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. 2014 Jun 10;9(6):e98415. doi: 10.1371/journal.pone.0098415

Figure 4. Monoclonal antibodies which recognize canine PD-L1.

Figure 4

(A) Cross-reactivities of antibovine PD-L1 monoclonal antibodies. Binding abilities of recently established anti-boPD-L1 monoclonal antibodies to canine PD-L1 were examined by flow cytometry. Three anti-boPD-L1 monoclonal antibody clones, 4G12-C1 (rat IgG2a), 5A2-A1 (rat IgG1), and 6G7-E1 (rat IgM), were tested and all the three clones were found to recognize canine PD-L1. Rat IgG2a, rat IgG1, and rat IgM were used as isotype-matched negative controls. (a) cPD-L1–EGFP–expressing Cos7 cells and (b) dog PBMCs stimulated with PMA/ionomysin for 3 days were stained with anti-boPD-L1 monoclonal antibodies (10 µg/mL) or isotype-matched control antibodies. Red line, 4G12-C1; blue line, 5A2-A1; green line, 6G7-E1; shaded area, rat IgG2a; vertical-striped area, rat IgG1; horizontal-striped area, rat IgM. (B) Blockade of cPD-1/cPD-L1 binding by anti-PD-L1 monoclonal antibody 6G7-E1. cPD-L1–EGFP–expressing cells were preincubated with anti-PD-L1 antibody and then cPD-1–Ig bindings were evaluated by flow cytometry. (a) Blocking effect of anti-PD-L1 monoclonal antibody 6G7-E1 on cPD-1/cPD-L1 binding. Five microgram per milliliter of isotype-matched control antibody (rat IgM) could not affect the cPD-1/cPD-L1 binding (left panel), whereas the same concentration of 6G7-E1 significantly blocked the Ig binding (right panel). (b) Representative histogram of the flow cytometric analysis. Shaded area, isotype control (5 µg/mL); solid line, anti-PD-L1 monocolonal antibody 6G7-E1 (5 µg/mL). (c) Dose-dependent blocking effect of 6G7-E1 on cPD-1/cPD-L1 binding. Cells were preincubated with 6G7-E1 or isotype control antibody at various concentrations (0.5, 1.0, 2.5, 5.0 µg/mL) and Ig binding was analyzed by flow cytometry. Each point indicates the average value of relative MFI obtained from three independent experiments (compared to no antibody control, error bar; SEM). Statistical significance was evaluated by Tukey’s test (*p<0.05, between the 0 µg/mL and the 1 µg/mL of anti-PD-L1 antibody treatment group and between the 1 µg/mL and the 5 µg/mL of anti-PD-L1 group. †p<0.05, between the each concentration of anti-PD-L1 group and the same concentration of isotype control group).