Anti–human LIGHT mAb binds to mouse LIGHT and costimulates mouse T cells.
(A) Anti-LIGHT mAb binds to mouse T cells according to flow cytometry. Resting BALB/c spleen cells or spleen cells activated by Con-A (2 μg/mL) for 24 hours were cross-linked with anti-CD3 and anti-CD28 hamster mAb followed by goat-anti–hamster IgG. After fixing with 3.7% formalin, the cells were stained with anti–human LIGHT mAb (clone 1.2.2) followed by PE-F(ab)′2 of goat anti–human IgG, along with FITC–anti-Thy1.2. In some samples, soluble human LIGHT (5 μg/sample) or TR6 without the Fc tag (5 μg/sample) was added as inhibitors during the staining process. The cells were analyzed by 2-color flow cytometry, and LIGHT expression on Thy1.2+cells is shown in the histograms. Shaded areas represent cells stained with an isotypic control mAb, and solid lines represent cells stained with anti-LIGHT mAb. (B) Anti-LIGHT mAb costimulates mouse T-cell proliferation. Nunc 96-well plates were coated with a suboptimal concentration of anti-CD3 (0.2 μg/mL) along with goat anti–human IgG (anti-HIgG, 5 μg/mL) overnight at 4°C. After washing, the wells were reacted with normal human IgG (10 μg/mL) or human mAb against human LIGHT (10 μg/mL) at 37°C for 2 hours. The wells were washed and BALB/c T cells were seeded into the wells at 4 × 105cells/well. 3H-thymidine uptake was measured between 48 and 64 hours. The samples were in triplicate, and the means ± SD of counts per minute are shown. The experiments were performed twice with similar results, and the data of a representative experiment are presented.
