NKT cell anergy is cell autonomous and does not exhibit dominant suppression. (A) NKT cell anergy is predominantly cell autonomous. Mice were injected with α-GalCer and sacrificed at 0 days (control [C]), 3 days, 7 days, or 1 month. DCs from the spleen and NKT cells from the liver were then enriched as described in Methods. NKT cells (1 × 10⁵ per well) and DCs (2 × 10⁴ per well) were then cultured in different combinations in the presence (+) or absence (–) of α-GalCer. Proliferation was assessed by [³H]thymidine incorporation, and IL-4 and IFN-γ levels in the supernatant were evaluated by ELISA. (B) NKT cells remain anergic upon adoptive transfer. Mice were injected with α-GalCer or vehicle and sacrificed on day 3. NKT cells were enriched as described in Methods, and 1 × 10⁷ cells were adoptively transferred into irradiated Jα18^–/– mice. Two weeks later, mice were sacrificed; splenocytes, normalized for numbers of NKT cells, were stimulated with vehicle or α-GalCer; and then proliferative and cytokine responses were measured. No IL-4 was detected in any of the cultures. Data represent the average of 3 mice per group. (C) Anergic NKT cells fail to exhibit dominant suppression. Spleen cells from naive mice and from mice injected 1 month earlier with α-GalCer were mixed at the indicated ratios and cultured with α-GalCer (100 ng/ml), and then proliferative and cytokine responses were measured. Proliferation results represent the mean ± SEM of triplicate wells, and cytokine results represent the mean ± SEM of duplicate wells. Representative data of 3 individual experiments are shown.