁺ T cells (n = 5) and in o, CD8⁺ TILs treated with PGE₂ for 24 h (n = 4). p, Violin Plot representation (Fold change to CTRL) of lipid droplets metabolic task in repeatedly activated CD8⁺ T cells treated with PGE₂ (n = 3). q, Microscopy image (representative of 4 biological replicates) and r, mean lipid droplets/cells in TILs upon 24 h PGE₂ (n = 4). s, mRNA relative expression of CPT1A, HIF2α, CREB3L3 in TILs treated with PGE₂ for 48 h (n = 5). t, Representative histogram of BODIPY-C11 lipid peroxidation staining in CTRL versus PGE₂ treated CD8⁺ TILs. u, Lipid peroxidation quantification in repeatedly activated CD8⁺ T cells treated with PGE₂ for 48 h (n = 3). v, Western blots of GPX4 protein expression in TILs treated or not with PGE₂ (representative of 2 biological replicates). w, mRNA relative expression of ACSL4, LPCAT3, FSP1 and GLS2 in TILs treated with PGE₂ for 48 h (n = 5). x, Lipid peroxidation quantification (n = 2) and y, Relative cell count (Fold change to CTRL) of TILs treated with PGE₂ +/− NAC or vitamin E (VITE) for 72 h (n = 6). Data are presented as mean ± S.D. Statistical comparisons were performed using paired (f,h,r,u) or unpaired two-tailed t test (e,k) or one-way ANOVA (a,c,g,j,l,m,s,w,y) with Dunnett post-hoc test for multiple comparisons. Results were pooled from 3 experiments with n = 5 mice per group in each experiment (l,m). Independent biological samples were used with exact numbers of biological replicates listed in each panel. p ≥ 0.05, not significant (ns).