(A) WT mice were injected i.v. with CpG, Cy5.5-labeled CpG or Cy5.5-labeled CpG complexed with DOTAP. After 30 minutes, up-take of CpG was examined by flow cytometry on gated conventional DCs (cDC; CD11c^hi, I-A^b+), plasmacytoid DCs (pDC; CD11c^int, B220⁺, Ly6C⁺, CD19⁻), macrophages (Mφ, CD11b^int, F4/80^hi, Ly6G⁻, SSC^hi), monocytes (Mo, CD11b⁺, F4/80⁺, Ly6C⁺, CD11c⁻, Ly6G⁻, SSC^low) or NK cells (NK1.1⁺, TCRβ⁻) in the spleen. Shown are representative FACS plots for one of two mice per group. (B) Splenic CD11c^hi DCs of WT mice were stained for intracellular IL-12p40 one hour after injection with the indicated TLR ligands. Shown are representative FACS plots for one of four mice per group analyzed on two separate days. (C) Control (Flox) or DC-MyD88 KO (Flox/Cre) mice were injected i.v. with indicated TLR ligands. After one hour, splenocytes were stained for intracellular TNFα. Shown are representative contour plots of gated CD11b⁺, F4/80⁺, Ly6C⁺, SSC^low, CD11c⁻, NK1.1⁻, B220⁻ cells from one of four mice per group analyzed on two separate days. (D) Induction of inflammatory cytokine mRNA in the spleen at one hour after injection i.v. with CpG complexed with DOTAP. “fold-induction” and fold difference were calculated as in Figure 1. Statistical comparison is between the DC-MyD88 KO mice and the control mice. *, P<0.05; **, P<0.01. Data are representative of two separate experiments.

(A) Control (Flox) or DC-MyD88 KO (Flox/Cre) mice were injected i.v. with CpG or CpG/DOTAP. After 12 hours, expression of CD86 (left), CD40 (middle), or I-A^b (right) on the surface of CD11c^hi DCs were assessed by flow cytometry. “Fold-induction” (mean+s.d. of four mice) of median fluorescence intensity (MFI) is relative to the expression levels in vehicle-treated mice. Statistical comparison is between the DC-MyD88 KO mice and the control mice. *, P<0.05; **, P<0.01. (B) Representative histograms of CD86 (left), CD40 (middle), or I-A^b on gated CD11c^hi DCs from control (WT or Flox), myd88^−/− or DC-MyD88 KO (Flox/Cre) mice 12 hours after i.v. injection of CpG or CpG/DOTAP.

Control (Flox) and DC-MyD88 KO (Flox/Cre) mice were immunized i.p. with OVA together with CpG (upper panel) or CpG/DOTAP (lower panel) and were bled 7 and 14 days after immunization. Sera were serially diluted and OVA-specific antibody isotypes (IgM on day7 and total IgG, IgG1, IgG2b and IgG2c on day 14) were measured by ELISA. Data shown are the end-point titers of all samples. Statistical significance was calculated with Mann-Whitney U-test. *, P<0.05 **, P<0.01. Similar results were obtained in three (CpG) and two (CpG/DOTAP) separate experiments.

(A and B) IL-12 expression in control (Flox) or DC-MyD88 KO (Flox/Cre) mice after i.v. injection with PBS or CpG. (A) Splenocytes isolated one hour after injection were stained for intracellular IL-12p40. Shown are representative FACS plots of gated CD11c^hi DCs for one of six mice per group analyzed on two separated days. (B) Amounts of IL-12p70 in the serum of mice, as measured by ELISA, at two hours after i.v. injection of CpG (n=5 for Flox/Cre mice). Amounts of IL-12p70 in PBS-treated mice all were under detection limit (not shown). (C) Induction of IL-12p40 mRNA in the spleen at one hour after i.v. injection of Pam3CSK4 (TLR1/2), LPS (TLR4), flagellin (TLR5), CpG (TLR9), or i.p. injection of Imiquimod (TLR7). (D) Induction of inflammatory cytokine mRNA in the spleen at one hour after i.v. injection of CpG. “fold-induction” (mean+s.d. of 3 mice) is relative to the abundance in vehicle-treated mice. Numbers on top of the bars are the fold difference between the induction levels in the control and the DC-MyD88 KO mice. Statistical comparison is between the DC-MyD88 KO mice and the control mice. *, P<0.05; **, P<0.01. Data are representative of two separate experiments.

(A and B) Mice were injected with the indicated TLR ligands. Five hours later, synthesis of IFNγ by splenic NK cells (NK1.1⁺, TCRβ⁻) was assessed by intracellular staining and flow cytometry. (A) Representative contour plots of control (Flox) or DC-MyD88 KO (Flox/Cre) mice from one of six mice per group analyzed on two separate days. (B) Representative contour plot of WT (C57BL/6), IL-12p35^−/−, Ifnar^−/− and myd88^−/− mice from one of four mice per group analyzed on two separate days.

(A) Purified CD4⁺ T cells from OT-II transgenic mice with a congenic marker (Thy1.1⁺) were labeled with CSFE and adoptively transferred into Thy1.2⁺DC-MyD88 KO and control mice. One day later (day 0), the mice were immunized with OVA mixed with CpG (upper panels) or OVA mixed with CpG/DOTAP (lower panels). On days 4 or 7, lymphocytes from the spleen were harvested, and re-stimulated in vitro with PMA and ionomycin for 4 hours. The proliferation of OT-II T cells (identified as CD4⁺, B220⁻, Thy1.1⁺) was tracked by CFSE dilution, and T_H1 effector function development was assessed by intracellular IFNγ staining and flow cytometry. Shown are representative dot-plots of intracellular IFNγ staining and CFSE dilution on day 4 of single mice immunized with OVA + CpG or OVA + CpG/DOTAP. Numbers in the gates indicate percentage of OT-II T cells that were IFNγ⁺. (B) Absolute numbers of OT-II T cells (left panel) and IFNγ⁺ OT-II T cells (right panel) in the spleen at day 4 after immunization with OVA + CpG or OVA + CpG/DOTAP and in vitro restimulation, as calculated from the counts of total splenocytes and the percentages of OT-II T cells and IFNγ⁺ OT-II T cells in the spleens. Data are expressed as mean+s.d. of four mice, and are representative of three separate experiments. Statistical comparison is between the DC-MyD88 KO mice and the control mice. *, P<0.05.