Immunization with BMDCs stimulated with TLR ligand combinations results in superior CTL priming in vivo. DCs were stimulated with poly-(I:C), R-848, a combination of both, or left unstimulated for 6 hours or 20 hours. SIINFEKL peptide (100 nM) was loaded onto the stimulated DCs for 1 hour. After removal of unbound peptide, 3 × 10⁵ BMDCs were injected intraperitoneally into C57BL/6 mice. Seven days after DC immunization, mice were killed and spleens were processed to single-cell suspensions and (A-B) stained for surface expression of CD8 and activation markers (CD44 and CD62L; not shown) as well as with H2-K^b SIINFEKL tetramer for specificity of the induced CD8⁺ T cells. The mean percentage with standard deviation of 3 independent experiments with 3 mice per group is shown. (C-D) To check for functionality in terms of peptide-specific lysis by CTLs an in vivo cytotoxicity assay was performed as described in “Flow cytometric analysis and in vivo cytotoxicity assay.” The mean percentage with standard deviation from 3 independent experiments is depicted. (E-F) To assess the IFN-γ production of the CD8⁺ T cells, splenocytes were restimulated in vitro for 6 hours with or without (data not shown; background levels were the same for all stimuli) SIINFEKL peptide in the presence of Brefeldin A (1 μg/mL) and analyzed by FACS. The mean percentage with standard deviation of IFN-γ–producing CD8⁺ cells is depicted. The results are summarized from 3 independent experiments. *P < .001 by ANOVA. **P < .05 by ANOVA.