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

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 antibodies used were polyclonal anti-human PD-1 (R&D Systems, Minneapolis, MN) and β-actin (Cell Signaling Technology, Danvers, MA).

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 antibodies used were polyclonal anti-human PD-1 (R&D Systems, Minneapolis, MN) and β-actin (Cell Signaling Technology, Danvers, MA).

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 antibodies used were polyclonal anti-human PD-1 (R&D Systems, Minneapolis, MN) and β-actin (Cell Signaling Technology, Danvers, MA).

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 antibodies used were polyclonal anti-human PD-1 (R&D Systems, Minneapolis, MN) and β-actin (Cell Signaling Technology, Danvers, MA).

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 antibodies used were polyclonal anti-human PD-1 (R&D Systems, Minneapolis, MN) and β-actin (Cell Signaling Technology, Danvers, MA).

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 antibodies used were polyclonal anti-human PD-1 (R&D Systems, Minneapolis, MN) and β-actin (Cell Signaling Technology, Danvers, MA).

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

Journal: Acta Pharmaceutica Sinica. B

Article Title: Anti-PD-L1 antibody enhances curative effect of cryoablation via antibody-dependent cell-mediated cytotoxicity mediating PD-L1 high CD11b + cells elimination in hepatocellular carcinoma

doi: 10.1016/j.apsb.2022.08.006

Figure Lengend Snippet: CRA induced higher tumoral PD-L1 expression but less infiltrated PD-L1 high CD11b + cells than MWA. Immunofluorescence staining of tumor biopsy specimens collected from CRA/MWA patients on Day 14 after ablation. Untreated samples were used as control group. PD-L1 + cells were stained with Alexa Fluor 594, CD11b + cells were stained with Alexa Fluor 488. Scale bar = 20 μm. Three separate fields from each patient and ten patients from each group were calculated. (B) The number of PD-L1 + tumor cells and PD-L1 + CD11b + cells in every field of view of 1 mm 2 ; Three separate fields from each patient and 10 patients for each group were calculated. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, one-way ANOVA with Tukey's post hoc tests. All data are mean ± SEM. (C–E) Schematic diagram of a complete animal experiment. H22 cells were inoculated subcutaneously into BALB/c mice. Twelve days later, cryoablation or microwave-ablation was performed. Two weeks later all mice were sacrificed ( n = 6 for each group). Tumor weight at end points in all 3 groups were shown. (F–H) Quantification of frequency of CD45 + , CD3 + T cells and CD11b + myeloid cells in 3 groups were shown. (I, J) Relative quantification and MFI of PD-L1 expressed in different cell sub-populations in three groups were revealed. n = 6 for each group, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ns, no significant difference, t- tests. All data are means ± SEM.

Article Snippet: For testing the affinity between PD-L1 antibodies and mouse CD16, atezolizumab (tecentriq), Avelumab (Bavencio), Mutant Anti-PD-L1-mIgG1e3 (InvivoGen, hpdl1-mab15, San Diego, CA, USA), Mouse IgG1 Isotype Control (R&D, MAB002, Emeryville, CA, USA), Human IgG1 (N298A) isotype control (InvivoGen, bgal-mab12, San Diego, CA, USA) were labeled with FITC fluorescence using the ZENON ALEXA FLUOR 488 MOUSE IG 1 KIT (ThermoFisher, Z25002, Carlsbad, CA, USA) as recommended by the manufacturer.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Anti-PD-L1 antibody enhances curative effect of cryoablation via antibody-dependent cell-mediated cytotoxicity mediating PD-L1 high CD11b + cells elimination in hepatocellular carcinoma

