Journal: Translational Oncology

Article Title: Peptide-based inhibitors targeting the PD-1/PD-L1 axis: potential immunotherapeutics for cancer

doi: 10.1016/j.tranon.2024.101892

Figure Lengend Snippet: Structure of the PD-1/PD-L1 complex (PDB: 4ZQK ) with the designed peptide groups marked on it; PD-1: blue, PD-L1: green. The PD-L1 fragments used to design three groups of peptides are marked as follows: purple - Group I, yellow - Group II, red - Group III. The amino acids in PD-L1 crucial for interaction with PD-1 are represented as sticks.

Article Snippet: Subsequently, 2-fold serial dilutions of PD-1-Fc (Sino Biological Company, China, #10084-H08H-B) in PBS-T, at concentrations from 8.00 to 0.5 µg/ml, were added to the precoated wells.

Techniques:

Journal: Translational Oncology

Article Title: Peptide-based inhibitors targeting the PD-1/PD-L1 axis: potential immunotherapeutics for cancer

doi: 10.1016/j.tranon.2024.101892

Figure Lengend Snippet: Binding of PD-1 to PD-L1 or to the indicated PD-L1-derived peptides analysed by indirect ELISA. Results are shown for at least three experiments performed independently in duplicate. Data are depicted as mean ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnet's post-hoc test. ****: p < 0.0001, ***: p < 0.001, **: p < 0.01, *: p < 0.05.

Article Snippet: Subsequently, 2-fold serial dilutions of PD-1-Fc (Sino Biological Company, China, #10084-H08H-B) in PBS-T, at concentrations from 8.00 to 0.5 µg/ml, were added to the precoated wells.

Techniques: Binding Assay, Derivative Assay, Indirect ELISA

Journal: Translational Oncology

Article Title: Peptide-based inhibitors targeting the PD-1/PD-L1 axis: potential immunotherapeutics for cancer

doi: 10.1016/j.tranon.2024.101892

Figure Lengend Snippet: Sensorgrams of the PD-L1 derived peptides interacting with PD-1, obtained using the SPR technique. ND - not determined (no binding detected or binding too weak to establish reliable constants).

Article Snippet: Subsequently, 2-fold serial dilutions of PD-1-Fc (Sino Biological Company, China, #10084-H08H-B) in PBS-T, at concentrations from 8.00 to 0.5 µg/ml, were added to the precoated wells.

Techniques: Derivative Assay, Binding Assay

Journal: Translational Oncology

Article Title: Peptide-based inhibitors targeting the PD-1/PD-L1 axis: potential immunotherapeutics for cancer

doi: 10.1016/j.tranon.2024.101892

Figure Lengend Snippet: Competitive inhibition of PD-1/PD-L1 complex formation by the peptides obtained in this study. Modified Jurkat cells (JE6–1-NF-κB::eGFP PD-1) were incubated with the PD-L1-derived peptides, with the final concentration used in the experiment ranging from 50.00 to 5.56 µM. Subsequently, the cells were probed with PD-L1-Fc followed by PE-labelled anti-human IgG antibody and analysed by flow cytometry. The bar diagram shows the fold induction of geometric mean fluorescent intensity (gMFI) for at least three experiments performed independently in triplicate. Data were normalized to the gMFI obtained for the reporter PD-1 cell line treated with PD-L1-Fc in the absence of peptides. Data are depicted as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Dunnet's post-hoc test. ****: p < 0.0001, ***: p < 0.001, **: p < 0.01, *: p < 0.05.

Article Snippet: Subsequently, 2-fold serial dilutions of PD-1-Fc (Sino Biological Company, China, #10084-H08H-B) in PBS-T, at concentrations from 8.00 to 0.5 µg/ml, were added to the precoated wells.

Techniques: Inhibition, Modification, Incubation, Derivative Assay, Concentration Assay, Flow Cytometry

Journal: Translational Oncology

Article Title: Peptide-based inhibitors targeting the PD-1/PD-L1 axis: potential immunotherapeutics for cancer

doi: 10.1016/j.tranon.2024.101892

Figure Lengend Snippet: Inhibitory properties of the peptides in the functional cellular reporter assay. The PD-1 reporter cells were stimulated with TCS CD86/PD-L1 in the absence or presence of the peptides. The inhibitory properties of the peptides were measured based on eGFP expression by flow cytometry and normalized to gMFI eGFP obtained for the PD-1 reporter/TCS CD86 cells treated with the peptides. The dotted line shows the normalized eGFP expression level from the co-culture of the PD-1 reporter cells with TCS CD86/PD-L1. Results are shown for three experiments performed independently in duplicate. Data are depicted as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Dunnet's post-hoc test. ****: p < 0.0001, ***: p < 0.001, **: p < 0.01, *: p < 0.05.

