Impaired CpG-ODN responses in Hmgb1^−/− cells. (A) Wt and Hmgb1^−/− immortalized or primary hematopoietic progenitor cells (HPCs) expressed the DC markers CD11b and CD11c following incubation with GM-CSF for 7 days, or expressed CD11b and F4/80 after culture with macrophage medium for 10 days. Although HPCs are uniformly round and nonadherent, following differentiation they become adherent and acquire a macrophage-like morphology. (IFLDCs indicates DCs derived from immortalized fetal liver. HPCs; PFLDCs, DCs derived from primary fetal liver HPCs.) (B) Protein levels of HMGB1, TLR9, and actin in wt and Hmgb1^−/− IFLDCs. (C) Wt and Hmgb1^−/− IFLDCs endocytosed comparable levels of CpG-ODN or GpG-ODN. Cells were treated with CpG-ODN-Cy5 (0.1 or 1.0 μg/mL for 1 hour, left panel), CpG-ODN-Cy5 (1018, 10 or 100 nM), or GpG-ODN-Cy5 (1019, 10 or 100 nM) for the indicated time points (right panel), and then subjected to FACS analysis. (D) Wild-type and Hmgb1^−/− IFLDMs were seeded at 1 × 10⁵/well in a 96-well plate (in triplicate) and treated with CpG-ODN (1018, 10 to 1000 nM), LPS (0.1 μg/mL), or PGN (10 μg/mL). After 24 hours, IL-6 secretion was assessed by ELISA (bars represent the average of triplicates ± SD). Experiments were replicated 3 times. (E) Wt and Hmgb1^−/− IFLDCs were seeded at 1 × 10⁵/well in a 96-well plate (in triplicate) and treated with CpG-ODN (1018, 1 to 1000 nM), LPS (0.1 μg/mL), or PGN (10 μg/mL). After 24 hours, IL-6, IL-12, and TNFα secretion was assessed by ELISA (bars represent the average of triplicates ± SD). Experiments were replicated 5 times. (F) Wt and Hmgb1^−/− IFLDCs were seeded at 1 × 10⁵/well in a 96-well plate (in triplicate) and treated with CpG-A (2216, 1 to 1000 nM, top panel) or CpG-ODN (1018, 0.1 or 1.0 μg/mL, bottom panel) in the presence or absence of DOTAP (10 μg/mL), or LPS (0.01 or 0.1 μg/mL, bottom panel), or left untreated. After 24 hours, IL-12 (top panel) or IL-6 (bottom panel) secretion was assessed by ELISA (bars represent the average of triplicates ± SD). (G) Wt and Hmgb1^−/− PFLDCs were seeded at 0.8 × 10⁵/well in a 96-well plate (in triplicate) and treated with CpG-ODN (1018, 100 or 1000 nM) or CpG-A (2216, 100 or 1000 nM) in the presence or absence of DOTAP (10 μg/mL), or LPS (0.1 μg/mL), or left untreated. After 24 hours, IL-6 and IL-12 secretion was assessed by ELISA (bars represent the average of triplicates ± SD). ND: not detected. (H) Wt and Hmgb1^−/− IFLDCs were treated with CpG-B (1018, 10 μg/mL), CpG-A (2216, 3.3 μg/mL), and poly(I:C) (10 μg/mL) in the presence or absence of DOTAP (10 μg/mL) for 24 hours. Type 1 IFN bioactivity in supernatant samples was detected by a biologic assay against vesicular stomatitis virus.⁴⁵  (I) Whole-cell lysates were prepared at 16 hours after treatment with CpG-ODN (1018, 10 μg/mL) or LPS (1 μg/mL) and the levels of iNOS, HMGB1, and actin were determined by IB. (n.s. = nonspecific band.) (J) Exogenous rHMGB1 restored cytokine production by Hmgb1^−/− IFLDCs in response to CpG-ODN. rHMGB1 (rH1, 0.2 μg/mL) was incubated in the presence or absence of CpG-ODN (1018) for 15 minutes. Wt and Hmgb1^−/− IFLDCs were treated with CpG-ODN (1018, 1 or 10 μg/mL) ± rHMGB1, LPS (0.1 μg/mL), or rH1 alone, or left untreated for 24 hours, and the levels of secreted IL-6 were determined by ELISA (bars represent the average of triplicates ± SD).