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 5 Tumor necrosis factor-alpha synergistically amplified interferon-gamma-induced JAK/STAT1/interferon regulatory factor 1 signaling activation by upregulating NF-κB-mediated interferon-gamma receptor 1/2 expression. A: The expression of NF-κB-mediated interferon-gamma receptor 1/2 (IFNGR1/2) were quantified by quantitative real-time PCR (qRT-PCR) in human umbilical-cord-mesenchymal stem cells (hUC-MSCs) treated with IFN-γ and tumor necrosis factor-alpha (TNF-α) alone or in combination for 4 h; B and C: IFNGR1 expression was quantified by flow cytometry or WB in hUC-MSCs treated with IFN-γ and TNF-α alone or in combination for 24 h; D and E: PD-L1 expression was quantified by qRT-PCR or flow cytometry in hUC-MSCs transfected with siRNA against NC, IFNGR1, or IFNGR2, and subsequently treated with IFN-γ and TNF-α for 4 h or 24 h; F: Activation of the JAK/STAT1/interferon regulatory factor 1 pathway and the expressions of PD-L1 and IFNGR1 were analyzed by western blot in hUC-MSCs after 24 h of incubation with IFN-γ and TNF-α in the presence or absence of IFNGR1 siRNA; G: Activation of the NF-κB pathway and expression of IFNGR1 were analyzed by western blot in hUC-MSCs after 24 h of incubation with TNF-α in the presence or absence of the NF-KB inhibitor BAY11-7082 (10 μM); H: IKKα, IKKβ, p65 and IκBα protein levels in the cytoplasm and nucleus of hUC-MSCs after TNF-α stimulation were measured through western blot; I: IFNGR1 expression was quantified by qRT-PCR in hUC-MSCs transfected with p65 siRNA and subsequently treated with IFN-γ and TNF-α for 4 h; J and K: PD-L1 expression was quantified by qRT-PCR or flow cytometry in hUC-MSCs transfected with p65 siRNA and subsequently treated with IFN-γ and TNF-α for 4 h or 24 h; L: hUC-MSCs were infected with IFNGR1 overexpressed lentivirus and transfected with siRNA of p65, and induced with IFN-γ and TNF-α for 4 h, and then PD-L1 expression was quantified by qRT-PCR; M: PGL4.10-IFNGR1 promoter plasmid, pIRES2-p65 plasmid, and Renilla luciferase plasmid were transfected together or separately into 293T cells. Cells were lysed 24 h after transfection and luciferase activity was measured; N: pIRES2-p65 plasmid and Renilla luciferase plasmid were transfected into 293T cells with the PGL4.10-IFNGR1 promoter plasmid or the p65 binding site mutant PGL4.10-IFNGR1 promoter plasmid. Cells were lysed 24 h after transfection and luciferase activity was measured. The predicted binding sites of p65 to the IFNGR1 promoter are above the graph. 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.