Generation of agonistic, but not antagonistic, anti-BTLA mAb BYK-1. (A) T cells isolated from WT, BTLA-KO, or HVEM-KO mice were stimulated with indicated doses of anti-CD3 mAb in the presence of 20 μg/mL immobilized control rat IgG (□) or BYK-1 (■). Proliferative activity was assessed by 3H-thymidine incorporation during the last 18 hours of a 3-day culture. (B) BTLA-expressing CHO cells were cultured with HVEM-mouse Ig fusion protein in the presence of 10 μg control Ig, BYK-1, or BYK-2 (filled histogram). Background staining without HVEM-Ig is also shown (open histogram). Binding of HVEM-Ig fusion protein was detected by a staining with fluorescein isothiocyanate–conjugated antimouse Ig Ab. (C) CD4+ T cells isolated from B6, BALB/c, or DBA/2 mice were stimulated with 1 μg/mL antimouse CD3 mAb. After 48 hours, T cells were stained with BYK-1 (filled histogram) or control Ig (open histogram), and analyzed by flow cytometry. Data are representative of 3 independent experiments.
