B cells but not accessory cells are the cellular targets of the adjuvant effect of CpG1668. (A) Allotype-chimeric mice with TLR9 gene deficiency restricted to all cells (in particular APCs) were generated by reconstituting irradiated C57BL/6 mice with BM from TLR9^−/− (Igh^b) mice (B right panel, TLR9^−/−) and by intraperitoneal injection of B-1b cells from C57BL/6 (Igh^a) mice. The control group was reconstituted with WT BM from C57BL/6 (Igh^b) mice (B left panel, WT) and by intraperitoneal injection of B-1b cells from C57BL/6 (Igh^a) mice. (B) Seven weeks after reconstitution, chimeric mice (4 per group) were immunized intraperitoneally with PBS, PS3 alone, or PS3 followed by injection 2 days later with 80 μg CpG1668. The PS3-specific IgM titers of both allotype (IgM^a and IgM^b) were determined by ELISA from blood samples collected 7 days after immunization. n.d. indicates not detected. (C) BM-chimeric mice with TLR9 gene deficiency restricted to B cells were generated by reconstituting irradiated C57BL/6 mice with 80% μMT BM and 20% TLR9^−/− BM (C right panels, TLR9^−/−). The control group received 80% μMT BM and 20% wild-type (WT) C57BL/6 BM (C left panel, WT) to generate a TLR9 proficient B-cell compartment. (D) Eight weeks after reconstitution, chimeric mice (6 per group) were immunized subcutaneously with PBS, PS3 alone, or PS3 followed by injection 2 days later with 80 μg CpG1668. The IgM titers were determined by ELISA from blood samples collected 7 days after immunization. Results are expressed as means ± SEM of the values collected in 4 to 6 individual mice.