Extended Data Fig. 4: Binding and functional characterization of anti-GREM1/2 monoclonal antibodies.
(a) Heatmap representing binding of 3-A1-3, 20-D1-5 and 14-D10-2 mAbs to human GREM1 and GREM2 as determined by ELISA. (b) Dose-dependent binding of the indicated anti-GREM1/2 mAbs to human GREM1 and GREM2 as determined by ELISA. Area under the curve (AUC) was calculated as the area under the titration curve from 1:2 serial dilutions of each antibody (2.5 - 0.02 µg) and GREM1 and GREM2 (0.5–0.008 µg/ml). (c–e) Neutralization capacity of the indicated mAbs using GREM1 and GREM2 in BMP4-induced luciferase activity by SL-0051 cells. Hundred percent BMP4 activity was determined as relative light units in the absence of GREM1 (c) or GREM2 (d) (N = 3, mean ± SEM; representative data from 1 out of 3 independent experiments with similar results). (e) The concentration of the indicated mAb necessary to restore 50% of BMP4 activity (EC50) was determined based on the values shown in (c) and (d); N = 6 independent replicates from 2 independent experiments. (f–h) Effect of anti-GREM1/2 mAb treatment in the adoptive T cell transfer myocarditis model. (f) Schematic representation of the experimental set up. (g) Quantification of heart-infiltrating CD45+ cells (h) and cytokine production of heart-infiltrating MYH6-specific CD4+ Vβ8+ T cells in Rag1−/− mice at day 28 post T cell adoptive transfer. N = 8, 10, 8 or 7 mice per group from 3 independent experiments. Box and whiskers show min to max, mean ± interquartile range; dots indicate individual mice. (i, j) Intracellular phospho-SMAD1/5/9 expression by CD45− CD31− PDPN+ CD56+ cardiac fibroblasts isolated from 8-week-old TCRM mice treated with IgG2b isotype control or GREM1/2-neutralizing antibody (14-D10-2) with representative histograms shown in (i). (j) Fold change of pSMAD1/5/9 mean fluorescence intensity (MFI) calculated relative to baseline pSMAD1/5/9 expression in CD56− fibroblasts (N = 4 mice per group from 2 independent experiments) Dots indicate individual mice, mean ± SEM is displayed. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparison test (g-h) or two tailed Student’s t test (j) with *, p < 0.05; **, p < 0.01; ***, p < 0.001.
