Enhanced activation of TCR transgenic CD4⁺ T cells in vitro and in vivo by BMDCs stimulated with TLR ligand combinations. (A) MACS-sorted transgenic OT-II CD4⁺ T cells (7 × 10³) were cocultured with titrated numbers of OVA_323-339 peptide–pulsed (0.3 μM) DCs activated with the indicated TLR ligands as described in “Generation and activation of mouse bone marrow–derived dedritic cells.” Proliferation by ³H-thymidine incorporation was analyzed on day 4 of the culture. ○ indicates BMDCs stimulated for 24 hours with medium; □, poly-(I:C) 50 μg/mL; ▵, R-848 1 μg/mL; and ⋄, poly (I:C) + R-848. (B) MACS-sorted OT-II transgenic CD4⁺ T cells (5 × 10⁴) were cocultured with 3 × 10³ OVA^323-339 peptide–pulsed DCs activated with the indicated TLR ligands as described in “Generation and activation of mouse bone marrow–derived detritic cells.” Culture supernatants were collected after 3 days of the coculture and IFN-γ secretion was measured by standard ELISA. (C) BMDCs were stimulated as described in “Generation and activation of mouse bone marrow–derived dedritic cells” for 24 hours and washed extensively before adding differentially stimulated BMDCs (5 × 10³/well) to cocultures of MACS-sorted transgenic CD4⁺ T cells from C57BL/6 mice (5 × 10⁴/well) and preactivated (αCD3/αCD28) CD4⁺CD25⁺ T_reg's. CD4⁺ T cells were activated with an α-CD3 mAb as proliferative stimulus. 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 10⁶ splenocytes from TCR transgenic OT-II mice recognizing the epitope ISQAVHAAHAEINEAGR and expressing the congenic marker CD90.1. Mice were injected with 3 × 10⁵ peptide-loaded DCs activated for 6 hours with the indicted TLR ligands as described in “Generation and activation of mouse bone marrow–derived dedritic cells.” After 4 days, the mice were killed and splenocytes were analyzed for expansion. Expression of CD90.1 and CD4 was analyzed on splenocytes of mice injected with BMDCs that were in vitro activated with the indicated TLR ligands. These results are representative of 2 independent experiments with 5 mice per group. The numbers are mean percentage with standard deviation after immunization with BMDCs that had been activated with the indicated stimuli from 2 experiments performed with 5 mice per group (n = 10). Analysis by ANOVA indicated a significant difference (P < .001).