Journal: PLoS ONE

Article Title: Contractile actomyosin arcs promote the activation of primary mouse T cells in a ligand-dependent manner

doi: 10.1371/journal.pone.0183174

Figure Lengend Snippet: (A) Representative 3D-SIM images of OT1 T cells fixed 7 min after surface attachment and stained with phalloidin (red) and anti-myosin 2A antibody (green). The merged images are shown in the bottom row. The cells were activated on glass surfaces coated with either OVA:H-2K b plus CD80, G4:H-2K b plus CD80, CD80 only, or streptavidin (SA) only. The pSMAC regions are labeled with yellow or white brackets. Scale bar, 5 μm. (B) Examples for FibrilTool analysis. 7–8 similar-sized trapezoidal ROIs covering the pSMAC portion of the IS were drawn to measure the anisotropy of actin arcs. (C) Average anisotropies of actin arcs in the pSMAC region of OT1 T cells activated on surfaces coated with OVA, G4, or CD80-only. (D) Total anisotropies of actin arcs from all ROIs measured in OT1 T cells activated on surfaces coated with OVA, G4, and CD80-only. (E) Histogram of total anisotropies from all ROIs measured on OVA- and G4-coated surfaces (p < 0.00001). Mean ± SEM. An unequal variance T-test (Welch's T-test) was used. ***, **** indicate p < 0.001, 0.0001.

Article Snippet: To activate OT1 T cells with pMHC monomers, 8-well coverglass chamber slides (Nunc LabTek II Chambered #1.5, Thermo Scientific, Waltham, MA) were incubated sequentially for one hour each with 1 mg/ml BSA-biotin, 1 mg/ml streptavidin (SA), and 10 μg/ml pMHCs plus 5 μg/ml mouse recombinant CD80 (Sino Biological, Beijing, China).

Techniques: Staining, Labeling

Journal: PLoS ONE

Article Title: Contractile actomyosin arcs promote the activation of primary mouse T cells in a ligand-dependent manner

doi: 10.1371/journal.pone.0183174

Figure Lengend Snippet: (A and B) Representative 3D-SIM images of OT1 T cells that had been pretreated with either DMSO or pnBB in DMSO, allowed to attach to the activating surface for 7 min, and then fixed and stained with phalloidin (red) and anti-myosin 2A antibody (green). The merged images are shown in the bottom row. The cells were activated on glass surfaces coated with either OVA:H-2K b plus CD80 (A) or G4:H-2K b plus CD80 (B). The pSMAC regions are labeled with yellow or white brackets. Scale bar, 5 μm. (C) Frequency of arc morphologies in OT1 cells pretreated with either DMSO or pnBB in DMSO and activated on OVA- or G4-coated surfaces. Arcs were scored as either Organized (i.e. present and concentric), Disorganized (i.e. present but either pointing inwards or entangled), or No arcs (i.e. not present). (D) Average anisotropies of actin arcs in the pSMAC region of OT1 cells pretreated with either DMSO or pnBB in DMSO and activated on OVA- or G4-coated surfaces. (E) Histogram of total anisotropies from all ROIs measured on OVA-coated surfaces with or without pnBB treatment (p < 0.0001). (F) Same as (E) but using G4-coated surfaces (p = 0.44). Mean ± SEM. **** indicates p < 0.0001.

Article Snippet: To activate OT1 T cells with pMHC monomers, 8-well coverglass chamber slides (Nunc LabTek II Chambered #1.5, Thermo Scientific, Waltham, MA) were incubated sequentially for one hour each with 1 mg/ml BSA-biotin, 1 mg/ml streptavidin (SA), and 10 μg/ml pMHCs plus 5 μg/ml mouse recombinant CD80 (Sino Biological, Beijing, China).

Techniques: Staining, Labeling

Journal: PLoS ONE

Article Title: Contractile actomyosin arcs promote the activation of primary mouse T cells in a ligand-dependent manner

doi: 10.1371/journal.pone.0183174

Figure Lengend Snippet: (A and B) Representative 3D-SIM images of OT1 T cells that had been pretreated with either DMSO or pnBB in DMSO, allowed to attach to the activating surface for 7 min, and then fixed and stained with phalloidin (red) and anti-pCasL antibody (green). The merged images are shown in the bottom row. The cells were activated on glass surfaces coated with either OVA:H-2K b plus CD80 (A) or G4:H-2K b plus CD80 (B). Scale bar, 5 μm. (C) Mean intensities of pCasL at the IS from 3D-SIM images of OT1 cells pretreated with either DMSO or pnBB in DMSO and activated on OVA- or G4-coated surfaces. Mean ± SEM. An unequal variance T-test (Welch's T-test) was used. * and ** indicate p < 0.05 and < 0.01, respectively.

