(A) Immunoblot analysis of caspase 1 and caspase 3 in liver iNKT, NK, and T cells from WT B6 mice. β-Actin was used as a loading control. The blot shown is representative of one of 6 experiments. (B) FACS-sorted iNKT cells from WT B6 mice were stimulated with OX86 for 30 minutes, and cleavage of caspase 1 and caspase 3 was determined by immunoblotting. β-Actin was used as a loading control. The blot shown is representative of one of 6 experiments. (C) WT B6 mice were treated with either OX86 or control IgG (200 μg, i.p.). Caspase 1 activation in liver iNKT cells was determined using a specific Ab that stains the cleaved active caspase 1 fragment 24 and 48 hours later. The FACS shown is representative of one of 6 individual experiments. (D) WT B6 mice were treated with OX86 (200 μg, i.p.), and groups of mice were also treated with PBS as a control, caspase 1 inhibitor (inh), or caspase 3 inhibitor (1 mg/kg, i.p.). The iNKT cells in the liver of treated mice were determined by FACS 2 weeks later. The FACS plot shown represents data for one of 10 animals. (E) Six-week-old OX40L-Tg mice were treated with the caspase 1 inhibitor or caspase 3 inhibitor (1 mg/kg), or PBS as a control. The iNKT cells in the liver of treated mice were determined by FACS 2 weeks later. The FACS plot shown is representative data of one of 10 animals. (F) WT B6 mice and caspase 1–KO mice were treated with OX86 or a control IgG (200 μg, i.p.), and iNKT cells in liver of the treated mice were analyzed by FACS 2 weeks later. Data shown are from one of 6 independent experiments. (G and H) The summary bar graphs show relative percentage and absolute number of iNKT cells in WT B6 and caspase 1–KO mice with or without OX86 treatment. Data shown are mean ± SD of 6 experiments. P values were calculated by unpaired 2-tailed Student’s t test between control and OX86-treated groups (G and H), *P < 0.05.