Enhanced activation of TCR transgenic CD8⁺ T cells in vitro and in vivo by BMDCs stimulated with TLR ligand combinations. (A) MACS-sorted transgenic St35 CD8⁺ T cells (5 × 10⁴) were cocultured with 4 × 10³, 1 × 10³, or 2.5 × 10² SGP-peptide–pulsed medium-activated, poly-(I:C)–activated, R-848–activated, or superactivated DCs and proliferation (³H-thymidine incorporation) was measured on day 3 of the culture. ○ indicates BMDCs stimulated for 24 hours with medium; ▿, poly-(I:C) 50 μg/mL; □, R-848 1 μg/mL; or ⋄, poly (I:C) + R-848. (B) MACS-sorted transgenic St35 CD8⁺ T cells (5 × 10⁴) were cocultured with DCs (1 × 10³) activated with the indicated TLR ligands as described in “Generation and activation of mouse bone marrow–derived dendritic cells” and culture supernatant was collected after 2 days of coculture and analyzed by IL-2 ELISA. (C) BMDCs were stimulated as described in “Generation and activation of mouse bone marrow–derived dendritic cells” for 24 hours and washed extensively before adding differentially stimulated BMDCs (5 × 10³/well) to cocultures of MACS-sorted transgenic CD8⁺ T cells from St35 mice (5 × 10⁴/well) and preactivated (αCD3/αCD28) CD4⁺CD25⁺ regulatory T cells (T_reg's). CD8⁺ T cells were stimulated with the adenoviral peptide SGP (0.5 nM). The inhibition in percentage is referenced to the respective wells without regulatory CD4⁺CD25⁺ T cells. All results are presented as mean and standard deviation of triplicate wells of 1 of 3 independent experiments. (D) C57BL/6 mice were adoptively transferred with splenocytes from 10⁵ TCR transgenic St35 mice recognizing the H2-D^b–restricted epitope SGPSNTPPEI and expressing the congenic marker CD45.1. Mice were injected with 3 × 10⁴ peptide-loaded DCs activated for 6 hours with the indicted TLR ligands, as in Figure 1, in vitro intraperitoneally 1 day later. The frequency, phenotype, and ex vivo production of IFN-γ was analyzed on day 5 after injection in CD45.1⁺ cells in the spleens of host animals. Expression of the congenic marker CD45.1 (on transgenic St35 CD8⁺ T cells) and CD44 (gated on CD8⁺) splenocytes of mice injected with DCs that were in vitro activated with the indicated TLR ligands is shown. One representative dot plot for each immunization is depicted. The numbers are the mean percentage with standard deviation after immunization with BMDCs that had been activated with the indicated stimuli from 2 independent experiments performed with 3 mice per group (n = 6). Analysis by ANOVA indicated a significant difference (P < .001) among the different groups.