Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A CRC cell lines were treated with the indicated concentrations of TER for 72 h. Cell viability assessed using the CCK assay is shown. B Human CRC cell lines were co-cultured with hPD-1 Jurkat-T cells and treated with the indicated concentrations of TER for 72 h. C PD-L1 protein expression in CRC cells co-cultured with hPD-1 Jurkat-T cells and treated with TER for 72 h. GAPDH was used as a loading control. The results are shown as the mean ± SEM. * <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the respective control.

Article Snippet: Next, 5 μL of 0.5 mg/mL biotinylated hPD-1 (#71109, BPS Bioscience) was added, and the plates were incubated for 2 h at room temperature.

Techniques: Cell Culture, Expressing, Control

Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A The viability of hPD-1 Jurkat-T cells and hPD-L1 CHO cells following treatment with the indicated concentrations of TER for 24 h. B Luciferase activity measured using a PD-1/PD-L1 blockade bioassay. hPD-1 Jurkat-T cells (effector cells) were co-cultured with hPD-L1-expressing aAPC/CHO-K1 cells (target cells) in the presence of indicated concentrations of TER. The luminescence signal indicates the level of TCR signaling activation. αPD-L1 was used as a positive control. * <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the respective control.

Article Snippet: Next, 5 μL of 0.5 mg/mL biotinylated hPD-1 (#71109, BPS Bioscience) was added, and the plates were incubated for 2 h at room temperature.

Techniques: Luciferase, Activity Assay, Bioassay, Cell Culture, Expressing, Activation Assay, Positive Control, Control

Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A The viability of hPD-L1 MC38 cells following treatment with the indicated concentrations of TER for 72 h. B CD8 + T cells were isolated from tumors of hPD-1 knock-in mice bearing hPD-L1 MC38 tumors. These tumor-infiltrating CD8 + T cells were co-cultured with hPD-L1 MC38 cells as target cells in the presence of TER for 72 h. Cell viability measured using the CCK assay is depicted. C PD-L1 expression in hPD-L1 MC38 cells, as assessed by western blot analysis using protein lysates from co-culture conditions. GAPDH was used as a loading control. D The levels of immune-related factors, including GrB, IL-2, and IFN-γ, measured in the co-culture supernatant by ELISA. * <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the respective control.

Article Snippet: Next, 5 μL of 0.5 mg/mL biotinylated hPD-1 (#71109, BPS Bioscience) was added, and the plates were incubated for 2 h at room temperature.

Techniques: Isolation, Knock-In, Cell Culture, Expressing, Western Blot, Co-Culture Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A Body weight of hPD-1 knock-in mice during the treatment period. The mice were treated with vehicle or TER (10 or 30 mpk) for the indicated time. B Spleen weight of mice at the endpoint of the experiment. C Tumor volume was measured over time in hPD-1 knock-in mice bearing hPD-L1 MC38 tumors treated with vehicle or TER (10 or 30 mpk). Representative images of excised tumors from each group are shown. D Tumor weight at the endpoint of the experiment. E Flow cytometry analysis of CD8 + T-cell populations in tumors from each treatment group. F PD-L1 expression in tumors from each group, as assessed by western blot analysis. GAPDH was used as a loading control. G IHC staining of tumor sections for immune-related markers, including CD8 + T cells and GrB. Representative images from each group are shown, and the quantitation of marker-positive cells per field is presented. * <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the respective control.

Article Snippet: Next, 5 μL of 0.5 mg/mL biotinylated hPD-1 (#71109, BPS Bioscience) was added, and the plates were incubated for 2 h at room temperature.

