Figure 1.

Rapamycin inhibits TLR-mediated IFN-α secretion by pDCs. (a) ELISA of IFN-α in supernatants of isolated pDCs pretreated for 3 h with various doses of rapamycin (Rap) and then stimulated with CpG-A (10 μg/ml; left) or treated with vehicle (Veh) or rapamycin and then stimulated with various doses of CpG-A (right). (b) ELISA of IFN-β, TNF, IL-6 and CXCL10 in supernatants of purified mouse pDCs pretreated with various doses of rapamycin and then stimulated for 24 h with CpG-A (10 μg/ml). (c) ELISA of IFN-α in supernatants of purified spleen pDCs cultured for 24 h with CpG-A (10 μg/ml), loxoribine (Loxo; 1 mM), YF-17D or vesicular stomatitis virus (VSV; all viruses at a multiplicity of infection of 1) after pretreatment with rapamycin (Rap) or vehicle. (d) Apoptotic death of isolated mouse pDCs pretreated with vehicle or 20 nM or 500 nM rapamycin, assessed by staining with annexin V and propidium iodide. Numbers in quadrants indicate percent cells in each. (e) Proliferation of and cytokine induction in naive CD4⁺CD62L⁺ OT-II T cells (1 × 10⁵ cells/ well) cultured with pDCs (5 × 10⁴) pretreated with ovalbumin peptide (10 μg/ml) and CpG-A (10 μg/ml) plus vehicle or rapamycin (100 nM for 3 h), assessed after 3 d by incorporation of [³H]thymidine (top left; proliferation) and ELISA of IFN-γ, IL-4, IL-10, IL-13 and IL-17. *, P < 0.05; **, P < 0.001; ***, P < 0.0001. Data are representative of at least three independent experiments (error bars, s.d.).