Figure 4: TLR9–MyD88 dependency of CpG adjuvant effect.

(a) Induction of skin test reactivity by CpG requires TLR9 as well as MyD88 signalling in pDCs. Wild-type (WT) C57BL/6 pDCs were treated overnight with 1 μg ml⁻¹ CpG (L-CpG), nc-ODN (L-nc-ODN) or GpC (L-GpC), pulsed with the HY peptide, and cotransferred with a minority fraction of suppressive CD8⁺ DCs. Parallel groups included pDCs from Tlr9^−/− or Myd88^−/− mice. Skin test reactivity to the eliciting peptide was measured at 2 weeks. (b) Suppression of skin test reactivity by CpG requires TLR9 but not MyD88 signalling, whereas the suppressive effect of GpC occurs independently of TLR9 and MyD88. pDCs from WT or genetically deficient mice were treated with 10 μg ml⁻¹ CpG (H-CpG), nc-ODN (H-nc-ODN) or GpC (H-GpC), and cotransferred with a majority fraction of immunogenic CD8⁻ DCs. In both a and b, the asterisks indicate the occurrence of a positive skin test reaction (*P<0.01; experimental versus control footpads; two-tailed paired t-test), and data are mean values±s.d. of three experiments. (c) IDO1 functional activity is induced by high-dose CpG in Myd88^−/− but not Tlr9^−/− mice, and by either GpC dosage in both Myd88^−/− and Tlr9^−/− mice. Functional IDO1 activity was measured in terms of kynurenine production by pDCs treated with either dosage of CpG, nc-ODN or GpC. Data are means±s.d. of three experiments. P<0.01 (treatment versus none; Student's t-test).