Techniques: Knock-In, Flow Cytometry, Expressing, Western Blot, Control, Immunohistochemistry, Quantitation Assay, Marker

Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A Body weight of hPD-1 knock-in mice during the treatment period. The mice were treated with vehicle or TER (30 mpk) and received either an isotype control or a CD8 depletion antibody. B Spleen weight of mice at the endpoint of the experiment. C Tumor volume was measured in hPD-1 knock-in mice bearing hPD-L1 MC38 tumors over time following treatment with vehicle or TER (30 mpk) with or without CD8 depletion. Representative images of excised tumors from each group are shown. D Tumor weight at the endpoint of the experiment. E Flow cytometry analysis confirming CD8 + T-cell depletion in tumors from each treatment group. The proportion of CD8 + cells among total live cells was quantified. F IHC staining of tumor sections for CD8 + T cells and GrB. Representative images from each treatment group are shown, and the quantitation of marker-positive cells per field is presented. * <0.05, ** p < 0.01, and **** p < 0.0001 compared with the respective control.

Article Snippet: Next, 5 μL of 0.5 mg/mL biotinylated hPD-1 (#71109, BPS Bioscience) was added, and the plates were incubated for 2 h at room temperature.

Techniques: Knock-In, Control, Flow Cytometry, Immunohistochemistry, Quantitation Assay, Marker

Journal: American Journal of Cancer Research

Article Title: Deglycosylation of PD-L1 by 2-deoxyglucose reverses PARP inhibitor-induced immunosuppression in triple-negative breast cancer

doi:

Figure Lengend Snippet: 2-DG deglycosylates PD-L1 in TNBC. A. Western blot analysis of PD-L1 protein expression in MDA-MB-231 cells treated with 2-DG, lactacystin (Lac), N-acetylglucosamine (GlcNac), or tunicamycin (TM). The dot indicates the glycosylated PD-L1 and the arrow indicates the deglycosyalted PD-L1. B. PDL1-overexpressing BT549 cells were treated with 2 or 10 mmol/L 2DG or with 1 µgml-1 TM for 12 hours and then subjected to immunoblotting with antibodies against PD-L1. BT549-4NQ cells were included as a negative control. C. Western blot analysis of PD-L1 protein expression in MB468, HCC1806, and BT549 cells treated with IFN-γ with or without 2-DG. D. BT549 cells were treated with indicated concentrations of 2-DG and IFN-γ or EGF for 12 hours, and then PD-L1 protein expression was determined by Western blot analysis. E. Western blot analysis of PD-L1 protein expression in MB468 and BT549 cells treated with 2 or 10 mmol/L 2-DG in combination with IFN-γ or EGF. TM treatment was used as the positive deglycosylation control. F. PD-L1 expression and PD-1 binding on the surface of BT549 cells were analyzed with FACS. The cells were treated with 10 mmol/L 2-DG, 1 µgml-1 TM, or 1 µgml-1 IFN-γ for 12 hours.

Article Snippet: Cells were incubated with 5 mg/mL recombinant human PD-1 Fc chimera protein (R&D Systems, Minneapolis, MN; cat. #1086-PD-050) at room temperature for 60 minutes.

Techniques: Western Blot, Expressing, Negative Control, Binding Assay

Journal: American Journal of Cancer Research

Article Title: Deglycosylation of PD-L1 by 2-deoxyglucose reverses PARP inhibitor-induced immunosuppression in triple-negative breast cancer

doi:

Figure Lengend Snippet: 2-DG deglycosylates PARPi-induced PD-L1 protein in TNBC. A. Western blot analysis of the PD-L1 protein expression of MDA-MB-231 cells treated with 2-DG and/or PARPi (talazoparib or olaparib). B. Western blot analysis of the PD-L1 protein expression of BT549 cells treated with 2-DG and/or PARPi (talazoparib or olaparib). C. PD-L1 expression and PD-1 binding on the surface of MDA-MB-231 cells treated with 2-DG and/or PARPi were analyzed with FACS. *P<0.05.

Article Snippet: Cells were incubated with 5 mg/mL recombinant human PD-1 Fc chimera protein (R&D Systems, Minneapolis, MN; cat. #1086-PD-050) at room temperature for 60 minutes.

