(a) Mice were primed with i.v. injections of vehicle, free glycolipid (C26:0), complexes or conjugates either once or three times at weekly intervals. One week later, all groups were re-stimulated with i.v. C26:0, and serum IFNγ was measured at 12 hours by ELISA. (b) Quantitation of iNKT cells as percentage of total T cells by tetramer staining of splenocytes harvested at 72 hours after priming and restimulation as in panel A. (c) Surface expression of inhibitory checkpoint receptors by iNKT cells was measured on spleen and liver on tetramer stained iNKT cells 48 hours after the second of two weekly injections of saline versus conjugates. (d) Upregulation of ligands for co-inhibitory receptors on spleen CD11c+ DCs after stimulation with free glycolipid, complexes or conjugates. Data are means ± 1 SE for groups of 3 – 5 mice, and are representative of at least three experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****p<0.0001, 2-way ANOVA with Sidak multiple comparisons test.