Figure 4. CD1d-mediated Treg-induced NKT cell apoptosis.

NKT cells and Tregs were isolated and co-cultured as described in Figure legend 2. NKT cell apoptosis was measured by AnnexinV staining with concurrent incubation of 7-AAD to assess cell necrosis. A) Representative dot plot of NKT cell (gated on CD1d-tetramer+ cells) apoptosis assay after co-cultured for 3 days. Apoptotic NKT cells are defined as Annexin-V⁺/7-AAD⁻. B) Time-course analysis of NKT cell apoptosis induced by Tregs isolated from wt and CD1dko mice. (n = 3 each group). C) Blocking of CD1d engagement completely abrogated Treg-mediated NKT cell apoptosis. Purified Tregs from wt mice were first incubated with anti-CD1d mAb or isotype control (10 g/ml, 1B1 clone, BD Biosciences) for 2 hours. After extensive washing, the Tregs were then co-cultured with NKT cells for 72 hours as described above. Mean (±SD) of three experiments were graphed. (n = 3/exp, and the experiments were repeated 3 times) *p<0.05 vs isotype control.