Both stimulatory and inhibitory activities of CpG-ODN are caused by specific interactions with TLR9. CD138⁻ spleen cells were obtained from hemophilic mice treated with 4 weekly doses of 200 ng FVIII and restimulated in vitro with stimulating (10 ng/mL) and inhibiting (1000 ng/mL) concentrations of FVIII in the presence of CpG-ODN or controls. Newly formed anti-FVIII ASC were detected by ELISPOT assay after 6 days of culture. (A) Representative ELISPOT assay. Each spot represents one anti-FVIII ASC. Cells were differentiated in the presence of no FVIII (0), 10 ng/mL FVIII (10), or 1000 ng/mL FVIII (1000). The differentiation cultures were supplemented with (A) medium only; (B) 100 ng/mL GpC-ODN (negative control of CpG-ODN); (C1) 100 ng/mL CpG-ODN only; (C2) 100 ng/mL CpG-ODN together with TLR9-blocking agent; (D1) 1000 ng/mL CpG-ODN only; and (D2) 1000 ng/mL CpG-ODN together with TLR9-blocking agent. (B) Quantitative evaluation of results presented in 4A for 10 ng/mL FVIII. Results of cultures differentiated in the presence of FVIII only (A) were set to 100% and presented as a dotted line. (B) One hundred ng/mL GpC-ODN (negative control of CpG-ODN); (C1) 100 ng/mL CpG-ODN only; (C2) 100 ng/mL CpG-ODN together with TLR9-blocking agent; (D1) 1000 ng/mL CpG-ODN only; and (D2) 1000 ng/mL CpG-ODN together with TLR9-blocking agent. Results of individual ELISPOT analyses and the median of all individual results for each group are presented. ***P < .001.