Cross-priming of a primary CD8⁺ T response by R848-activated pDCs. Naive 129sv mice were immunized by 2 consecutive intravenous injections (at days 0 and 7) of pDCs or cDCs purified from mice injected with either PBS alone or OVA (9 mg) and 10 μg R848. Spleen from immunized mice were analyzed 1 week after the last immunization. (A) Splenocytes were labeled with anti-CD90, anti-CD8α mAbs, and H2-K^b-SIINFEKL-pentamer and analyzed by flow cytometry. First row: CD8α⁺ CD90⁺cells were gated among total T cells. Second row: K^b-SIINFEKL-pentamer⁺ cells were gated among CD8⁺ T cells. Percentages of gated cells are indicated in each quadrant. (B,C) Splenocytes were restimulated with OVA peptide (1 μg/mL) for 24 hours and with brefeldin A for the last 4 hours. Cells were then labeled with anti-CD5, anti-CD8α mAbs, and H2-K^b-SIINFEKL-pentamer. Intracellular anti–IFN-γ staining was performed before analysis by flow cytometry. CD8α⁺ cells were gated among total T cells and percentage of IFN-γ⁺ within CD5⁺ T cells is shown. IFN-γ is plotted against H2-K^b-SIINFEKL-pentamer to visualize costaining (B). Pooled results showing percentages of IFN-γ⁺ cells among CD8⁺ T cells from 2 independent experiments are plotted (C). (D) Specific lysis of OVA_257-264–loaded cells was assayed by in vivo killing. Results from individual mice are shown for each group.