Techniques: Western Blot, Expressing, Binding Assay

Journal: American Journal of Cancer Research

Article Title: Deglycosylation of PD-L1 by 2-deoxyglucose reverses PARP inhibitor-induced immunosuppression in triple-negative breast cancer

doi:

Figure Lengend Snippet: 2-DG decreases PD-L1 translocation and stabilization. A. Left, The quantitative binding of PD-1 Fc protein on PD-L1-overexpressing MDA-MB-231 cells was assessed at the indicated times. Cells were treated with 10 mmol/L 2-DG, 1 µgml-1 tunicamycin (TM), or 10 µmol/L olaparib. Right, Images of PD1 Fc protein on PD-L1-overexpressing MDA-MB-231 cells from 0 to 72 hours. B. Western blot analysis of PD-L1 protein expression in PD-L1-overexpressing MDA-MB-231 cells and MDA-MB-231-4NQ cells treated with 2 mmol/L 2-DG IR800. C. Western blot analysis of PD-L1 protein expression in PD-L1-overexpressing MDA-MB-231 cells. Cells were treated with 20 mM cycloheximide (CHX) with or without 2 mmol/L 2-DG at the indicated times. The intensity of PD-L1 protein expression was quantified using a densitometer. *P<0.05. D. Confocal microscopy image showing HSP90B1 and PD-L1 expression in PD-L1-overexpressing MDA-MB-231 cells after treatment with 2-DG. Scale bar, 20 mm.

Article Snippet: Cells were incubated with 5 mg/mL recombinant human PD-1 Fc chimera protein (R&D Systems, Minneapolis, MN; cat. #1086-PD-050) at room temperature for 60 minutes.

Techniques: Translocation Assay, Binding Assay, Western Blot, Expressing, Confocal Microscopy

Journal: Advanced Science

Article Title: Sulfisoxazole Elicits Robust Antitumour Immune Response Along with Immune Checkpoint Therapy by Inhibiting Exosomal PD‐L1

doi: 10.1002/advs.202103245

Figure Lengend Snippet: Schematic illustration depicting the mechanism of action of combination therapy using sulfisoxazole (SFX) and anti‐PD‐1 ( α PD‐1). SFX, an FDA‐approved ETA antagonist, inhibits cancer exosome biogenesis and synergistically enhances the antitumor effect of α PD‐1. 1) Tumors actively secrete exosome with PD‐L1 (exosomal PD‐L1), which inhibits T cell activation as an immune escape mechanism in α PD‐1 monotherapy. 2) SFX inhibits exosome biogenesis in tumors, leading to enhanced antitumor efficacy of α PD‐1.

Article Snippet: [ ] In brief, to evaluate the binding of exosomal PD‐L1 to PD‐1, 96‐well ELISA plates were coated with 4 µg mL −1 human PD‐1 protein (BPS Bioscience, Cat# 71106, San Diego, CA, USA) overnight at 4 °C.

Techniques: Activation Assay

Journal: Advanced Science

Article Title: Sulfisoxazole Elicits Robust Antitumour Immune Response Along with Immune Checkpoint Therapy by Inhibiting Exosomal PD‐L1

doi: 10.1002/advs.202103245

Figure Lengend Snippet: Sulfisoxazole (SFX) inhibits cancer exosome (EXO) biogenesis and suppresses exosomal PD‐L1. a) TEM images of MDA‐MB231‐derived EXOs immunogold‐labeled with α PD‐L1 antibodies. Arrowheads indicate 5 nm gold particles. Scale bar, 50 nm. b) Quantification of secreted EXOs in the presence of different concentrations of SFX ( n = 3). c) Immunoblot for the indicated proteins in cell lysates and EXOs from MDA‐MB231 cells. Exosomal proteins, obtained from equal number of cells (1 × 10 7 ), were loaded per lane ( n = 3). d) PD‐1 binding to MDA‐MB231‐derived EXOs, obtained in the presence of SFX (200 × 10 −6 m ) or IFN‐ γ (10 ng mL −1 ). e,f) Immunoblot of human CD63 and PD‐L1 in circulating EXOs and tumor lysates from MDA‐MB231 xenograft models. β ‐actin was used as the loading control for tumor lysates. (Vehicle; n = 5 and SFX; n = 4). Significance was determined using an unpaired two‐tailed Student's t ‐test. *** p < 0.001, ** p < 0.01, and * p < 0.05. Error bar, standard deviation (SD).

