Interferon-gamma and tumor necrosis factor-alpha synergistically enhance the immunosuppressive capacity of human umbilical-cord-derived mesenchymal stem cells by increasing PD-L1 expression

Figure 3 Tumor necrosis factor-alpha synergistically enhanced interferon-gamma-induced programmed cell death protein 1 ligand 1 expression in human umbilical-cord-mesenchymal stem cells. A: Programmed cell death protein 1 ligand 1 (PD-L1) expression was measured by flow cytometry in human umbilical-cord-mesenchymal stem cells (hUC-MSCs) treated with 0, 10, 20, 40, 60, 80 or 100 ng/mL tumor necrosis factor-alpha (TNF-α) for 24 h; B: PD-L1 expression was measured by flow cytometry in hUC-MSCs treated with 100 ng/mL TNF-α for 0, 2, 4, 6, 8, 12, 24, or 48 h; C: Proliferation was measured by CCK8 in hUC-MSCs treated with 0, 10, 20, 40, 60, 80 or 100 ng/mL of TNF-α for 24 h; D: Apoptotic hUC-MSCs were measured by flow cytometry in hUC-MSCs treated with 0, 10, 20, 40, 60, 80 or 100 ng/mL TNF-α for 24 h. The histogram shows the quantification results pertaining to the percentage of apoptotic cells (Annexin V⁺ cells) in each group; E: PD-L1 expression was quantified by flow cytometry in hUC-MSCs after 24 h of incubation with IFN-γ (20 ng/mL) and 0, 10, 20, 40, 60, 80 or 100 ng/mL TNF-α; F: PD-L1 expression was measured by quantitative real-time PCR in hUC-MSCs treated with IFN-γ (20 ng/mL), TNF-α (10 ng/mL), or a combination of IFN-γ (20 ng/mL) and TNF-α (10 ng/mL) for 4 h; G: PD-L1 expression was analyzed by western blot in hUC-MSCs treated with IFN-γ (20 ng/mL), TNF-α (10 ng/mL), or a combination of IFN-γ (20 ng/mL) and TNF-α (10 ng/mL) for 24 h. Data were represented as mean ± SEM of n = 3. C: Control; Iso: Isotype; ^bP < 0.001; ^cP < 0.0001; ^dP > 0.05. IFN-γ: Interferon-gamma; PD-L1: Programmed cell death protein 1 ligand 1; TNF-α: Tumor necrosis factor-alpha.