pDCs cross-prime OT-I T cells in vivo. 129sv mice were injected intravenously with OVA (9 mg) and either PBS, 10 μg of R848, 100 μg CpG in DOTAP, 10³ hemagglutinin units of heat-inactivated Influenza A virus (IAV), or 10⁶ pfu of BV. A control group received only PBS. Two hours later, splenic DC subsets were purified by magnetic sorting and their capacity to cross-prime OT-I T cells in vitro (A,C) and in vivo (B,D) was assessed. (A,C) Various numbers of purified cDCs (A, left panel) and pDCs (A, right panel; C) were cultured with 2.5 × 10⁴ CFSE-labeled OT-I T cells. OT-I T-cell proliferation was analyzed by flow cytometry 72 hours later. Proliferating T cells were assessed by CFSE dye dilution by OT-I T cells as described in Figure 5. Percentages of proliferating T cells are plotted against the number of DCs per well. (A) cDCs (left panel) and pDCs (right panel) were purified from mice injected with either OVA alone (○) or with R848 (●) or with CpG (■). (C) pDCs were purified from mice injected with either OVA alone (○) or with BV (●) or with IAV (■). Left panel: 1 representative experiment of 3 is depicted. Right panel: statistical analysis of OT-I T-cell proliferation, when cocultured with 2 × 10⁵ purified pDCs, from 3 independent experiments, including 1 or 2 mice per group in each experiment. (B,D) Purified pDCs were injected into CD45.1 hosts that had received 2 × 10⁶ CFSE-labeled OT-I T (CD45.2) cells 24 hours before. Seven days later, splenocytes from these mice were incubated 4 hours in the presence of brefeldin A with or without OVA_257-264 peptide. Cells were stained using anti-CD45.1, CD45.2, CD3ϵ, and CD8α mAbs, and OT-I T cells were characterized as CD45.1⁻, CD45.2⁺, CD8α⁺, and CD3ϵ⁺ cell populations. (B) OT-I T-cell analysis from mice that received pDCs purified from mice injected with PBS or with OVA and PBS, CpG, or R848 as indicated. CD44 expression and intracellular expression of IFN-γ by OT-I T cells were assessed as described in “Adoptive transfer.” First row: CFSE profiles of OT-I T cells (percentages of OT-I T cells showing CFSE dye dilution and gated in M1 are indicated); second row: CD44 expression on OT-I T cells according to CFSE profile; third row: IFN-γ intracellular staining on OT-I T cells according to CFSE profile; last row: intracellular staining with control isotype. (D) CFSE profiles of OT-I T cells from mice that received pDCs purified from mice injected with PBS or with OVA and PBS, BV, or IAV. First row: percentages of OT-I T cells showing CFSE dye dilution and gated in M1. Second row: CD44 expression by OT-I cells according to CFSE profiles. Third row: IFN-γ intracellular staining on OT-I cells according to CFSE profile; last row: intracellular staining with control isotype. One representative experiment of 4 is depicted.