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Fig. 6. CD11c⁺ bone marrow (BM) cells and FMS-like tyrosine kinase-3 ligand dendritic cells (FL-DC) are activated in response to the KOS strain of herpes simplex virus type 1 (HSV-1) in the absence of Toll-like receptor 9 (TLR9). (A) Magnetic cell-sorting (MACS)-enriched CD11c⁺ ex vivo BM cells (2.5 × 10⁶/ml) from WT or TLR9 knockout (KO) mice were stimulated with HSV-1 KOS strain [1 × 10⁶ plaque-forming units (pfu)/ml] or CpG-2216 in the presence of IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF). (B) Total FL-DC (2.5 × 10⁶/ml) from WT or TLR9 KO mice were stimulated with HSV-1 F strain (1 × 10⁶ pfu/ml), HSV-1 KOS strain (1 × 10⁶ pfu/ml), or CpG-2216 in the presence of IL-3 and GM-CSF. Nonstimulated (open histogram) and HSV-1 KOS-stimulated (shaded histogram) total FL-DC cells were stained with antibodies directed to CD69 or CD40. (C) Sorted plasmacytoid DC (pDC) and conventional DC (cDC) from FL-DC cultures of WT and TLR9 KO mice were stimulated with HSV-1 F strain (2.5 × 10⁶ pfu/ml), HSV-1 KOS strain (2.5 × 10⁶ pfu/ml), or CpG-2216 in the presence of IL-3 and GM-CSF. All supernatants were tested by ELISA for IFN-a production. Error bars represent the range of duplicate samples; asterisks indicate that cytokines were below the detection limit of the ELISA.

Fig. 7. Total or MACS-enriched CD11c⁺ and CD11c^- spleen cells produce IFN-ain response to HSV-1 in the absence of TLR9, but the IFN-aresponse of highly purified spleen pDC depends on TLR9. (A) Total spleen cells (2.5 × 10⁶/ml), (B) MACS-enriched CD11c^- spleen cells (2.5 × 10⁶/ml), or (C) MACS-enriched CD11c⁺ spleen cells (2.5 × 10⁶/ml) from WT or TLR9 KO mice were stimulated with HSV-1 or CpG-2216 in the presence of IL-3 and GM-CSF. (D) To identify the source of IFN-awithin the splenic CD11c⁺-selected fraction, we sorted highly purified pDC (CD11c⁺, CD45RA^high, or B220^high) and cDC (CD11c⁺, CD45RA^neg, or B220^neg) with a MoFlo instrument (DakoCytomation, Glostrup, Denmark) and stimulated the cells with HSV-1 or CpG-2216 in the presence or absence of IL-3 and GM-CSF. All supernatants were tested by ELISA for IFN-aproduction. One representative experiment of at least three is shown. Error bars represent the range of duplicate samples; asterisks indicate that cytokines were not detectable by ELISA.