Article Snippet: To activate OT1 T cells with pMHC monomers, 8-well coverglass chamber slides (Nunc LabTek II Chambered #1.5, Thermo Scientific, Waltham, MA) were incubated sequentially for one hour each with 1 mg/ml BSA-biotin, 1 mg/ml streptavidin (SA), and 10 μg/ml pMHCs plus 5 μg/ml mouse recombinant CD80 (Sino Biological, Beijing, China).

Techniques: Staining

Journal: Cell Reports Medicine

Article Title: Development and Preliminary Clinical Activity of PD-1-Guided CTLA-4 Blocking Bispecific DART Molecule

doi: 10.1016/j.xcrm.2020.100163

Figure Lengend Snippet: MGD019 Molecular Structure and Bispecific Binding to PD-1 and CTLA-4 (A) MGD019 is a tetravalent bispecific (2 × 2) Fc-bearing DART molecule. (B) Binding of MGD019 (red diamonds), parental PD-1 mAb retifanlimab (blue squares), parental CTLA-4 mAb 4B6 (green triangles), or isotype control (black circles) to Jurkat/PD-1 cells and blockade of PD-L1 binding to the cells. (C) Binding to Jurkat/CTLA-4 cells and blockade of B7-1 binding to the cells. (D) Re-activation of β-galactosidase (β-gal) upon co-engagement of PD-1 and CTLA-4 by MGD019 in PathHunter PD-1 + CTLA-4 + assay. Error bars depict standard errors of the mean (SEMs). (E) Binding to in vitro -stimulated, PD-1 + /CTLA-4 + primary T cells and blockade of B7.1 binding to Jurkat PD-1 + /CTLA-4 + cells. (F) Blockade B7.1 binding to Jurkat PD-1 + /CTLA-4 + by MGD019 or CTLA-4 mAbs alone or in the presence of a 10× concentration of competing PD-1 mAbs (open red diamonds and purple crosses, respectively). (G) Interaction of MGD019 with single- and dual-expressing cells. Average (EC 50 ) values of PD-1 (blue) and CTLA-4 (yellow) ligand binding blockade . Representative experiments out of ≥3 independent repeats are shown in (B)–(F). See also and and .

Article Snippet: Human B7-1 protein (biotinylated) , BPS Bioscience , Cat# 71114.

Techniques: Binding Assay, Activation Assay, In Vitro, Concentration Assay, Expressing, Ligand Binding Assay

Journal: Cell Reports Medicine

Article Title: Development and Preliminary Clinical Activity of PD-1-Guided CTLA-4 Blocking Bispecific DART Molecule

doi: 10.1016/j.xcrm.2020.100163

Figure Lengend Snippet:

Article Snippet: Human B7-1 protein (biotinylated) , BPS Bioscience , Cat# 71114.

Techniques: Recombinant, Purification, Cell Culture, Lysis, Luciferase, Enzyme-linked Immunosorbent Assay, Software

Journal: Scandinavian journal of immunology

Article Title: Human Soluble CD80 is generated by alternative splicing, and recombinant soluble CD80 binds to CD28 and CD152 influencing T-cell activation.

doi: 10.1111/j.1365-3083.2007.02009.x

Figure Lengend Snippet: Figure 1 The distribution of sCD80 levels in 153 apparently healthy individuals. The concentration of sCD80 was evaluated using an ELISA and recombinant CD80-Ig as a standard.

Article Snippet: Western blot was also performed, with transfer of the proteins onto a Hybond-P membrane (Amersham Biosciences), and detection by using a biotinylated polyclonal anti-CD80 antibody (R&D Systems) and avidin-HRP conjugate (Zymed, San Francisco, CA, USA).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Scandinavian journal of immunology

Article Title: Human Soluble CD80 is generated by alternative splicing, and recombinant soluble CD80 binds to CD28 and CD152 influencing T-cell activation.

doi: 10.1111/j.1365-3083.2007.02009.x

Figure Lengend Snippet: Figure 2 Alternatively spliced CD80 mRNA. (A) Splice variants of human CD80 mRNA. The markers: *** or ### represent the spliced nucleotides for s1CD80 or s2CD80 respectively. The sequence data are available from GenBank under accession numbers ban- kit512201 and bankit508046. (B) Schematic presentation of the alternative splicing of CD80 mRNA. CD80mRNA consists of four exons: IgV, IgC, transmembrane (TM) and intracellular. The transmembrane domain is spliced out in s1CD80, while both the IgC and TM domains are spliced out in s2CD80. (C) The expression of different CD80 mRNA isoforms in fresh and stimulated B cells, T cells and monocytes is shown. Purified T cells, B cells and monocytes were stimulated for 24 h by ConA, LPS or IFN-c respectively. RNA integrity and cDNA synthesis was veri- fied by amplifying GAPDH cDNA. Mw, molecular weight markers; T, T lymphocytes; Ta, activated T lymphocytes; M, monocytes; Ma, activated monocytes; B, B lymphocytes; Ba, activated B lymphocytes.

