Treg cells inhibit the apoptosis of anti-CD3–stimulated CD8⁺ T cells by regulating PD-L1 expression levels. (A) Expression levels of PD-L1 (top histograms) and PD-1 (bottom histograms) at day 0 (□) and after 48 hours (■) of anti-CD3 stimulation were compared on the indicated CD8⁺ T-cell subsets. *Significant change (P < .05) compared with nonstimulated control. (B) Treg cells and anti–PD-L1–blocking mAb inhibit CD8⁺ T-cell AICD but do not prevent anti–PD-1–induced apoptosis. Pooled data from 7 separate experiments where CD8⁺ T cells were stimulated for 48 hours with coated anti-CD3 mAb alone, in the presence of CD4⁺CD25⁻ T cells or in the presence of Treg cells. Blocking anti–PD-L1 or agonist anti–PD-1 antibodies were added as indicated. The percentage of apoptosis change was calculated on the basis of annexin V⁺ cells (mean ± SD) detected when CD8⁺ T cells were stimulated alone. (C) Pooled data from 12 donors showing the expression of PD-1 and PD-L1 on anti-CD3–stimulated CD8⁺ T cells in the presence of CD4⁺CD25⁻ T cells (□) or Treg cells (■). (D) M (squares), E (triangles), or TE (circles) CD8⁺ T-cell subsets were sorted from 3 different donors and stimulated in the presence of CD4⁺CD25⁻ T cells (open symbols) or Treg cells (filled symbols). Percentages of PD-1 and PD-L1 expression in each subset are shown. P values were calculated using a paired Student t test. (E) Correlation between the inhibition of PD-L1 expression on CD8 T cells and the inhibition of CD8 T-cell apoptosis in the presence of Treg cells. (F) Pooled data from 14 donors showing the expression of PD-1 and PD-L1 on anti-CD3–stimulated CD4⁺CD25⁻ T cells (□) or Treg cells (■).