Figure 3: Type I IFNs are determinant for the immunomodulatory activities of pDCs treated with CpG or GpC.

(a) Splenic CD11c⁺mPDCA-1⁺ DCs from Ifnar^−/− mice were combined with CD8⁺ DCs, pulsed with the P815AB peptide and transferred intravenously into recipient hosts that were assayed at 2 weeks for footpad reactivity to the peptide in saline. Transferred pDCs were either untreated or treated overnight with 1 (pDC/L-CpG) μg ml⁻¹ of CpG 1826 or with 1 μg ml⁻¹ of the modified, non-CpG ODN 1826 (pDC/L-GpC). Data are mean values±s.d. of four experiments. (b) After overnight exposure to 10 (pDC/H-CpG) μg ml⁻¹ CpG or 10 μg ml⁻¹ of GpC (pDC/H-GpC), pDCs from Ifnar^−/− mice were assayed for suppressive activity of immunogenic peptide presentation by the CD8⁻ DC fraction. The asterisk (*P<0.001; experimental versus control footpads; two-tailed paired t-test) indicates the occurrence of a positive skin test reaction as a result of immunogenic peptide presentation. Data are mean values±s.d. of four experiments. (c,d) IL-23 and TGF-β dependency of the immunostimulant and immunosuppressive activity of pDCs treated with CpG. Splenic CD11c⁺ DCs were fractionated according to mPDCA-1 or CD8 expression, pulsed with the P815AB peptide and transferred into recipient DBA/2 hosts to be assayed at 2 weeks for skin test reactivity to the eliciting peptide. (c) The pDCs were treated overnight with 1 μg ml⁻¹ CpG and cotransferred with a minority fraction of suppressive CD8⁺ DCs. A portion of the pDCs was coexposed to anti-IL-23 p19 (20 μg ml⁻¹) or anti-TGF-β (10 μg ml⁻¹). (d) Alternatively, the pDCs were treated with 10 μg ml⁻¹ CpG, with or without anti-TGF-β or anti-IL-23 p19, and cotransferred with a majority fraction of immunogenic CD8⁻ DCs. The asterisks indicate the occurrence of a positive skin test reaction. Data are mean values±s.d. of three experiments. (*P<0.01; experimental versus control footpads; two-tailed paired t-test). (e) IDO1 protein expression in pDCs treated with low- or high-dose CpG, low- or high-dose GpC, and control or anti-TGF-β (+) antibody was assayed by immunoblot analysis of whole-cell lysates. Blots were stripped and reprobed with anti-β-tubulin. One of three experiments. (f) Functional IDO1 activity was measured in terms of kynurenine levels in supernatants from pDCs treated as above, with either GpC or high-dose CpG. The reference IDO1 inhibitor, 1-methyl-tryptophan (1-MT), was also used in place of anti-TGF-β. Data are means±s.d. of three experiments. P<0.01 (treatment versus control; Student's t-test).