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

Figure 4.

Figure 4.

Potential of monoclonal AIGAs to neutralize IFN-γ signaling. (A) In vitro neutralization by 19 monoclonal AIGAs and AMG811 in HeLa GAS reporter cells (2 × 104 cells) treated with 4 ng/ml IFN-γ plus serial threefold dilutions of mAb, beginning at 1 μg/ml, for 20 h at 37°C. Luciferase activity was measured to estimate the percentage of neutralization. The assays were performed at least three times, independently. The results were pooled and are shown as the mean and SD (n = 3–6 per mAb). (B) Correlation between the binding affinity (KD, M) and neutralizing potential (log IC50, ng/ml) of the neutralizing mAbs. 2E10 was not included, because no log IC50 value was available for this mAb. A two-tailed Pearson’s correlation analysis was performed. (C) Assessment of the neutralizing potential of three selected mAbs (2A6, 2B6, and 2F2; serial threefold dilutions and beginning at 1 μg/ml) before and after mutation, in HeLa GAS reporter cells. The results are shown as the mean and SD for three independent experiments. (D) Analysis of competitive binding to sites on IFN-γ for three selected mAbs (2A6, 2B6, and 2F2; 5 μg/ml), before and after mutation, in an in-tandem cross-competition BLI assay. (E) Measurement of HLA-DR expression in THP-1 cells (1 × 105 cells), incubated with and without IFN-γ (20 ng/ml), in the presence of the various mAbs (3 μg/ml), for 24 h at 37°C. Representative histogram (left), as in Fig. S3, and quantitative results (right) for HLA-DR–positive THP-1 cells. The binding sites (I, II, and III) on IFN-γ are indicated above the names of the mAbs. On the right, the results are expressed as the mean and SD for three independent experiments. P values are indicated for comparisons with IgG control (ctrl.) in two-tailed unpaired Student’s t tests. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001. (F) Monoclonal AIGAs inhibit CXCL-10 production. Assessment of CXCL-10 production in a PBMC activation assay involving incubation with the selected monoclonal AIGAs (E1, 2B6, and 1E8; 1 μg/ml) and BCG + IL-12 (10 ng/ml) for 48 h at 37°C. NA, nonactivation. Dashed lines indicate the background signal. Each dot corresponds to a healthy donor of PBMCs. The data are presented as floating bars from minimum to maximum, with a line indicating the mean of four independent experiments.