Article Snippet: Subsequently, 2-fold serial dilutions of PD-1-Fc (Sino Biological Company, China, #10084-H08H-B) in PBS-T, at concentrations from 8.00 to 0.5 µg/ml, were added to the precoated wells.

Techniques: Functional Assay, Reporter Assay, Expressing, Flow Cytometry, Co-Culture Assay

Journal: Translational Oncology

Article Title: Peptide-based inhibitors targeting the PD-1/PD-L1 axis: potential immunotherapeutics for cancer

doi: 10.1016/j.tranon.2024.101892

Figure Lengend Snippet: Structures A and B show, respectively, the complexes between PD-1 (blue) and PD-L1 (green), and between PD-1 (blue) and the PD-L1(111–127) fragment (red) trimmed from the PD-1/PD-L1 complex (PDB code: 4ZQK). Structures C-F show the complexes of peptide ( L11 ) obtained from docking NMR structures to PD-1 by using the UNRES force field: (C) cluster from family 1 (lowest energy) – RMSD 3.84 Å, (D) cluster from family 1 (centroid) – RMSD 3.93 Å, (E) cluster from family 2 (lowest energy) – RMSD 4.86 Å, (F), cluster from family 2 (centroid) – RMSD 4.84 Å.

Article Snippet: Subsequently, 2-fold serial dilutions of PD-1-Fc (Sino Biological Company, China, #10084-H08H-B) in PBS-T, at concentrations from 8.00 to 0.5 µg/ml, were added to the precoated wells.

Techniques:

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:

Article Title: Human Immunodeficiency Virus Type 1 Nef Induces Programmed Death 1 Expression through a p38 Mitogen-Activated Protein Kinase-Dependent Mechanism ▿

doi: 10.1128/JVI.00485-08

Figure Lengend Snippet: Nef is necessary for PD-1 upregulation during HIV-1 infection. (A) Flow cytometric analysis of cell surface expression of PD-1 on human PBMCs mock infected or infected with HIV-1 (NL4-3) with different viral genes deleted as indicated. Seventy-two hours postinfection, the cells were stained for CD4/FITC, CD24HSA/APC (infection marker), and PD-1/PE. The histograms depict the PD-1 expression staining gated on CD4+/CD24HSA+ T cells. The shaded histograms represent staining with isotype control, the thin-line histograms represent the uninfected control, and the thick-line histograms represent staining with PD-1 antibody.

Article Snippet: The following directly conjugated antibodies were used: CD3-phycoerythrin (PE)/fluorescein isothiocyanate (FITC)/allophycocyanin (APC)/Pacific Blue (PB), CD4-PE/FITC/PB, CD8-PE/FITC/PB, and streptavidin-FITC or PE-Cy5 with their respective isotype control antibodies (BD Biosciences, San Jose, CA); CD4-APC, CD8-APC, and PD-1-FITC/PE/APC with their respective isotype control antibodies (eBiosciences, San Diego, CA); and biotinylated anti-human PD-1 and PD-L1 (R&D Systems).

Techniques: Infection, Expressing, Staining, Marker

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Nef Induces Programmed Death 1 Expression through a p38 Mitogen-Activated Protein Kinase-Dependent Mechanism ▿

doi: 10.1128/JVI.00485-08

Figure Lengend Snippet: Nef is sufficient for PD-1 upregulation. (A and B) Analysis of PD-1 expression in transfected cells. Flow cytometric analysis of PD-1 was performed on Jurkat cells transfected with HIV-1 viral genes as indicated and pGFP. The cells were collected 3 days later, and PD-1 expression was measured. Gates were set to include green fluorescent protein (GFP)-positive cells only. The data are representative of three or more separate studies. The shaded histograms represent staining with isotype control, and the open histograms represent staining with PD-1 antibody. FSC-A, forward scatter area.

Article Snippet: The following directly conjugated antibodies were used: CD3-phycoerythrin (PE)/fluorescein isothiocyanate (FITC)/allophycocyanin (APC)/Pacific Blue (PB), CD4-PE/FITC/PB, CD8-PE/FITC/PB, and streptavidin-FITC or PE-Cy5 with their respective isotype control antibodies (BD Biosciences, San Jose, CA); CD4-APC, CD8-APC, and PD-1-FITC/PE/APC with their respective isotype control antibodies (eBiosciences, San Diego, CA); and biotinylated anti-human PD-1 and PD-L1 (R&D Systems).

