Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE blockade of PD-1/PD-L1 interaction in coculture cell-based luciferase assay. (A, B) Cytotoxicity assay performed using Cell Counting Kit-8 (CCK) assay. The hPD-1/NFAT Jurkat T cells (A) and hPD-L1/TCR CHO-K1 cells (B) after treatment with SRE for 24 hours. (C, D) The PD-1/PD-L1 blockade bioassay was performed using the Bio-Glo™ luciferase assay system. After addition of hPD-1/NFAT Jurkat T cells and SRE (C) and anti-PD-1 antibodies (αPD-1) (D) , hPD-L1/TCR CHO-K1 cells were seeded for 20 hours. Data are presented as the mean ± SD. * p < 0.05 and *** p < 0.001 compared to the control.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Luciferase, Cytotoxicity Assay, Cell Counting

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE-induced activation of T cells and cytotoxic effect of T cell-mediated cancer cells. (A, B) The cell viability was performed using the CCK-8 assay. Splenocytes were isolated from hPD-L1 MC38 cell-bearing hPD-1 knockin mice. Murine CRC hPD-L1 MC38 cells (A) and hPD-1 mice splenocytes (B) were treated with SRE for 72 hours. (C) Cocultured hPD-L1 MC38 cell viability tested by crystal violet staining; (D) Lactate dehydrogenase (LDH) released by damaged cells, detected via LDH cytotoxicity assay; (E) Relative interleukin-2 (IL-2) level, determined using the mouse IL-2 ELISA set. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Activation Assay, CCK-8 Assay, Isolation, Knock-In, Staining, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE elevated the activation of hPD-1 + CD8 + T cells and the CD8 + T cell-mediated killing effect on hPD-L1 MC38 cancer. (A) Cocultured hPD-L1 MC38 cell viability, tested by crystal violet staining. Cocultured hPD-L1 MC38 cells detected with fluorescence microscopy (× 200) (B) and determined by fluorescent-activated cell sorting analysis (C) . (D) LDH released from damaged cells; (E) Relative perforin 1 (PRF1) level, determined with use of the mouse PRF1 ELISA kit. Data are presented as the mean ± SD. ** p < 0.01 and *** p < 0.001 compared to the vehicle group.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Activation Assay, Staining, Fluorescence, Microscopy, FACS, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: Sanguisorbae Radix extract reduced tumor growth in the hPD-L1 MC38 cell allograft hPD-1 mouse model. (A) Body weight (grams); (B) Tumor volume after 18 days; (C) Tumor weight after 18 days; (D) Images of tumor tissues (bar indicates 5 mm); (E) hPD-L1 MC38 tumor-bearing mice 18 days after treatment; (F) Representative microscopic images (×400) of CD8 and PRF1-positive area of tumor tissues calculated using immunohistochemical analysis. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the vehicle group.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Immunohistochemical staining, 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: 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: 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: 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