Article Snippet: [ ] In brief, to evaluate the binding of exosomal PD‐L1 to PD‐1, 96‐well ELISA plates were coated with 4 µg mL −1 human PD‐1 protein (BPS Bioscience, Cat# 71106, San Diego, CA, USA) overnight at 4 °C.

Techniques: Derivative Assay, Labeling, Western Blot, Binding Assay, Two Tailed Test, Standard Deviation

Journal: Advanced Science

Article Title: Sulfisoxazole Elicits Robust Antitumour Immune Response Along with Immune Checkpoint Therapy by Inhibiting Exosomal PD‐L1

doi: 10.1002/advs.202103245

Figure Lengend Snippet: Sulfisoxazole (SFX) synergistically enhances the antitumor effect of an immune checkpoint inhibitor. a) Schematic illustration of the therapeutic schedule for CT26 tumor‐bearing mice. b) Antitumor effects of SFX, α PD‐1, and SFX + α PD‐1 ( n = 10). c) Photographs of the tumors harvested on day 21 ( n = 10). d) Tumor weight after treatment ( n = 10). e) Quantification of exosomal PD‐L1 in mouse plasma after therapeutic regime ( n = 10). f, g) Cytokine levels in plasma were quantified using ELISA ( n = 6). Significance was determined using an ANOVA with Tukey correction. *** p < 0.001, ** p < 0.01, and * p < 0.05. Error bar, standard deviation (SD).

Article Snippet: [ ] In brief, to evaluate the binding of exosomal PD‐L1 to PD‐1, 96‐well ELISA plates were coated with 4 µg mL −1 human PD‐1 protein (BPS Bioscience, Cat# 71106, San Diego, CA, USA) overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Advanced Science

Article Title: Sulfisoxazole Elicits Robust Antitumour Immune Response Along with Immune Checkpoint Therapy by Inhibiting Exosomal PD‐L1

doi: 10.1002/advs.202103245

Figure Lengend Snippet: Combination of sulfisoxazole (SFX) and α PD‐1 elicits adaptive immunity against tumor. a) Schematic illustration of the therapeutic schedule for CT26 tumor‐bearing mice. b) Representative histogram of CD45 + CD4 + cells in tumor microenvironment (TME). c) Quantification of CD45 + CD4 + cells in the TME ( n = 3). d) Representative histogram of CD45 + CD8 + cells in TME. e) Quantification of CD45 + CD4 + cells in TME ( n = 5). f) Representative dot plot of CD45 + CD3 + CD8 + cytotoxic T cells in the TME. g) Quantification of CD45 + CD3 + CD8 + cytotoxic T cells in the TME ( n = 9). h) Quantification of perforin + T cells in CD8 + T cells from tumor‐draining lymph nodes (DPBS; n = 12, SFX; n = 12, α PD‐1; n = 14 and SFX + α PD‐1; n = 11). i) Quantification of CD4 + T cells, CD8 + T cells, and perforin + T cells in CD8 + T cells from spleen (DPBS; n = 9, SFX; n = 10, α PD‐1; n = 11 and SFX+ α PD‐1; n = 9). Significance was determined using an ANOVA with Tukey correction or an unpaired two‐tailed Student's t ‐test. *** p < 0.001, ** p < 0.01, and * p < 0.05. Error bar, standard deviation (SD).

Article Snippet: [ ] In brief, to evaluate the binding of exosomal PD‐L1 to PD‐1, 96‐well ELISA plates were coated with 4 µg mL −1 human PD‐1 protein (BPS Bioscience, Cat# 71106, San Diego, CA, USA) overnight at 4 °C.