Techniques: Expressing, Transfection, Staining

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Nef Induces Programmed Death 1 Expression through a p38 Mitogen-Activated Protein Kinase-Dependent Mechanism ▿

doi: 10.1128/JVI.00485-08

Figure Lengend Snippet: Nef expression induces PD-1 production. (A) Jurkat T cells were transiently transfected with a PD-1/Luc reporter construct (10 μg) and equal amounts of either empty vector or vectors containing accessory genes as indicated. Forty-eight hours posttransfection, PD-1 transcriptional activity was examined by luciferase assay as described in Materials and Methods. Values and bars represent means (n = 3) and standard deviations. (B and C) Characterization of PD-1 expression. Total RNA and proteins were extracted from the samples in panel A and analyzed for PD-1 expression. (B) Northern blot of 20 μg of total RNA isolated from transfected cells (top).Shown is hybridization with α-32P-labeled human PD-1 cDNA probe. The same blot was subsequently hybridized with β-actin cDNA probe (bottom) as a loading control. (C) Western blot analysis of PD-1 expression from the transfected cells using specific PD-1 antibody (top) or β-actin antibody (bottom). HIV-1-infected samples were used as a positive control.

Article Snippet: The following directly conjugated antibodies were used: CD3-phycoerythrin (PE)/fluorescein isothiocyanate (FITC)/allophycocyanin (APC)/Pacific Blue (PB), CD4-PE/FITC/PB, CD8-PE/FITC/PB, and streptavidin-FITC or PE-Cy5 with their respective isotype control antibodies (BD Biosciences, San Jose, CA); CD4-APC, CD8-APC, and PD-1-FITC/PE/APC with their respective isotype control antibodies (eBiosciences, San Diego, CA); and biotinylated anti-human PD-1 and PD-L1 (R&D Systems).

Techniques: Expressing, Transfection, Construct, Plasmid Preparation, Activity Assay, Luciferase, Northern Blot, Isolation, Hybridization, Labeling, Western Blot, Infection, Positive Control

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Nef Induces Programmed Death 1 Expression through a p38 Mitogen-Activated Protein Kinase-Dependent Mechanism ▿

doi: 10.1128/JVI.00485-08

Figure Lengend Snippet: HIV-1 Nef stimulates upregulation of PD-1 in infected cells. (A) Phenotypic analysis of PD-1 on HIV-specific and positive CD4+ T cells using class II tetramer and p24Gag staining in viremic patients. PBMCs from the HIV-1-positive and -negative patients were stained directly ex vivo and were assessed by five-color flow cytometry on gated CD3+/CD4+ lymphocytes. Representative dot plots (panel-I) show positive/negative class II tetramer staining in HIV-infected individuals gated on CD3+ T cells. The inset boxes indicate the tetramer-positive cells. The percentage of tetramer-positive cells is indicated in each plot. Further representative dot plots (panel-II and -III) show the staining of HIV-1 p24Gag-positive and -negative cells from the tetramer-positive and -negative cells. The overlay histograms (panel-IV) represent the MFI of PD-1 expression. The shaded histograms represent the tetramer-negative/HIV-1-positive cells, and the open histograms represent tetramer-positive/HIV-1-positive cells. (B) Cell surface expression of PD-1 on human PBMCs infected with HIV-1Wt or HIV-1ΔNef virus. Infected cells (after 2 days and 6 days of infection) were analyzed for PD-1 expression in the CD3+/CD4+/CD24HSA+ populations. (C) Longitudinal analysis of PD-1 on human PBMCs infected with wild-type virus at different time periods as indicated. PD-1 expression on CD3+/CD4+/CD24HSA+ cells was measured in HIV-1-infected and -uninfected control cells. Representative data show the MFI of PD-1 expression (n = 3). The bars show mean values. Error bars show standard deviations.

Article Snippet: The following directly conjugated antibodies were used: CD3-phycoerythrin (PE)/fluorescein isothiocyanate (FITC)/allophycocyanin (APC)/Pacific Blue (PB), CD4-PE/FITC/PB, CD8-PE/FITC/PB, and streptavidin-FITC or PE-Cy5 with their respective isotype control antibodies (BD Biosciences, San Jose, CA); CD4-APC, CD8-APC, and PD-1-FITC/PE/APC with their respective isotype control antibodies (eBiosciences, San Diego, CA); and biotinylated anti-human PD-1 and PD-L1 (R&D Systems).

