Journal: Molecular Therapy Oncolytics

Article Title: A PD-L1-Based Cancer Vaccine Elicits Antitumor Immunity in a Mouse Melanoma Model

doi: 10.1016/j.omto.2019.06.002

Figure Lengend Snippet: Antibody Responses Induced by DPDL1E Vaccination in C57BL/6 and BALB/c Mice (A) Mice (n = 8) were immunized with DPDL1E three times at 2-week intervals. One week after the third immunization, the antibody titers were measured by ELISA using His-tagged PDL1 recombinant protein as a coating antigen. DTT-immunized serum was used as a negative control. (B) The levels of PD-L1- and DTT-specific antibody subclasses induced by DPDL1E vaccination. Sera were isolated from C57BL/6 and BALB/C mice immunized with the DPDL1E vaccine. The levels of the indicated antibody subclasses were measured by ELISA. (C) The sera from DPDL1E-immunized mice inhibited binding of PD-L1 to PD-1. PD-L1 mAb at 20 μg/mL was used as a positive control, and sera from DTT-immunized mice and PBS were used as a negative control and blank control, respectively. (D) The inhibition efficiency of sera at different concentrations was tested and compared with the control group. (E) A standard curve was created (relative inhibition versus concentration of PD-L1 mAb) to calculate the effective anti-PD-L1 concentration in vitro . ****p < 0.0001.

Article Snippet: The plates were incubated at room temperature for 1 h. After washing, 100 μL (2.5 μg/mL) biotin-labeled PD-1 protein (Acrobiosystems, Bethesda, MD, USA) was added to the wells and incubated for 1 h at 37°C.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Negative Control, Isolation, Binding Assay, Positive Control, Inhibition, Concentration Assay, In Vitro

Journal: Molecular Therapy Oncolytics

Article Title: A PD-L1-Based Cancer Vaccine Elicits Antitumor Immunity in a Mouse Melanoma Model

doi: 10.1016/j.omto.2019.06.002

Figure Lengend Snippet: The DPDL1E Vaccine Decreased the Ratio of PD-1+ Cells and Increased the Ratio of PD-L1+ Cells in Tumors (A) PD-L1+ cells were stained with anti-CD274-APC and anti-CD45-FITC. (B) PD-1+ cells were stained with anti-CD279-APC and anti-CD45-FITC. (C) LAG3+ cells were stained with anti-CD223-PE, anti-CD3-FITC, and anti-CD8-PerCP-Cy5.5. (D) LAG3+PD-1+ cells were stained with anti-CD279-APC, anti-CD223-PE, anti-CD3-FITC, and anti-CD8-PerCP-Cy5.5. All samples were detected using FCM, and the data were analyzed using FlowJo software. *p < 0.05, **p < 0.01, ***p < 0.001. Statistically significant differences were assessed using Student’s t test.

Article Snippet: The plates were incubated at room temperature for 1 h. After washing, 100 μL (2.5 μg/mL) biotin-labeled PD-1 protein (Acrobiosystems, Bethesda, MD, USA) was added to the wells and incubated for 1 h at 37°C.

Techniques: Staining, Software

Journal: bioRxiv

Article Title: The natural stilbenoid (–)-hopeaphenol inhibits cellular entry of SARS-CoV-2 USA-WA1/2020, B.1.1.7 and B.1.351 variants

doi: 10.1101/2021.04.29.442010

Figure Lengend Snippet: Identification of stilbenoids as Spike-ACE2 inhibitors by AlphaScreen. A, Demonstration of AlphaScreen-based fluorescence due to interactions of His-tagged Spike RBD (from USA-WA1/2020) and Fc-tagged ACE2 peptides. B, Dose-response curves of stilbenoids and resveratrol on fluorescence inhibition due to disruption of RBD/ACE2 interactions. C, Demonstration of AlphaScreen-based fluorescence due to interactions of His-tagged PD-1 and Fc-tagged PD-L1 peptides. D, Dose-response curves of stilbenoids and control inhibitor BMS-1166 on fluorescence inhibition due to disruption of PD-1/PD-L1 interactions.

Article Snippet: 0.5 nM of human PD-L1-Fc (Sino Biological) was incubated with 5 nM HIS-tagged human PD-1 (Sino Biological) in the presence of 5 μg/mL protein A AlphaScreen acceptor bead and 5 μg/mL nickel chelate donor bead in a total volume of 10 μL of 20 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% Tween in white, opaque low-volume 384-well plates.

Techniques: Amplified Luminescent Proximity Homogenous Assay, Fluorescence, Inhibition

Journal: Oncology Letters

Article Title: Role of programmed death ligands in effective T-cell interactions in extranodal natural killer/T-cell lymphoma

doi: 10.3892/ol.2014.2356

Figure Lengend Snippet: Representative immunohistochemical streptavidin-peroxidase staining in extranodal natural killer/T-cell lymphoma (upper row) and rhinitis tissues (lower row). The positive cases of (A and D) programmed death 1, (B and E) PD-L1 and (C and F) PD-L2 (magnification, ×200). PD-L, programmed death ligand.

