Fig. 3: YTHDF2 prevents mitochondrial stress and T cell exhaustion.
a Volcano plots of genes with differential mRNA expression in activated Ythdf2F/F or Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h) (n = 2 independent samples per genotype). Significantly upregulated or downregulated genes are indicated as red or blue dots. Putative YTHDF2 targets enriched in both RIP-seq and m6A-seq are marked with yellow circles. b GO enrichment analysis of upregulated genes in Ythdf2CKO compared with Ythdf2F/F CD8 T cells after priming (anti-CD3/CD28, 5 μg/ml, 24 h). c Mitochondrial membrane potential and mitochondrial mass were measured by MitoTracker Orange (MO) and MitoTracker Green (MG) staining in activated Ythdf2F/F (n = 5 independent samples) and Ythdf2CKO (n = 5 independent samples) CD8 T cells. Mitochondrial fitness was evaluated according to the MO/MG ratio. d Mitochondrial ROS was measured by MitoSOX staining in activated CD8 T cells from Ythdf2F/F (n = 5) or Ythdf2CKO (n = 5) mice. e MitoSOX staining in MC38 tumor-infiltrating CD8 T cells from Ythdf2F/F (n = 5) and Ythdf2CKO (n = 5) mice. f Quantification of the MG MFI in MC38 tumor-infiltrating CD8 T cells from Ythdf2F/F (n = 5) or Ythdf2CKO (n = 5) mice. g, h Quantification of Tim3+ PD-1+ (g), Zombie NIR+ (h) frequencies among primed Ythdf2F/F (n = 5 independent samples) and Ythdf2CKO (n = 5 independent samples) CD8 T cells (anti-CD3/CD28, 5 μg/ml, 48 h) in the presence of 10 mΜ NAC or veh. i Heatmap showing the relative expression of representative genes (mitochondrion-related and up-regulated in Ythdf2CKO) in activated Ythdf2F/F and Ythdf2CKO CD8 T cells from RNA-seq data. Putative YTHDF2 targets are depicted by filled circles. Error bars, mean ± s.e.m. One-sided Fisher’s exact test with P values adjusted by the Benjamini–Hochberg method (a, b). Two-way ANOVA (g, h) or two-tailed unpaired Student’s t-test (c–f).
