Figure 3

Blockade of the PD-1/PD-L1 pathway by anti-PD-L1 antibody. Blockade of the programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) pathway by anti-PD-L1 antibody decreased apoptosis of human peripheral T cells from septic patients induced by TNFα and by T-cell receptor ligation. (a) Peripheral blood mononuclear cells were obtained from septic shock patients and cultured at 4 × 10⁵ per well in plates precoated with 10 ng/ml TNFα (Peprotech) alone or with anti-PD-L1 antibody(10 μg/ml) or isotype (10 μg/ml) for 18 hours to analyze apoptosis by flow cytometry. Alternatively, peripheral blood mononuclear cells were treated with 10 μg/ml anti-CD3 and 5 μg/ml anti-CD28 alone or with anti-PD-L1 antibody (10 μg/ml) or isotype (10 μg/ml) for 72 hours. Cells were doubled-stained with annexin V and propidium iodide (PI) with gating on CD3⁺ cells and were analyzed by fluorescence-activated cell sorting. Apoptosis was calculated as the percentage of annexin V-positive/PI-negative cells after gating on CD3⁺ cells. *P < 0.05 compared with control group. (b) Representative micrographs from six independent experiments. Representative data showing apoptosis of CD3⁺ T cells.