PMC

Schematic image of this study. γ‐Interferon (IFN‐γ) stimulation upregulates expression levels of programmed cell death ligand 1 (PD‐L1) mediated by the activation of macrophage migration inhibitory factor (MIF)‐CD74 interactions, which is induced by the upregulation of cell surface CD74 expression by IFN‐γ stimulation

Expressions of CD74 and programmed cell death ligand 1 (PD‐L1) in melanoma tissue samples. A, Representative immunohistochemical staining of CD74 (left panels) and (PD‐L1; right panels) in melanoma tissue samples. Magnification, 200×. Positive staining is visualized by red staining. Upper left panel: metastatic melanoma sample stained positively for CD74 in both the cytoplasm and cell membrane. Upper right panel: the same metastatic melanoma sample in the upper left panel stained positively for PD‐L1 in the cell membrane. Positive staining in the cytoplasm was considered to be negative in the present study. Lower left panel: primary melanoma sample stained negatively for CD74. Lower right panel: same primary melanoma sample in the lower left panel stained negatively for PD‐L1. B, Correlation analysis of CD74 and PD‐L1 expression status in all 97 samples (upper table), 64 primary samples (middle table), and 33 metastatic samples (lower table). The χ² values were 12.44 (P < .01), 13.97 (P < .01), and 9.03 (P < .01) in all samples, primary lesions, and metastatic lesions, respectively, suggesting a high correlation between the expression levels of CD74 and PD‐L1

Macrophage migration inhibitory factor (MIF)‐CD74 interaction regulates activity of several signaling molecules in the WM1361A melanoma cell line. A, Immunocytochemical analysis. WM1361A cells were confirmed to express programmed cell death ligand 1 (PD‐L1) (left panel), MIF (middle panel), and CD74 (right panel) in untreated cultures. B, Flow cytometry analysis. WM1361A cells showed cell surface expression of CD74. Mean fluorescence intensity of each condition was as follows. Isotype control, 0.47; nontreated (NT), 0.71; 4‐iodo‐6‐phenylpyrimidine (4‐IPP) 200 μmol/L, 0.65. C, Cell viability analysis. 4‐IPP treatment suppressed the proliferation of WM1361A cells in a dose‐dependent manner. D, Protein array. 4‐IPP treatment for 48 h decreased expression of phosphorylated (phospho‐)AMPKa2 (T172) (a), phospho‐STAT2 (Y689) (b), phospho‐STAT6 (Y461) (c), β‐catenin (d), and phospho‐c‐JUN (S63) (e), and increased expression of phospho‐P53 (S392) (f) and phospho‐STAT3 (S727) (g). Right panel, relative dot density of protein array analysis

γ‐Interferon (IFN‐γ) stimulation upregulates the expression of CD74 in melanoma cells. A375 and SB2 cells were treated with/without IFN‐γ and/or 4‐iodo‐6‐phenylpyrimidine (4‐IPP). A, Cell viability analysis. A375 (upper panel) and SB2 (lower panel). Treatment with IFN‐γ 100 IU/mL alone did not affect the cell proliferation in either cell line. However, 4‐IPP 100 μmol/L treatment alone or combined with IFN‐γ 100 IU/mL suppressed cell proliferation. B, Quantitative real‐time PCR analysis to measure mRNA levels of CD74 in A375 cells (upper panel) and SB2 cells (lower panel). Stimulation with IFN‐γ‐ upregulated the expression of CD74, which was not affected by further treatment with 4‐IPP. Shown are representative data from 1 of 3 experiments. C, Western blot analysis of CD74 protein expression in A375 cells (upper panel) and SB2 cells (lower panel). Stimulation with IFN‐γ upregulated the expression of CD74. Further treatment of A375 cells with 4‐IPP showed further upregulation of CD74 expression, possibly due to a compensatory mechanism. MIF, macrophage migration inhibitory factor. D, Flow cytometry analysis of cell surface CD74 protein in A375 cells (upper panel) and SB2 cells (lower panel). IFN‐γ stimulation upregulated the expression of cell surface CD74 protein level in both cell lines. Further treatment of A375 cells with 4‐IPP showed further upregulation of CD74 expression. Mean fluorescence intensity (MFI) of each condition was as follows. A375 cells: isotype control, 0.26, nontreated (NT), 0.30; IFN‐γ 100 IU/mL, 0.48; IFN‐γ 100 IU/mL and 4‐IPP 100 μmol/L, 0.69. SB2 cells: isotype control, 0.16; NT, 0.19; IFN‐γ 100 IU/mL, 0.34; IFN‐γ 100 IU/mL and 4‐IPP 100 μmol/L, 0.25

