TLR9 stimulation accelerates Tfh cell, GC B cell, and antibody responses to FVIII. (A) Experimental timeline. S129/C57BL/6 HA mice received repeated (4, 6, or 8) weekly injections of FVIII (1.5 IU) with or without ODN1826 (50 μg) IV (n = 5-27 per group). Blood samples and the spleens were collected for flow cytometry analysis, ELISA, and Bethesda assays, 1 week after the last injection. (B) Tfh cell (CD4⁺CXCR5⁺PD-1⁺Bcl-6⁺FoxP3⁻) and GC B cell (CD19⁺CD95⁺GL7⁺) frequencies after 4, 6, or 8 weekly injections of FVIII (1.5 IU) with or without ODN1826 (50 μg) IV. (C) Inhibitor and FVIII-specific IgG1 titers after 4, 6, or 8 weekly injections of FVIII (1.5 IU) with or without ODN1826 (50 μg) IV. (D) Correlations of Tfh or GCB cell frequencies and inhibitor titers. P values and 95% CIs were determined for Pearson correlation coefficients. (E) Inhibitor titers (mean ± SD) in S129/C57BL/6 HA mice that received 4 weekly injections of FVIII only or FVIII and ODN1826 administered separately on 2 consecutive days (first ODN1826, then FVIII on the following day). (B and C) Shown are mean ± SD and P values from analysis of variance. ELISA, enzyme-linked immunosorbent assay.