Leukemia/α-GalCer vaccination is ineffective in the presence of established acute leukemia despite NKT-cell and DC activation. Mice were challenged with C1498 cells intravenously 1 week before vaccination with α-GalCer–pulsed or unpulsed irradiated leukemia cells. (A) Kaplan-Meier graph showing survival of mice. Symbols represent treatment groups: unvaccinated (●), therapeutic α-GalCer vaccination (▪), and treatment with unpulsed irradiated leukemia cells (▲). This graph represents 3 experiments, each with 5 mice per group. (B-E) Mice were inoculated with C1498 cells 7 or 14 days before vaccination with irradiated α-GalCer–pulsed leukemia cells. (B) Representative flow cytometry plots showing identification of splenic NKT cells (CD3⁺ α-GalCer–loaded CD1d tetramer⁺ [tet]). (C) Frequency of splenic NKT cells after vaccination in mice with and without established acute leukemia. (D) Serum IL-4 levels and (E) serum IFN-γ levels 2 hours after vaccination. (F-G) The splenic langerin⁺CD8α⁺ DC population in lang-EGFP mice was analyzed 24 hours after vaccination. (F) Representative flow cytometry plots showing identification of splenic langerin⁺CD8α⁺ DCs (CD11c⁺GFP⁺). (G) Frequency of splenic langerin⁺CD8α⁺ DCs. (H-I) The expression of CD40 and CD86 on langerin⁺CD8α⁺ DCs, respectively. (J-K) The CD8α⁺ DC population in C57BL/6 mice was analyzed 24 hours after vaccination. The expression of CD40 and CD86 on CD8α⁺ DCs, respectively. (L) Serum IL-12p70 was quantified 5 hours after vaccination. These results are indicative of 2 independent experiments, each with 5 mice per group. **P < .01; ***P < .001 (1-way ANOVA with a Bonferroni posttest). FSC-A, forward scatter area; MFI, mean fluorescent intensity; SSC, side scatter.