Techniques: Infection, Staining, Ex Vivo, Flow Cytometry, Expressing, Virus

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Nef Induces Programmed Death 1 Expression through a p38 Mitogen-Activated Protein Kinase-Dependent Mechanism ▿

doi: 10.1128/JVI.00485-08

Figure Lengend Snippet: p38 MAPK activation by Nef is required for the transcriptional upregulation of PD-1. (A) p38 MAPK activation by Nef. Shown is Western blot analysis of protein extracted from Jurkat cells transfected with vector control (5 μg) or pNef (5 μg) and immunoblotted with total p38 MAPK- and phospho (P)-p38 MAPK-specific antibodies. The histograms represent FACS analysis of phospho-p38 MAPK expression. (B) Blockade of PD-1 expression by p38 MAPK inhibition. Human PBMCs (1 × 106) were infected with NL4-3Wt virions and treated with or without increasing doses of p38 MAPK inhibitor (RWJ67657) as indicated. Four days postinfection and posttreatment, equal number of cells were assayed for surface PD-1 expression in a CD3+/CD4+ population by flow cytometry. The data are representative of two independent experiments. Observations of similar suppression of PD-1 were obtained. Wt, wild type; Inhi, inhibitor. (C) Jurkat T cells negative for p38 MAPK activity by siRNA or a dominant-negative phenotype with pNef. At 48 h after transfection, the surface levels of PD-1 expression were determined by flow cytometry using a PD-1-specific antibody. The shaded histograms show the isotype-matched control antibodies, and the open histograms represent PD-1 expression. Nef-induced PD-1 induction in clone p38 siRNA (clone 61) (top) and p38 MAPK-DN cells was inhibited (bottom). Similar results were obtained in two independent experiments. The transfection efficiency was monitored by cotransfection of a pCMV plasmid encoding green fluorescent protein, which also served as a marker for gating on transfected cells. (D) Jurkat T cells were transiently transfected with the reporter construct PD-1/Luc and the empty vector or pNef and cultured for 2 days in the presence or absence of p38 inhibitor, and luciferease activity was measured as described in Materials and Methods. Values and bars represent means (n = 3) and standard deviations. AU, arbitrary units.

Article Snippet: The following directly conjugated antibodies were used: CD3-phycoerythrin (PE)/fluorescein isothiocyanate (FITC)/allophycocyanin (APC)/Pacific Blue (PB), CD4-PE/FITC/PB, CD8-PE/FITC/PB, and streptavidin-FITC or PE-Cy5 with their respective isotype control antibodies (BD Biosciences, San Jose, CA); CD4-APC, CD8-APC, and PD-1-FITC/PE/APC with their respective isotype control antibodies (eBiosciences, San Diego, CA); and biotinylated anti-human PD-1 and PD-L1 (R&D Systems).

Techniques: Activation Assay, Western Blot, Transfection, Plasmid Preparation, Expressing, Inhibition, Infection, Flow Cytometry, Activity Assay, Dominant Negative Mutation, Cotransfection, Marker, Construct, Cell Culture

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Nef Induces Programmed Death 1 Expression through a p38 Mitogen-Activated Protein Kinase-Dependent Mechanism ▿

doi: 10.1128/JVI.00485-08

Figure Lengend Snippet: Activation of p38 by Nef correlates with PD-1 expression and inversely with CD4 counts in HIV+ patients. (A) MFI of PD-1 expression on tetramer-positive CD4+ cells and viral-RNA counts. The FACS plots are gated on CD3+/CD4+/p24.17-DR1 tetramer-positive T cells (n = 20). (B) MFI of PD-1 expression on total CD4+ (Tet−/HIV+) and HIV-1-specific CD4+ (Tet+/HIV+) T cells (n = 12) from the infected patients. The lines show mean values. (C) Correlation between MFI of PD-1 expression and the serum Nef level. There is a correlation between the serum Nef concentration and PD-1 expression (n = 20). (D and F) Intracellular staining for phospho-p38 MAPK in HIV-1 patients was determined by FACS analysis; the plots are gated on CD3+/CD4+ T cells or CD3+/CD4+/tetramer+ T cells. (D) There is an inverse correlation between phospho-p38 MAPK expression and CD4 T-cell counts (n = 15). (E) There is no correlation between p38 MAPK activation and PD-1 expression on total CD4 T cells (n = 15). However, a positive correlation exists with PD-1 expression (MFI) on HIV-specific CD4+ T cells (F). These relationships were evaluated using the Spearman correlation test using the Prism 4 GraphPad software.

Article Snippet: The following directly conjugated antibodies were used: CD3-phycoerythrin (PE)/fluorescein isothiocyanate (FITC)/allophycocyanin (APC)/Pacific Blue (PB), CD4-PE/FITC/PB, CD8-PE/FITC/PB, and streptavidin-FITC or PE-Cy5 with their respective isotype control antibodies (BD Biosciences, San Jose, CA); CD4-APC, CD8-APC, and PD-1-FITC/PE/APC with their respective isotype control antibodies (eBiosciences, San Diego, CA); and biotinylated anti-human PD-1 and PD-L1 (R&D Systems).

Techniques: Activation Assay, Expressing, Infection, Concentration Assay, Staining, Software