Increased PD-L1 expression in activated neutrophils inhibits cellular apoptosis by maintaining AKT phosphorylation. (A) PD-L1 expression was increased and rates of apoptosis were significantly decreased in human healthy control neutrophils stimulated with IFN-γ (10 ng/mL) and/or LPS (1 μg/mL) for 21 hours (n = 6; *P < .05, 1-way analysis of variance [ANOVA]). (B) Twenty-one hours after isolation, PD-L1 expression was increased in neutrophils from patients with sepsis, when compared with resting control neutrophils, and correlated negatively with rates of apoptosis (n = 14; P < .05, by linear regression analysis). (C) Genetic silencing of PD-L1 using siRNA in human control neutrophils stimulated with IFN-γ (10 ng/mL) and/or LPS (1 μg/mL) for 21 hours significantly increased apoptosis (n = 5). *P < .05, 1-way ANOVA. (D) Twenty-one hours after isolation, human septic neutrophils transfected with PD-L1 siRNA demonstrated significantly higher rates of apoptosis than those transfected with scrambled control siRNA (n = 5). *P < .05, 1-way ANOVA. (E) Representative immunoblot shows that AKT phosphorylation (S473) is increased in human healthy control neutrophils challenged with IFN-γ (10 ng/mL) and LPS (1 μg/mL; n = 3) 21 hours after isolation. (F) Representative immunoblots measuring AKT phosphorylation in neutrophil lysates from healthy donors vs that in patients with sepsis (n = 3). The representative blots for PD-L1 and β-actin correspond to the data points shown in panel B. (G) Genetic silencing of PD-L1 in human septic neutrophils with siRNA decreased AKT phosphorylation 21 hours after isolation (n = 3). (H) HEK293 cells were transfected with empty GFP plasmid or GFP-tagged PD-L1. Immunoprecipitated GFP alone and GFP-tagged PD-L1 lysates demonstrate that PD-L1 complexed with p85 (n = 3).