doi: 10.1016/j.apsb.2022.08.006

Figure Lengend Snippet: CRA had better curative effect than MWA in combining with anti-PD-L1 antibody therapy by inducing antitumor immune activity. (A) Schematic diagram of a complete animal experiment was shown. H22 cells were inoculated subcutaneously into BALB/c mice. 12 days later, cryoablation or microwave-ablation was proceeded, with anti-mouse PD-L1 antibody being infused via tail injection every 3 days, 10 mg/kg. Two weeks later all mice were sacrificed. (B, C) Tumor weight at end point in all 6 groups was shown. ( n = 6 for each group, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ns, no significant difference, t- tests. All data are mean ± SEM). (D) The H&E staining of tumor tissue sections. Red lines represented the edge of live tumor zone and necrosis zone. (Scale bar = 500 μm). (E) Mouse model was constructed refering to the methods of Fig. 2 A and overall survival of mice from 6 groups was revealed. (Survival curve was analyzed by log-rank test P > 0.05 was considered no significant difference). (F, G) Gene-expression analysis of CD45 + immune cells from CRA+anti-PD-L1 group and untreated group. PCA map and heatmap of the different expressed genes between two groups was shown. Blue color indicates down-regulated genes, and red color indicates up-regulated genes. (H) GSEA of immune response pathways in the transcriptome analysis, which presented as the normalized enrichment score (NES). (I) Heatmap of mean fold-change of genes in cytokines, chemokines, and so on. Red and blue colors indicate up-regulated or down-regulated genes, respectively.

Article Snippet: For testing the affinity between PD-L1 antibodies and mouse CD16, atezolizumab (tecentriq), Avelumab (Bavencio), Mutant Anti-PD-L1-mIgG1e3 (InvivoGen, hpdl1-mab15, San Diego, CA, USA), Mouse IgG1 Isotype Control (R&D, MAB002, Emeryville, CA, USA), Human IgG1 (N298A) isotype control (InvivoGen, bgal-mab12, San Diego, CA, USA) were labeled with FITC fluorescence using the ZENON ALEXA FLUOR 488 MOUSE IG 1 KIT (ThermoFisher, Z25002, Carlsbad, CA, USA) as recommended by the manufacturer.

Techniques: Activity Assay, Injection, Staining, Construct, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: Anti-PD-L1 antibody enhances curative effect of cryoablation via antibody-dependent cell-mediated cytotoxicity mediating PD-L1 high CD11b + cells elimination in hepatocellular carcinoma

doi: 10.1016/j.apsb.2022.08.006

Figure Lengend Snippet: Anti-PD-L1 antibody enhanced infiltration of CD8 + T cells by increasing proportion of cDC1 cells that secrete CXCL9. (A, B) Flow cytometry plots of CD3 + CD8 + T cells gated on CD45 + cells and CD39 + IFN- γ + gated on CD3 + CD8 + cells in all 6 groups. (C–E) Relative quantification of CD3 + CD8 + T cells gated on CD45 + cells and CD39 + IFN- γ + cells gated on CD3 + CD8 + T cells. (F) Immunofluorescence staining of tumor tissue sections. CD8 + T cells were stained with Alexa Fluor 594, CXCL9 was stained with Alexa Fluor 488. Scale bar = 200 μm. (G) The percentages of CD8 + T cells and CXCL9 + spots in every field of view found in all different 6 groups; three separate fields from each mice and three mice from each group were calculated. ∗ P < 0.05, ∗∗ P < 0.01,∗∗∗ P < 0.001, one-way ANOVA with Tukey's post hoc tests. All data are means ± SEM. (H,I) Representative flow cytometry analysis of the frequency of CXCL9 gated from different cell types in tumor microenvironment. (J, K) Analysis of flow cytometric quantification of CD11c + CD11b − cDC1 cells gated on tumor infiltrating CD45 + cells. (L, M) H22 cells were inoculated subcutaneously into BALB/c mice. 12 days later, cryoablation or microwave-ablation was proceeded, with anti-mouse CXCL9 neutralizing antibody (10 mg/kg) or anti-mouse PD-L1 antibody (10 mg/kg) being infused via tail injection every 3 days. Anti-mouse IgG was used as negative control. Tumor volume and frequency of infiltrated CD8 + T cells were revealed. n = 6 for each groups, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ns, no significant difference, t- tests. All data are means ± SEM.