γ‐Interferon (IFN‐γ) stimulation upregulates the expression of programmed cell death ligand 1 (PD‐L1) in melanoma cells through macrophage migration inhibitory factor (MIF)‐CD74 interaction. A375 and SB2 cells were treated with 4‐iodo‐6‐phenylpyrimidine (4‐IPP) for 48 h under IFN‐γ stimulatory conditions. A, Quantitative real‐time PCR analysis to measure mRNA levels of PD‐L1 in A375 cells (upper panel) and SB2 cells (lower panel). IFN‐γ stimulation upregulated expression of PD‐L1, which was suppressed by further treatment with 4‐IPP. *P < .05; **P < .01. Shown are representative data from 1 of 3 experiments. B, Western blot analysis of PD‐L1 protein expression in A375 cells (upper panel) and SB2 cells (lower panel). IFN‐γ stimulation upregulated the expression of PD‐L1, which was suppressed by further treatment with 4‐IPP. C, Flow cytometry analysis of cell surface PD‐L1 protein in A375 cells (upper panel) and SB2 cells (lower panel). IFN‐γ stimulation upregulated the expression of cell surface PD‐L1 protein level in both cell lines. Further treatment with 4‐IPP showed downregulation of CD74 expression. Mean fluorescence intensity of each condition was as follows. A375 cells: isotype control, 0.86; nontreated (NT), 0.87; IFN‐γ 100 IU/mL, 2.57; IFN‐γ 100 IU/mL; and 4‐IPP 100uM, 1.77. SB2 cells: isotype control, 0.90; nontreated (NT), 0.91; IFN‐γ 100 IU/mL, 3.90; IFN‐γ 100 IU/mL and 4‐IPP 100 μmol/L, 1.16. D, Immunocytochemistry of A375 cells (left panels) and SB2 cells (right panels) PD‐L1 was negative under normal culture conditions (upper panels). IFN‐γ stimulation upregulated the membrane staining of PD‐L1 (middle panels), and cell staining was suppressed by further treatment with 4‐IPP (lower panels) in both cell lines. Shown are representative data from 1 of 3 experiments

Macrophage migration inhibitory factor (MIF)‐CD74 interaction regulates expression of programmed cell death ligand 1 (PD‐L1) in the WM1361A melanoma cell line. WM1361A cells were treated with 4‐iodo‐6‐phenylpyrimidine (4‐IPP) for 72 h. A, Quantitative real‐time PCR analysis. The mRNA level of PD‐L1 was suppressed in a dose‐dependent manner by 74.4% when treated with 50 μmol/L 4‐IPP, and suppressed by 60.7% with 200 μmol/L 4‐IPP. **P < .01. B, Western blot analysis. The expression level of PD‐L1 protein was suppressed in a dose‐dependent manner. C, Flow cytometry analysis. Cell surface PD‐L1 expression was positive in untreated condition. 4‐IPP treatment decreased its expression level. Mean fluorescence intensity of each condition was: isotype control, 5.74; nontreated (NT), 17.9; 4‐IPP 200 μmol/L, 12.5. D, Membrane staining of PD‐L1 was suppressed when treated with 200 μmol/L 4‐IPP. Number of PD‐L1 positive cells from 5 different randomly selected areas were counted using a high‐powered field (400× magnification), and the average of the 5 sums was calculated. The mean number of PD‐L1‐positive cells were 21.8 in the untreated condition and 4.6 in the 4‐IPP 200 μmol/L treated condition. E, WM1361A cells were transfected with siRNA targeting CD74, and the expression levels of CD74 (upper panel) and PD‐L1 (lower panel) were analyzed by quantitative real‐time PCR 48 h after transfection. Transfections of 2 sequences of siRNA targeting CD74 decreased the expression levels of CD74 by 40.7% (siCD74‐1) and 3.8% (siCD74‐2). E, Flow cytometry analysis. Expression of cell surface CD74 (upper panel) and PD‐L1 (lower panel) were analyzed 72 h after the transfections of 2 sequences of siRNA targeting CD74. Both sequences decreased expression level of CD74 and PD‐L1. Mean fluorescence intensity of each condition was follows. CD74: siNT, 0.51; siCD74‐1, 0.43; siCD74‐2, 0.40. PD‐L1: siNT, 7.20; siCD74‐1, 4.78; siCD74‐2, 4.12. Shown are representative data from 1 of 3 experiments