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: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A ) Overview of the pooled CRISPR screen. The genome-scale Brunello library was introduced into Cas9 expressing Caco2 cells, followed by selection of transduced cells. After 10 days, pooled Caco2 cells were infected with HAstV1 for 24h, followed by staining using anti-HAstV capsid antibody. Uninfected cells were sorted to determine the sgRNA counts by next-generation sequencing. ( B ) FcRn protein levels in Cas9-Caco2 cells disrupted for FCGRT or DPP4 using independent sgRNAs per gene or targeted with a control anti-GFP sgRNA. Two independent replicates are shown. ( C ) Representative histogram for surface expression of DPP4 in naïve Caco2 cells stained with anti-DPP4 antibody or isotype control antibody. ( D ) Representative histogram showing DPP4 expression in fixed, permeabilized Caco2 cells disrupted for FCGRT or DPP4 or B2M using gene specific sgRNAs or targeted with a control anti-GFP sgRNA. ( E ) Percentage of DPP4 expressing Caco2 cells disrupted for DPP4 using sgRNAs or targeted with a control anti-GFP sgRNA. (n=3) ( F ) Percentage of anti-HAstV capsid antibody stained Caco2 cells treated with PBS (n=5) or isotype antibody (n=5) or various concentrations of anti-DPP4 antibody (n=6) prior to HAstV1 infection. ( G ) Percentage of anti-HAstV capsid antibody-stained control (n=6), FCGRT (n=6) or DPP4 (n=6) knockout Caco2 cells transfected with HAstV1 RNA at 48h post-transfection. Results were analyzed using Kruskal-Wallis test with Dunn’s post-test (F and G) from two to three independent experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns=not significant. Bars indicate mean of all data points.

Article Snippet: To assess the neutralization of HAstV infection by soluble DPP4 and soluble FcRn, 4×10 4 FFU/ml of HAstV1 were incubated with 5μg/ml recombinant human DPP4/CD26 protein (Acro Biosystems; DP4-H5266) or 5μg/ml recombinant FCGRT/B2M complex (BPS Bioscience, 71283-1) in growth media at 37°C.

Techniques: CRISPR, Expressing, Selection, Infection, Staining, Next-Generation Sequencing, Control, Knock-Out, Transfection

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A ) Enrichment [-log10 Robust Rank Aggregation (RRA)] scores of positively-selected sgRNAs in HAstV1-negative cells sorted 24hpi of a genome-wide CRISPR-Cas9 Caco2 cell library compared to HAstV1-positive cells, calculated by MAGeCK. ( B,C ) Cas9-Caco2 cells disrupted for FCGRT (n=6-9) or B2M (n=9) using two independent sgRNAs per gene or targeted with a control anti-GFP sgRNA (n=9) were infected with HAstV1or HAstV8, then stained with anti-HAstV capsid antibody at 24hpi. ( D ) Mean fluorescent intensity (MFI) of DPP4 in negative and positive cell populations of Caco2 cells infected with HAstV1 (n=6) or HAstV8 (n=6) infected at 24hpi. ( E ) Cas9-Caco2 cells were disrupted for DPP4 (n=9) using two independent sgRNAs per gene or targeted with a control anti-GFP sgRNA (n=9), then assessed for infection using anti-HAstV capsid antibody 24hpi with HAstV1 or HAstV8. (F ) Percentage of anti-HAstV capsid antibody-stained Caco2 cells treated with PBS (n=4-5) or isotype control (n=4-6) or anti-DPP4 polyclonal antibody (n=6-7) for 12h hours prior to infection with HAstV1 or HAstV8. Results from three independent experiments were analyzed using the Kruskal-Wallis test with Dunn’s post-test (B to F). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns=not significant. Bars indicate mean of all data points.

Article Snippet: To assess the neutralization of HAstV infection by soluble DPP4 and soluble FcRn, 4×10 4 FFU/ml of HAstV1 were incubated with 5μg/ml recombinant human DPP4/CD26 protein (Acro Biosystems; DP4-H5266) or 5μg/ml recombinant FCGRT/B2M complex (BPS Bioscience, 71283-1) in growth media at 37°C.

Techniques: Genome Wide, CRISPR, Control, Infection, Staining

Journal: bioRxiv

Article Title: The neonatal Fc receptor and DPP4 are human astrovirus receptors

doi: 10.1101/2024.07.12.603331

Figure Lengend Snippet: ( A ) Schematic of the CRISPR activation surfaceome screen. The human surfaceome library was introduced into dCas9 expressing Caco2 cells, followed by selection of transduced cells. After 10 days, pooled Caco2 cells were infected with HAstV1 or HAstV8 for 24h, followed by staining using anti-HAstV capsid antibody. We sorted top 3% of the HAstV capsid positive cells to determine the sgRNA counts by next-generation sequencing. ( B ) Representative histogram showing expression of DPP4 in dCas9-Caco2 cells transduced with sgRNAs for DPP4 overexpression. ( C ) FcRn protein levels in dCas9-Caco2 transduced with sgRNA for overexpressing FcRn. Two independent replicates are shown. ( D ) FcRn protein levels in 293T and 293T-FCGRT cells. Two independent replicates are shown. ( E ) Representative histogram showing surface expression of DPP4 in 293T cells stained with anti-DPP4 antibody or isotype control antibody. ( F ) Abundance of DPP4 and HAstV capsid positive 293T and 293T-DPP4 cells infected with HAstV1. ( G, H ) HEK293T or HEK293T-DPP4 cells were transfected with FCGRT (n=5) , DPP4 (n=5) and/or B2M (n=5) plasmids then 48h later were infected and stained with anti-HAstV capsid antibody at 24 hpi. Results were analyzed using Kruskal-Wallis test with Dunn’s post-test (G and H) from three independent experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns=not significant. Bars indicate mean of all data points.

Article Snippet: To assess the neutralization of HAstV infection by soluble DPP4 and soluble FcRn, 4×10 4 FFU/ml of HAstV1 were incubated with 5μg/ml recombinant human DPP4/CD26 protein (Acro Biosystems; DP4-H5266) or 5μg/ml recombinant FCGRT/B2M complex (BPS Bioscience, 71283-1) in growth media at 37°C.

Techniques: CRISPR, Activation Assay, Expressing, Selection, Infection, Staining, Next-Generation Sequencing, Transduction, Over Expression, Control, Transfection

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