Fig. 6

PD-1 reduces the number and length of mitochondrial cristae. a Representative micrographs showing magnified mitochondria from T_CTRL, T_ACT, and T_{ACT + PD1}-stimulated cells (48 h). b-d Percentage of mitochondria without cristae (b), average number of cristae per mitochondrion in each cell (c), and length of cristae in each mitochondrion (d) in CD8⁺ T cells stimulated for 48 h, as indicated. e Relative CHCHD3 mRNA levels in shRNA_CTRL- or shRNA_CHCHD3-transduced CD8⁺ T cells. Data are mean ± SEM (n = 3). f Representative immunoblot showing CHCHD3 protein levels in shRNA_CTRL- or shRNA_CHCHD3-transduced cells. The densitometric CHCHD3/β-actin ratio was calculated, using the value for shRNA_CTRL cells as reference (n = 2). g, h Percentage of shRNA_CTRL- or shRNA_CHCHD3-transduced CD8⁺ T cells showing polarized mitochondria, as determined by TMRM staining (g), and producing IFNγ (h). Each pair of points represents an independent donor. For a, b and d, n = 127 (T_CTRL), 170 (T_ACT) and 222 (T_{ACT + PD1}) mitochondria analyzed; for c, n = 17 (T_CTRL), 23 (T_ACT) and 33 (T_{ACT + PD1}) cells. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA with Bonferroni’s post-test (b-d) or two-tailed paired Student’s t-test (e, g-h)