Inhibition of invariant chain synthesis enhances the generation of CD4⁺ T-cell responses and cytotoxic T-cell (CTL) responses in mice immunized with OVA mRNA-transfected DCs. (A) CD4⁺ T-cell proliferation assay. Mice were immunized intravenously with 2.5 × 10⁵ DCs transfected with either OVA mRNA or influenza matrix (Flu M1) mRNA. Where indicated, the OVA mRNA-transfected DCs were also transfected with Ii AS (AE40) or control (SE40) ODNs. Splenocytes were harvested after 8 days and CD4⁺ T cells were isolated using StemSep Murine CD4⁺ Negative Isolation Column (StemCell Technologies, Vancouver, BC, Canada). CD4⁺ T cells were cocultured with I-A^b-restricted OVA or VSV peptide-pulsed DCs for 3 days. ³H-thymidine incorporation was measured for 17 hours prior to harvest. Data are representative of 3 experiments. (B) Cytotoxicity assay. Mice were immunized with 2.5 × 10⁵ OVA mRNA-transfected DCs transfected with Ii AS (AE40) or control (SE40) ODNs, as indicated. Splenocytes isolated 8 days after immunization were either tested directly for OVA CTLs (primary response) or first incubated in vitro in the presence of OVA mRNA-transfected DCs and then tested for OVA CTLs (secondary response).²⁹  RMA-S cells pulsed with the MHC class I-restricted OVA or VSV peptides were used as targets. Data are representative of 5 experiments. (C) CTL responses were measured 8 or 35 days after immunization. (D) At 85 days after immunization with Ii AS (AE40) or control ODN (SE40)-treated OVA mRNA-transfected DCs, mice were reimmunized with OVA mRNA-transfected DCs and OVA CTLs were measured. Measurements were done in triplicates. Variability is indicated by the error bars.