Article Snippet: The antigen retrieval was conducted in 0.01 mol/l citrate (pH 6.0) and the slides were incubated overnight with rabbit anti-human PD-L1 polyclonal antibody (1:120; Proteintech, Chicago, IL, USA), rabbit anti-human PD-L2 polyclonal antibody (1:150) and mouse anti-human PD-1 monoclonal antibody (mAb; 1:100) (both ZSGB-BIO, Beijing, China) and phosphate-buffered saline was used as a blank control.

Techniques: Immunohistochemical staining, Staining

Journal: Oncology Letters

Article Title: Role of programmed death ligands in effective T-cell interactions in extranodal natural killer/T-cell lymphoma

doi: 10.3892/ol.2014.2356

Figure Lengend Snippet: PD-1 expression in (A) CD4 + and (B) CD8 + T-cell subsets in 20 ENKL patients was significantly increased compared with that in 10 HVs (P<0.05). Representative PD-1 expression in (C) CD4 + and (D) CD8 + T-cell subsets in six ENKL patients was (E) downregulated with chemotherapy. (F) T-helper cell type 1 cytokine (IL-2 and IFN-γ) mean production levels in the serum of 20 ENKL patients were significantly lower than those in 10 HVs (P<0.05). PD1, programmed death 1; ENKL, extranodal natural killer/T-cell lymphoma; HVs, healthy volunteers; IL-2 interleukin 2; IFN-γ, interferon γ.

Article Snippet: The antigen retrieval was conducted in 0.01 mol/l citrate (pH 6.0) and the slides were incubated overnight with rabbit anti-human PD-L1 polyclonal antibody (1:120; Proteintech, Chicago, IL, USA), rabbit anti-human PD-L2 polyclonal antibody (1:150) and mouse anti-human PD-1 monoclonal antibody (mAb; 1:100) (both ZSGB-BIO, Beijing, China) and phosphate-buffered saline was used as a blank control.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Role of programmed death ligands in effective T-cell interactions in extranodal natural killer/T-cell lymphoma

doi: 10.3892/ol.2014.2356

Figure Lengend Snippet: (A) Purity of CD8 + T cells separated by magnetic-activated cell sorting was 99%. (B) Purity of CD8 + PD-1 + T cells was 96.2% following the stimulation of allogeneic CD8 + T cells with phytohemagglutinin for 48 h. (C) SNK-6 cells were used as the control group and, following the coculture of SNK-6 cells and CD8 + T cells for 72 h, a significant inhibitory effect of PD-L1 on allogeneic CD8 + T-helper type 1 cytokine (IL-2 and IFN-γ) secretion was observed; (A and B) P<0.05. (D) CD8 + T-cell apoptosis in groups A and B was not altered significantly compared with activated CD8 + T cells at 72 h (P>0.05). (E) SNK-6 cells were used as the control group and cells harvested at 0, 24, 48 and 72 h were analyzed by flow cytometry gating CFSE + events. The proliferation index was not significantly different among the groups (P>0.05). PD-1, programme death 1; PD-L. programmed death ligand; IL-2 interleukin 2; IFN-γ, interferon γ; CFSE, carboxy-fluorescein succinimidyl ester.

Article Snippet: The antigen retrieval was conducted in 0.01 mol/l citrate (pH 6.0) and the slides were incubated overnight with rabbit anti-human PD-L1 polyclonal antibody (1:120; Proteintech, Chicago, IL, USA), rabbit anti-human PD-L2 polyclonal antibody (1:150) and mouse anti-human PD-1 monoclonal antibody (mAb; 1:100) (both ZSGB-BIO, Beijing, China) and phosphate-buffered saline was used as a blank control.

Techniques: FACS, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Discovery of galectin-8 as an LILRB4 ligand driving M-MDSCs defines a class of antibodies to fight solid tumors

doi: 10.1016/j.xcrm.2023.101374

Figure Lengend Snippet:

Article Snippet: The plate was then washed with PBST (PBS, 0.05% Tween 20) (MA0015, Meilunbio) (T8220, Solarbio) and blocked with 3% BSA (97061-420, VWR) at 37°C for 90 min. After repeated washing, hFc-tagged recombinant proteins, including CD3ε (10977-H02H; Sino Biological), CTLA-4 (CT4-H5255; Acro Biosystems), CD28 (CD8-H525a; Acro Biosystems), CD96 (TAE-H5252; Acro Biosystems), LAG-3 (LA3-H5255; Acro Biosystems), TIM-3 (TM3-H5258; Acro Biosystems), CD40 (CD0-5253; Acro Biosystems), ICOS (ICS-H5258; Acro biosystems), OX40 (OX0-H5255; Acro Biosystems), TIGHT (TIT-H5254; Acro Biosystems), LY86 (10242-H02H; Sino Biological), LILRB4 (16742-H02H; Sino Biological), CD27 (CD7-H5254; Acro biosystems), PD-1 (10377-H02H; Sino Biological), and CD8b (11031-HCCH; Sino Biological), were added into each well.

Techniques: Recombinant, Derivative Assay, Microarray, Enzyme-linked Immunosorbent Assay, Lysis, Blocking Assay, Staining, Transfection, Marker, Flow Cytometry, shRNA, Plasmid Preparation, Negative Control, Silver Staining, Bicinchoninic Acid Protein Assay, Selection, Extraction, Labeling, RNA Expression, Cell Culture, Software