Article Snippet: Western blot was also performed, with transfer of the proteins onto a Hybond-P membrane (Amersham Biosciences), and detection by using a biotinylated polyclonal anti-CD80 antibody (R&D Systems) and avidin-HRP conjugate (Zymed, San Francisco, CA, USA).

Techniques: Sequencing, Alternative Splicing, Expressing, cDNA Synthesis, Molecular Weight

Journal: Scandinavian journal of immunology

Article Title: Human Soluble CD80 is generated by alternative splicing, and recombinant soluble CD80 binds to CD28 and CD152 influencing T-cell activation.

doi: 10.1111/j.1365-3083.2007.02009.x

Figure Lengend Snippet: Figure 3 Demonstration of Rs1CD80 using Western blot and Coomas- sie staining. Western blot (left part) and Coomassie staining (right part) of purified Rs1CD80 under reducing conditions (lanes 3 and 4). Lanes 1 and 5 represent molecular weight markers (Invitrogen) and lanes 2 and 6 commercially available CD80-Ig (R&D Systems).

Article Snippet: Western blot was also performed, with transfer of the proteins onto a Hybond-P membrane (Amersham Biosciences), and detection by using a biotinylated polyclonal anti-CD80 antibody (R&D Systems) and avidin-HRP conjugate (Zymed, San Francisco, CA, USA).

Techniques: Western Blot, Staining, Molecular Weight

Journal: Lipids in Health and Disease

Article Title: Docosahexaenoic acid prevents dendritic cell maturation and in vitro and in vivo expression of the IL-12 cytokine family

doi: 10.1186/1476-511X-9-12

Figure Lengend Snippet: DHA prevents cytokine production in LPS-treated DC . CD11c+ DC (2 × 10 6 /ml for IL-10 or 1 × 10 6 /ml for TNFα, IL-6 and CCL-4) were treated with various concentrations of DHA for 24 h followed by LPS (0.1 μg/ml) for an additional 24 h. Supernatants were harvested and assayed for IL-6, CCL-4, IL-10 and TNFα by ELISA. Two independent cultures were tested in triplicate. Results are expressed as mean +/- SD. * p < 0.01, # p < 0.05, compared to LPS-treated samples (no DHA).

Article Snippet: FITC-conjugated anti-mouse CD80, CD86, CD40, MHCII; recombinant mouse IL-10; capture and biotinylated anti-mouse IL-12/p70, IL-6, TNFα, IL-10 and CCL-4; and TMB Substrate Reagent Set were purchased from BD PharMingen (San Diego, CA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: Glyco-engineered anti-human programmed death-ligand 1 (PD-L1) antibody shows an enhanced binding to FcγRIIIa. A competitive FcγRIIIa AlphaLISA was performed for the three anti-PD-L1 variants. Thereby the test antibody competes with antibody-conjugated acceptor beads for binding to FcγRIIIa-conjugated donor beads. The chemiluminescent signal as a result of close proximity of the donor and acceptor beads was plotted against increasing concentrations of αPDL1 NG (open circles), αPDL1 WT (gray triangles), and αPDL1 GE (black squares). Statistics: mean and SD were plotted in the graph. Data are representative of two independent experiments.

Article Snippet: Test antibodies were incubated in presence of 1 µg/ml PD-1-Biotin (tebu-bio/BPS Bioscience, #71109) or 0.5 µg/ml CD80-Biotin (tebu-bio/BPS Bioscience, #71114).

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: Glyco-engineered anti-human programmed death-ligand 1 (PD-L1) antibody and its normal and non-glycosylated counterparts show comparable antigen binding characteristics. The three anti-PD-L1 variants αPDL1 NG (open circles), αPDL1 WT (gray triangle), and αPDL1 GE (black squares) were tested for PD-L1 antigen binding and their capacity to block interaction with PD-L1 ligands in enzyme-linked immunosorbent assays (ELISA). (A) PD-L1 antigen binding ELISA. OD 450–620 values were plotted against increasing concentrations of test antibody to assess binding to plate-bound human PD-L1. (B) Competitive ELISA measuring binding of soluble programmed death 1 to plate-bound PD-L1 in presence of test antibody. OD4 450–620 values were plotted against increasing concentrations of test antibody. (C) Competitive ELISA measuring binding of soluble CD80 to plate-bound PD-L1 in presence of test antibody. OD 450–620 values were plotted against increasing concentrations of test antibody. Statistics: mean and SD were plotted in all graphs. Data are representative of two independent experiments.

Article Snippet: Test antibodies were incubated in presence of 1 µg/ml PD-1-Biotin (tebu-bio/BPS Bioscience, #71109) or 0.5 µg/ml CD80-Biotin (tebu-bio/BPS Bioscience, #71114).

