Figure 3

Representative flow histograms of lymphocyte gating strategy and detection of apoptosis and IFN-γ. Peripheral blood mononuclear cells (PBMCs) from a septic patient were plated overnight with isotype control antibody, anti-programmed cell death 1 (PD-1) or anti-programmed cell death ligand 1 (PD-L1) antibody. A: Cells were immunostained for CD4 and CD8 T cells and TUNEL assay performed. The lymphocyte fraction in the PBMCs was identified by characteristic forward and side scatter properties and CD4 and CD8 T cells identified by cell-specific antibodies. Apoptosis in CD4 T cells incubated in inactive isotype control antibody was 7.5%. Anti-PD-1 and anti-PD-L1 decreased CD4 apoptosis to 1.25 and 2.0%, respectively. A similar protective effect was seen in CD8 T cells. B: PBMCs from a septic patient were incubated overnight with isotype control antibody, anti-PD-1 or anti-PD-L1 antibody. The following morning, cells were stimulated with PMA/ionomycin plus brefeldin for 5 h, washed, immunostained with phenotypic markers to CD3 and CD56, fixed and stained for intracellular interferon (IFN)-γ. The lymphocyte gate was identified by characteristic forward and side scatter properties. Natural killer (NK) cells were identified as CD3 negative CD56 positive. The percentage of NK cells that were positive for IFN-γ that were incubated in inactive isotype control antibody was 9.9%. Treatment with anti-PD-1 and anti-PD-L1 increased the percentage of NK cells that were IFN-γ positive to 34.8 and 21.8%, respectively.