Chemokines superinduce IFN-I production by TLR9-activated pDCs. (A) Purified pDCs from heathy donors (HDs; n = 3) were left unstimulated (Unstim.) or cultured with CpG (0.25 μM) alone or with different concentrations of CXCL4 as indicated. IFN-α secretion was quantified by ELISA. (B) Purified pDCs (n = 4–49) were left unstimulated or cultured with CpG (0.25 μM) alone or with the indicated chemokines (10 μg/ml). IFN-α secretion was quantified by ELISA. (C and D) Purified pDCs from HDs (n = 4–13) were cultured for 24 h with medium only (Unstim.), with UV-treated U937 supernatant (C) or hu gDNA (D) alone or with CXCL4, CXCL10, CCL5, CXCL12, CXCL7, and CXCL9. IFN-α production was quantified by ELISA. (E) PBMCs and pDC-depleted PBMCs (PBMC-ΔpDCs; n = 5) were cultured with CpG in the presence of the indicated chemokines, and IFN-α production was quantified by ELISA. (F and G) BMpDCs (n = 3) from WT and TLR9-deficient mice were cultured for 4 h with medium only (Unstim.) or with CpG-B alone or with CXCL4. The expression of IFNβ (F) and IL-6 (G) was measured by Q-PCR. (H) BMpDCs (n = 3) from WT mice were left unstimulated or cultured with CpG-C(C274) alone or with mCCL3, mCXCL4, mCXCL10, mCXCL12, and mCCL5 for 4 h. The expression of mouse IFN-α was measured by Q-PCR. All results are represented as means ± SEM. Statistical significance was evaluated using Student’s t test in F–H. All other statistical significance was evaluated using Mann–Whitney U test, and only comparisons that are significant are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.