Techniques: Two Tailed Test, Standard Deviation

Journal: Science Advances

Article Title: Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles

doi: 10.1126/sciadv.aar2766

Figure Lengend Snippet: ( A and B ) PD1 blockade prevents the inhibition of PD-L1 high GSC EVs on PBMCs. Percent change in CD69 expression for CD4 + (A) and CD8 + (B) T cells. PD1 blocking antibody (10 μg/ml) or isotype control (10 μg/ml) was added at day 0 ( n = 7 PBMC donors, means ± SD). ( C and D ) PD1 blockade furthermore prevents the inhibition of PD-L1 high GSC EVs on CD3 + isolated cells. CD3 + CD4 + (C) and CD3 + CD8 + (D) cells ( n = 3) after treatment. ( E ) PD-L1–carrying, palmtdT-labeled PD-L1 high GSC EVs can bind to wells coated with recombinant PD1, whereas PD1 antibody blockade inhibits EV binding. Representative confocal images are shown on the left, whereas quantification is provided on the right. Spots per field of view (FOV) on the y axis represent palmtdT-positive dots. Scale bar, 50 μm; ×500 magnification inserts; quadruplicates as means ± SD. One-way ANOVA, with post hoc Bonferroni’s correction, was used to differentiate multiple groups (**** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05).

Article Snippet: Human PD1 Fc fusion protein (2 μg/ml; BPS Bioscience) was coated on high protein-binding 96-well plates (#3590, Costar).

Techniques: Inhibition, Expressing, Blocking Assay, Isolation, Labeling, Recombinant, Binding Assay

Journal: Science Advances

Article Title: Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles

doi: 10.1126/sciadv.aar2766

Figure Lengend Snippet: ( A ) Glioma GSCs up-regulate PD-L1 in vitro in response to activated PBMC supernatants. PBMCs were stimulated with anti-CD3, and supernatants were collected and co-incubated with GSCs (G44, a PD-L1 low GSC) in the presence or absence of anti–IFN-γ. PD-L1 expression was measured by flow cytometry. DMEM, Dulbecco’s modified Eagle’s medium. ( B ) IFN-γ–mediated increase of PD-L1 expression levels in PD-L1 High and PD-L1 low GSCs as shown by Western blots of four different GSCs. ( C and D ) EVs derived from IFN-γ–treated PD-L1 low GSCs inhibit anti-CD3–stimulated T cell activation, and this can be partially reversed by PD1 blockade. Inhibition potential was measured by the percentage change of CD69 + levels on anti-CD3–stimulated CD3 + CD4 + (C) or CD3 + CD8 + (D) cells, isolated from five human volunteers (means ± SD). Representative dot plots for (C) and (D) can be found in fig. S4C. ( E ) PD-L1 low EVs up-regulated indoleamine 2,3-dioxygenase (IDO) mRNA in PBMCs treated with PD-L1 low EVs. Quantitative polymerase chain reaction (qPCR) expression levels are shown ( n = 3). ( F ) PD-L1 low EVs cause interleukin-10 (IL-10) up-regulation in PBMCs. IL-10 cytokine (left) and qPCR expression levels (right) are shown ( n = 3). ( G and H ) Immunosuppressive molecules IDO and IL-10 primarily derive from the CD3-negative population. IDO (G) and IL-10 (H) mRNA levels are shown after CD3 + magnetic-activated cell sorting ( n = 3). Data sets consist of EVs from four different glioblastoma cell lines with means ± SD. One-way ANOVA, with post hoc Bonferroni’s correction, was used to differentiate multiple groups (**** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05). Student’s t test was used to differentiate between two groups, and one-way ANOVA with post hoc Bonferroni’s correction was used for multiple groups (**** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05).

Article Snippet: Human PD1 Fc fusion protein (2 μg/ml; BPS Bioscience) was coated on high protein-binding 96-well plates (#3590, Costar).

Techniques: In Vitro, Incubation, Expressing, Flow Cytometry, Modification, Western Blot, Derivative Assay, Activation Assay, Inhibition, Isolation, Real-time Polymerase Chain Reaction, FACS