Article Snippet: For testing the affinity between PD-L1 antibodies and mouse CD16, atezolizumab (tecentriq), Avelumab (Bavencio), Mutant Anti-PD-L1-mIgG1e3 (InvivoGen, hpdl1-mab15, San Diego, CA, USA), Mouse IgG1 Isotype Control (R&D, MAB002, Emeryville, CA, USA), Human IgG1 (N298A) isotype control (InvivoGen, bgal-mab12, San Diego, CA, USA) were labeled with FITC fluorescence using the ZENON ALEXA FLUOR 488 MOUSE IG 1 KIT (ThermoFisher, Z25002, Carlsbad, CA, USA) as recommended by the manufacturer.

Techniques: Flow Cytometry, Immunofluorescence, Staining, Injection, Negative Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Anti-PD-L1 antibody enhances curative effect of cryoablation via antibody-dependent cell-mediated cytotoxicity mediating PD-L1 high CD11b + cells elimination in hepatocellular carcinoma

doi: 10.1016/j.apsb.2022.08.006

Figure Lengend Snippet: Less CD11b + myeloid cells infiltration was observed in CRA + anti-PD-L1 groups than in MWA + anti-PD-L1 groups. (A–F) Representative flow cytometry plots of CD11b + (A), CD11b + GR1 high (B), and CD11b + F4/80 + cells (E) gated on tumor infiltrating CD45 + cells in all groups. Flow cytometric quantification of CD11b + (C), CD11b + GR1 high (D), and CD11b + F4/80 + (F) in all groups. (G, H) Flow cytometric quantification of CD11b + F4/80 + (TAMs) population, and expression of iNOS and CD206 in CD11b + F4/80 + cell populations in all groups ( n = 6 for each groups, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ns, no significant difference, t- tests. All data are means ± SEM). (I) Immunofluorescence staining of tumor tissue sections. CD11b + cells were stained with Alexa Fluor 594, CCL2 was stained with Alexa Fluor 488. Scale bar = 200 μm. (J, K) The percentages of CD11b + cells and the percentages of CCL2 + spots in every field of view found in all different 6 groups; Three separate fields from each mice and three mice from each group were calculated. ∗ P < 0.05, ∗∗ P < 0.01,∗∗∗ P < 0.001, one-way ANOVA with Tukey's post hoc tests. All data are means ± SEM.

Article Snippet: For testing the affinity between PD-L1 antibodies and mouse CD16, atezolizumab (tecentriq), Avelumab (Bavencio), Mutant Anti-PD-L1-mIgG1e3 (InvivoGen, hpdl1-mab15, San Diego, CA, USA), Mouse IgG1 Isotype Control (R&D, MAB002, Emeryville, CA, USA), Human IgG1 (N298A) isotype control (InvivoGen, bgal-mab12, San Diego, CA, USA) were labeled with FITC fluorescence using the ZENON ALEXA FLUOR 488 MOUSE IG 1 KIT (ThermoFisher, Z25002, Carlsbad, CA, USA) as recommended by the manufacturer.

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Anti-PD-L1 antibody enhances curative effect of cryoablation via antibody-dependent cell-mediated cytotoxicity mediating PD-L1 high CD11b + cells elimination in hepatocellular carcinoma

doi: 10.1016/j.apsb.2022.08.006

Figure Lengend Snippet: Anti-PD-L1 antibody triggered NK cell-mediated ADCC effect to eliminating CD11b + myeloid cells. (A, B) Representative flow cytometry plots of CD49b + NK cells gated on CD45 + cells and granzyme B + cells gated on NK cells in all groups. (C) Flow cytometric quantification of CD49b + (NK) population, and expression of granzyme B + populations in all groups. n = 6 for each group, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ns, no significant difference, t- tests. All data are mean ± SEM. (D) H22 cells were inoculated subcutaneously into BALB/c mice. Twelve days later, cryoablation or microwave-ablation was proceeded, with corresponding antibody being infused via tail injection every 3 days. ADCC blocking assay employed by anti-mouse CD16 neutralizing antibody. Anti-mouse IgG was used as negative control. Tumor volume was shown. n = 6 for each groups, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ns, no significant difference, t- tests. All data are mean ± SEM. (E) Immunofluorescence staining of tumor tissue sections. CD11b were stained with Alexa Fluor 647, CD49b was stained with Alexa Fluor 488, Tunel was stained by Alexa Fluor 594. Scale bar = 20 μm.

