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. 2022 Jul 14;219(9):e20212126. doi: 10.1084/jem.20212126

Figure 5.

Figure 5.

Epitope-orientated mechanism of the signal blockade by mAbs. (A and B) Evaluation, by ELISA, of the reactivity between 10 nM biotin–IFN-γ and immobilized 20 nM IFN-γR1 in the presence of mAbs (0, 5, 10, 20, and 100 nM, left to right). The assays were performed at least three times, independently. The results were pooled and are shown as the mean and SD (n = 3–7 per mAb). (A) Bar graph showing the signal for biotin–IFN-γ binding to IFN-γR1 following the addition of the biotin–IFN-γ–mAb mixture. (B) Bar graph showing the signal for the binding of the mAb to IFN-γR1 via biotin–IFN-γ. (C and D) IFN-γ–mediated binding of mAbs (40 nM) to THP-1 cells (1 × 105 cells). The amount of antibody binding to the cell was determined by flow cytometry with anti-human IgG–PE antibodies. Representative histogram (C) and quantitative results (D) for the mean fluorescence intensity (MFI) of IgG–PE from THP-1 cells in the presence and absence of IFN-γ (20 nM). The binding sites (I, II, and III) on IFN-γ are indicated above the mAbs. The results are shown as the mean and SD for three independent experiments. (E) Labeling of IFN-γR1 (purple) and IFN-γR2 (green) in the plasma membrane (PM) of live cells with Rho-11– (red) and AT643-conjugated (blue) anti-GFP nanobodies, for the assessment of IFN-γR1/IFN-γR2 dimerization by single molecule cotracking (magenta circle). Heterodimerization (top) was detected as the orthogonal labeling of IFN-γR1 and IFN-γR2 with mXFPm/MIRho11 and mXFPe/ENAT643, respectively. IFN-γR2 homodimerization (bottom) was detected as the labeling of mXFPe-IFN-γR2 with a mixture of ENRho11 and ENAT643. EN, enhancer; MI, minimizer. (F) Quantification of the heterodimerization of IFN-γR1 and IFN-γR2 with anti-GFP nanobodies labeled with Rho11 and AT643. Relative cotracking of Rho11–IFN-γR1 and AT643–IFN-γR2 in the presence of IFN-γ (10 nM) and mAb (20 nM). The data presented are means ± SEM; IgG Ctrl. (n = 20), E1 (n = 18), 2B6 (n = 19), 2A102 (n = 16), 1E8 (n = 19), 2G7 (n = 19). Each data point corresponds to a cell. (G) Quantification of the homodimerization of IFN-γR2. The data presented are means ± SEM; IgG Ctrl. (n = 20), E1 (n = 19), 2B6 (n = 20), 2A102 (n = 18), 1E8 (n = 22), 2G7 (n = 19). Each data point represents a cell. The P values in panels F and G were calculated in two-tailed unpaired Student’s t tests. **, P < 0.01; ****, P < 0.001. The binding sites (I, II, and III) on IFN-γ are indicated above the mAbs. (H and I) Evaluation of the synergistic effect on neutralization of mAbs (serial threefold dilutions and beginning at 1 μg/ml in total) based on binding sites (I, II, and III) and neutralizing capacity in HeLa GAS reporter cells (2 × 104 cells) within 4 ng/ml IFN-γ. In vitro neutralizing potential of groups of neutralizing mAbs (H) Site I, E1; site II, E5; site III, 1E8 (I); site I: E1; site II: 2B6; site III: 1E8 (J). In vitro neutralizing potential of groups of non-neutralizing mAbs. Site II: 2C10 and 1D5; site III: 2G7 and 2A101. 2A6 is a neutralizing mAb. (K) In vitro neutralizing potential of a group of neutralizing and non-neutralizing mAbs. Site I: E1 with neutralization; site II: 2C10, and site III: 2G7 without neutralization. (L and M) In vitro dose-dependent neutralizing potential of neutralizing mAbs (E1 or 2B6; serial threefold dilutions and beginning at 1 μg/ml) in the presence of a non-neutralizing mAb (2G7; serial threefold dilutions and beginning at 1, 3, or 9 μg/ml) with one, three, or ninefold dilutions. Control IgG, IgG ctrl. The results in H–M are shown as the mean and SD for three independent experiments. Two-tailed paired Student’s t tests were used to compare 1 μg/ml mAb treatments in H and K–M. ns, non-significant; *, P < 0.05; ***, P < 0.005.