pDCs cross-prime naive T cells. (A) A total of 10⁴ purified splenic cDCs (left panel) or pDCs (right panel) were incubated for 2 hours in the presence of various concentrations of OVA with either medium alone (○, CM), R848 (●, 1 μg/mL), or CpG (■, 10 μg/mL). (B) A total of 10⁴ purified splenic cDCs (gray dotted line) or pDCs (black lines) were incubated for 2 hours in the presence of various concentrations of beads carrying the OVA_257-264 peptide (beads-pOVA, left panel) or beads carrying OVA protein (beads-OVA, right panel) with either medium alone (○, CM), R848 (●, 1 μg/mL), or CpG (■, 10 μg/mL). (A,B) Cells were washed twice and cocultured with 2 × 10⁴ OT-I CD8⁺ transgenic T cells for 72 hours. T-cell proliferation was measured according to ³H-labeled thymidine incorporation during the final 6 hours of culture. Results are expressed as mean cpm plus or minus SD for duplicate wells. One representative experiment of 3 (with CpG) or 7 (with R848) is depicted. (C) For statistical analysis, cumulative data from 3 to 7 independent experiments are plotted. Each dot represents the maximum OT-I response obtained in the presence of a given DC subset and of various doses of OVA. Results are expressed as a percentage of OVA cross-presentation obtained with nonstimulated cDCs (cDC = 100%). ns indicates not significant. *P < .05, **P < .01.