Article Snippet: For testing the affinity between PD-L1 antibodies and mouse CD16, atezolizumab (tecentriq), Avelumab (Bavencio), Mutant Anti-PD-L1-mIgG1e3 (InvivoGen, hpdl1-mab15, San Diego, CA, USA), Mouse IgG1 Isotype Control (R&D, MAB002, Emeryville, CA, USA), Human IgG1 (N298A) isotype control (InvivoGen, bgal-mab12, San Diego, CA, USA) were labeled with FITC fluorescence using the ZENON ALEXA FLUOR 488 MOUSE IG 1 KIT (ThermoFisher, Z25002, Carlsbad, CA, USA) as recommended by the manufacturer.

Techniques: Flow Cytometry, Expressing, Injection, Blocking Assay, Negative Control, Immunofluorescence, Staining, TUNEL Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Anti-PD-L1 antibody enhances curative effect of cryoablation via antibody-dependent cell-mediated cytotoxicity mediating PD-L1 high CD11b + cells elimination in hepatocellular carcinoma

doi: 10.1016/j.apsb.2022.08.006

Figure Lengend Snippet: Bavencio induces more PD-L1 high CD11b + myeloid cells killing than Tecentriq via ADCC effect. (A) Flow cytometry revealed the affinity of different anti-PD-L1 antibodies with humanized Fc fragment or murine Fc fragment to CD16 molecule on mouse NK cells. (B) Residual tumor and autologous spleen were collected from HCC mouse models treated with CRA. Then CD11b + myeloid cells from tumor or spleen were sorted, as well as tumor cells for target cells. NK cells were firstly incubated with antibodies for 30 mins for CD16 and Fc fragment conjunction, then cocultured with target cells with E:T ratio at 2:1 for 24 h in vitro . Golgi-stop (#554724, BD) was added at 1:1000 to stop cytokine secretion. (C, D) Flow cytometry analysis of effector molecules revealed the degree of ADCC effect activated by different antibodies. (E, F) Flow cytometric quantification of effector molecules gate on NK cells in all groups. n = 3 for each group, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ns, no significant difference, t- tests. All data are means ± SEM. (G) In vivo experiment of tumor-bearing mice treated with CRA combined with different antibodies. H22 cells were inoculated subcutaneously into BALB/c mice. Twelve days later, cryoablation or microwave-ablation was proceeded, with avelumab (bavencio) or atezolizumab (tecentriq) or human IgG1 being infused via tail injection every 3 days. Each antibody was injected at 10 mg/kg. Tumor volume was shown. n = 6 for each group, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ns, no significant difference, t- tests. All data are means ± SEM. (H) Immunofluorescence staining of tumor tissue sections. CD11b + cells were stained with Alexa Flour 647, tunel was stained with Alexa Flour 594，CD49b was stained with Alexa Fluor 488 and nuclei were stained with DAPI. Scale bar = 200 μm.

Article Snippet: For testing the affinity between PD-L1 antibodies and mouse CD16, atezolizumab (tecentriq), Avelumab (Bavencio), Mutant Anti-PD-L1-mIgG1e3 (InvivoGen, hpdl1-mab15, San Diego, CA, USA), Mouse IgG1 Isotype Control (R&D, MAB002, Emeryville, CA, USA), Human IgG1 (N298A) isotype control (InvivoGen, bgal-mab12, San Diego, CA, USA) were labeled with FITC fluorescence using the ZENON ALEXA FLUOR 488 MOUSE IG 1 KIT (ThermoFisher, Z25002, Carlsbad, CA, USA) as recommended by the manufacturer.

