T-independent boost of the primary Ab response to the PS3 vaccine by CpG1668. (A) Kinetics of CpG1668 administration. C57BL/6 mice (5 per group) were immunized with PBS or PS3 and injected with CpG1668 at the time of immunization (d0); 1 day before immunization (d−1); 1, 2, or 3 days after immunization (d1, d2, and d3). The anti-PS3 IgM titers were determined by ELISA from sera collected at day 7 after immunization. (B) Effect of delayed administration of CpG1668 on the PS3-specific ASC frequency in the spleen. Mice were injected subcutaneously with PBS or PS3 and injected 2 days later with either CpG1668 or CpG1720, as a negative control. Splenocytes were collected at day 5 after immunization, and PS3-specific ASCs were determined by ELISPOT. (C) Visualization of PS3-specific ASCs in ELISPOT wells from splenocytes of PS3-immunized C57BL/6 mice injected CpG1668 either at day 0 (top) or 2 days later (bottom). (D-E) WT and CD3 KO mice (5 per group) were immunized with PS3 and injected or not with CpG1668: at the time of immunization (d0) or 2 days (d2) after immunization. Spleens from each group were collected 5 days after immunization, and ASC numbers were determined by ELISPOT (D). Sera from each group were collected 5 days after immunization, and titers were determined by ELISA (E). Results are expressed as means ± SEM of the values collected in 5 individual mice.