Fig. 3.

Kinetic analysis of invariant natural killer T (iNK T) cell anergy following treatment with a multi-dose (MD) protocol of glycolipid. Non-obese diabetic (NOD) mice (4–6 weeks old) received MD treatment [4 µg/dose every other day for 3 weeks, intraperitoneally (i.p.)] of glycolipid or vehicle and were then rested for 10 days or 1 month. (a) Splenocytes were isolated after the in vivo treatment and restimulated in vitro with glycolipid (100 ng/ml) for 72 h. The proliferative capacity and levels of cytokine secretion by the splenic iNK T cells were assayed by [³H]-thymidine incorporation and enzyme-linked immunosorbent assay (ELISA), respectively. (b) After 10 days or 1 month of rest, mice were reinjected with an additional dose (4 µg, i.p.) of glycolipid. Splenocytes were harvested 2 h after the final injection, cultured in vitro for 3 h in the presence of a protein transport inhibitor, GolgiStop, and T cell receptor (TCR)-β⁺tetramer⁺ iNK T cells were stained for intracellular levels of interleukin (IL)-2, interferon (IFN)-γ and IL-4. Data are representative of three independent experiments yielding similar results and are expressed as mean ± standard deviation (s.d.); five mice/treatment group/time-point. *Significant (P < 0·05) reduction between primary and recall responses (vehicle/α-galactosylceramide C26:0 (α-GalCer) versusα-GalCer/α-GalCer, vehicle/C20:2 versus C20:2/C20:2).