Techniques: Flow Cytometry, Incubation, In Vitro, In Vivo, Injection, Immunofluorescence, Staining, TUNEL Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Anti-PD-L1 antibody enhances curative effect of cryoablation via antibody-dependent cell-mediated cytotoxicity mediating PD-L1 high CD11b + cells elimination in hepatocellular carcinoma

doi: 10.1016/j.apsb.2022.08.006

Figure Lengend Snippet: Bavencio did not induce ADCC effect to tumor cDC1 cells or spleen derived myeloid cells. (A) Flow cytometry analysis of effector molecules revealed the degree of ADCC effect induced by different antibodies. NK cells and tumor derived cDC1 cells were co-cultured at E:T = 2:1 for 24 h. Golgi-stop (#554724, BD) was added for suppressing cytokines release at 1:1000. (B, C) Flow cytometric quantification of effector molecules gate on NK cells cocultured with tumor cDC1 cells. (D, E) Flow cytometric quantification of effector molecules gate on NK cells cocultured with spleen derived myeloid cells. n = 3 for each group, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ns, no significant difference, t- tests. All data are means ± SEM. (F, G) Relative quantification and MFI of PD-L1 expressed in different cell subpopulations in tumor microenvironment or spleen were revealed. n = 3 for each group, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ns, no significant difference, t- tests. All data are mean ± SEM.

Article Snippet: For testing the affinity between PD-L1 antibodies and mouse CD16, atezolizumab (tecentriq), Avelumab (Bavencio), Mutant Anti-PD-L1-mIgG1e3 (InvivoGen, hpdl1-mab15, San Diego, CA, USA), Mouse IgG1 Isotype Control (R&D, MAB002, Emeryville, CA, USA), Human IgG1 (N298A) isotype control (InvivoGen, bgal-mab12, San Diego, CA, USA) were labeled with FITC fluorescence using the ZENON ALEXA FLUOR 488 MOUSE IG 1 KIT (ThermoFisher, Z25002, Carlsbad, CA, USA) as recommended by the manufacturer.

Techniques: Derivative Assay, Flow Cytometry, Cell Culture

Journal: Acta Pharmaceutica Sinica. B

Article Title: Anti-PD-L1 antibody enhances curative effect of cryoablation via antibody-dependent cell-mediated cytotoxicity mediating PD-L1 high CD11b + cells elimination in hepatocellular carcinoma

doi: 10.1016/j.apsb.2022.08.006

Figure Lengend Snippet: Bavencio induced enhanced ADCC effect than mutant Fc antibodies in eliminating human HCC infiltrating PD-L1 + CD33b + myeloid cells in vitro . (A) Surgically resected human HCC tumor and autologous PBMCs were collected from three individuals. Three different parts of each tumor was collected considering intratumoral heterogeneity. CD33 + myeloid cells and CD45 - tumor cells from tumor were sorted as target cells, and CD56 + NK cells from PBMCs for effector cells. NK cells were co-cultured with target cells as previously described. (B) Quantification of flow cytometry revealed PD-L1 expressed on tumor cell, T cell and myeloid cells in tumor microenvironment for three patients. (C) Representative images for IFN- γ Elispot assay revealed the different antibodies induced ADCC effect. Target cells derived from one of the three part of tumor in HCC-1 candidate. PHA was regarded as positive control, no antibodies added and Isotype IgG1 was control group. (D) The number of IFN- γ spots/1 × 10 5 cells in each group. n = 3 for each patient, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ns, no significant difference, t- tests. All data are mean ± SEM.

Article Snippet: For testing the affinity between PD-L1 antibodies and mouse CD16, atezolizumab (tecentriq), Avelumab (Bavencio), Mutant Anti-PD-L1-mIgG1e3 (InvivoGen, hpdl1-mab15, San Diego, CA, USA), Mouse IgG1 Isotype Control (R&D, MAB002, Emeryville, CA, USA), Human IgG1 (N298A) isotype control (InvivoGen, bgal-mab12, San Diego, CA, USA) were labeled with FITC fluorescence using the ZENON ALEXA FLUOR 488 MOUSE IG 1 KIT (ThermoFisher, Z25002, Carlsbad, CA, USA) as recommended by the manufacturer.

Techniques: Mutagenesis, In Vitro, Cell Culture, Flow Cytometry, Enzyme-linked Immunospot, Derivative Assay, Positive Control