Th17 generation by IFN-α–conditioned pDCs. (A) Intracellular TLR7 and TLR9 expression and surface expression of TLR4, MHC class II, CD86, and IFNAR in FACS-purified pDCs from Flt3L (FL-pDC; dashed line) or Flt3L + IFN-α cultures (IFN-pDC; solid line). Shaded area represents isotype control staining. (B) Cytokine secretion from FACS-purified, CpGA-stimulated pDCs from Flt3L (FL) or Flt3L + IFN-α cultures (FL + IFN), determined by ELISA. Error bars represent SEM of values obtained from triplicate wells. (C) Th differentiation stimulated by FACS-purified pDCs from Flt3L or Flt3L + IFN-α cultures in the presence of optimal Th17-inducing conditions and absence of CpGA. pDCs were cocultured with CD4⁺ CD25⁻ CD62L⁺ CD44⁻ naive T cells from OT-II mice and OVA peptide 323-339 in the presence of the indicated cytokines and antibodies. Results show intracellular staining for IL-17 and Foxp3 at 4 days, after restimulation with phorbol myristate acetate and ionomycin. (D) Th differentiation stimulated by FACS-purified CpGA-activated pDCs from Flt3L (FL-pDC) or Flt3L + IFN-α cultures (IFN-pDC) with or without exogenous TGF-β, as indicated. pDCs were cocultured with naive T cells from OT-II mice and OVA peptide 323-339 in the presence of TGF-β (1 ng/mL) with or without exogenous IFN-α (3 ng/mL) or IL-6 blocking antibodies (10 μg/mL), as indicated. Th differentiation was determined as indicated in panel C. (E) Th induction in vivo by CpGA-activated pDCs from Flt3L (FL-pDC) or Flt3L + IFN-α cultures (IFN-pDC). Rag1^−/− mice received naive OT-II CD4⁺ T cells intravenously and CpGA-stimulated, OVA peptide-pulsed pDCs subcutaneously. Rag1^−/− mice injected with only T cells were included as controls (NT). Inguinal lymph nodes were collected at 7 days, and Th induction was determined as indicated in the left panel by intracellular staining of IL-17 and IFN-γ. Absolute number of IL-17⁺ IFN-γ⁻ CD4⁺ lymph node cells is shown in the right panel. n = 6. (A-E) Results represent 3 independent experiments.