Techniques: Binding Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Competitive ELISA

Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: Glyco-engineered anti-human programmed death-ligand 1 (PD-L1) antibody induces strongest NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) against PD-L1 + cancer cells, but none against B cells and monocytes. (A) The NK cell line KHYG-1-CD16aV as effector cells was incubated with europium-loaded PD-L1 + DU-145 cancer cells as target cells in an effector to target ratio of 10:1 in the presence of increasing concentrations of αPDL1 NG (white circles), αPDL1 WT (gray triangles), or αPDL1 GE (black squares) for 5 h to determine the lysis of target cells in an in vitro cytotoxicity assay. The percentage of specific target cell lysis is plotted against the antibody concentration used. The dashed line indicates % of lysis in the medium control. (B,C) The NK cell line KHYG-1-CD16aV as effector cells were incubated with calcein-labeled primary B cells (B) or monocytes (C) as target cells in an effector to target ratio of 10:1 in the presence of increasing concentrations of αPDL1 WT (gray bars) or αPDL1 GE (black bars) for 4 h to determine the killing of target cells in a flow cytometry based in vitro cytotoxicity assay. The relative frequency of dead 7-AAD + of calcein + target cells was plotted against the antibody concentration used. As a positive control (striped bars) either obinutuzumab (αCD20) was used to induce B cell lysis or staurosporine was used to induce monocyte lysis. Statistics: mean and SD were plotted in all graphs. Data are representative of two independent experiments. Significance was tested against the medium control (* p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001. Abbreviation: ns., not significant).

Article Snippet: Test antibodies were incubated in presence of 1 µg/ml PD-1-Biotin (tebu-bio/BPS Bioscience, #71109) or 0.5 µg/ml CD80-Biotin (tebu-bio/BPS Bioscience, #71114).

Techniques: Incubation, Lysis, In Vitro, Cytotoxicity Assay, Concentration Assay, Labeling, Flow Cytometry, Positive Control

Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: Glyco-engineered anti-human programmed death-ligand 1 (PD-L1) antibody induces strong CD8 T cell activation in a mixed leukocyte reaction. The three anti-PD-L1 variants αPDL1 NG (open circles), αPDL1 WT (gray triangles), and αPDL1 GE (black squares) were tested for their effect on T cell activation in a mixed leukocyte reaction (MLR). The medium control (black crosses) served as a negative control. T cells as responder cells were isolated from a single healthy donor (donor A). Monocyte-derived dendritic cells as stimulator cells were generated from different healthy donors. (A) IL-2 enzyme-linked immunosorbent assays on day 2 of MLR. The determined concentrations of IL-2 in the culture supernatants were plotted. (B) The activation status of CD8 and CD4 T cells in the MLR was determined on day 5 by flow cytometric analysis. The relative frequencies of CD25 + and CD137 + in CD8 and CD4 T cells were plotted. (C) The proliferation of CFSE-labeled CD8 T cells in the MLR was determined on day 5 by CFSE dilution measured by flow cytometric analysis. Representative plots of CD25 expression and CFSE signal intensity of CD8 T cells are shown. Statistics for (A,B) : besides individual data points ( n = 8 for IL-2 secretion, n = 16 for CD25 expression, and n = 7 for CD137), mean and SEM were plotted in all graphs (* p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001. Abbreviation: ns, not significant).

Article Snippet: Test antibodies were incubated in presence of 1 µg/ml PD-1-Biotin (tebu-bio/BPS Bioscience, #71109) or 0.5 µg/ml CD80-Biotin (tebu-bio/BPS Bioscience, #71114).

Techniques: Activation Assay, Negative Control, Isolation, Derivative Assay, Generated, Labeling, Expressing

Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: Glyco-engineered anti-human programmed death-ligand 1 (PD-L1) antibody induces increased CD8 T cell activation in presence of cancer cells. The three anti-PD-L1 variants αPDL1 NG (light gray bar), αPDL1 WT (dark gray bar), and αPDL1 GE (black bar) were tested for their effect on T cell activation (donor A) in allogeneic mixed leukocyte reaction (MLRs) in absence or presence of the cancer cell lines HSC-4 (horizontal stripes), ZR-75-1 (plaid), and Ramos (vertical stripes). MLR without addition of test antibody (medium; white bar) served as negative control. The relative frequencies of CD25 + in CD8 T cells were plotted. Statistics: mean and SD were plotted in all graphs. Data are representative of two independent experiments. Significance was tested against the medium control without presence of cancer wells (* p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001).

Article Snippet: Test antibodies were incubated in presence of 1 µg/ml PD-1-Biotin (tebu-bio/BPS Bioscience, #71109) or 0.5 µg/ml CD80-Biotin (tebu-bio/BPS Bioscience, #71114).

Techniques: Activation Assay, Negative Control