Poly(I:C) and CpG promote functional CTL responses in the absence of CD4⁺ T-cell help. (A) C57BL/6 mice were left untreated or were immunized by a single intravenous injection of BOVAp either alone or in combination with zymosan, poly(I:C), LPS, poly(U), R837, R848, CpG-B, or CpG-A. CpG was administered either naked or mixed with DOTAP (CpG(DOTAP)). The percentage of OVA_257-264–specific lysis was determined by in vivo killing assay 7 days after immunization. Control mice were immunized with control ODNs or BOVAp mixed with DOTAP. Specific lysis was calculated as follows: % specific lysis = 100 − [100 × (% CFSE^high-primed mice/% CFSE^low-primed mice)/(% CFSE^high-naive mice/% CFSE^low-naive mice)]. (B) Frequency of OVA_257-264–specific CTLs detected in the spleen 7 days after intravenous injection of BOVAp alone or with poly(I:C), CpG-B(DOTAP), or CpG-A(DOTAP). OVA_257-264–specific CTLs (OVATetr⁺) were quantified by tetramer staining. (C) C57BL/6 mice received a single intravenous or subcutaneous injection of BOVAp either alone or with poly(I:C) or CpG-B(DOTAP). Seven days after injection, the CTL response was quantified by in vivo killing assay. (D-E) Poly(I:C) and CpG induce CTL responses against BOVAp in the absence of CD4⁺ T-cell help. C57BL/6 and MHC-IIko mice were either left untreated or immunized intravenously with BOVAp alone or with poly(I:C) (D-E), CpG-B(DOTAP) (E), or anti-CD40 mAb (D). The specific CTL response was analyzed on day 7 by in vivo killing assay (D) and tetramer staining (E). (F) CTL response induced by poly(I:C) and beads coated with either E7_49-57 (BE7p), HY_738-746 (BHYp), or LCMV_33-41 (BLCMVp) peptides. Female C57BL/6 mice were either left untreated or immunized by a single intravenous injection of the corresponding beads either alone or in combination with poly(I:C). (G) CTL response induced by poly(I:C) and soluble OVA_257-264 peptide. C57BL/6 mice were left untreated or immunized by a single subcutaneous injection of either BOVAp or soluble OVA_257-264 peptide (OVAp) alone or in combination with poly(I:C). (F-G) The CTL response was quantified in the spleen by in vivo killing on day 7 after priming. Dots represent individual mice. All results are representative of at least 2 independent experiments.