Repeated alpha-galactosylceramide (α-GalCer) administration decreases the proportion of NKT cells in multiple organs. (a) Flow cytometry analysis of liver mononuclear cells (MNCs) in the vehicle (left) and α-GalCer (right) groups, stained with anti-TCR αβ and anti-NK1.1 antibodies. In both panels, black squares show NKT cells. Flow cytometry analysis of (c) liver, (e) kidney, and (g) spleen MNCs in the (left) vehicle and (right) α-GalCer groups. Isolated MNCs were double stained with anti-TCR αβ antibody and α-GalCer-loaded CD1d tetramer; invariant NKT (iNKT) cells are shown in black squares. The percentages of (b) whole NKT cells in the liver and iNKT cells in the (d) liver, (f) kidney, and (h) spleen are shown (n = 4 in each group). *p < 0.05 compared with the vehicle group.