Immunization with Hsp70L1-OVA_257-264 hybrid induces OVA peptide-specific Th1 responses and CTL. Splenocytes from immunized mice were used to assess OVA_257-264 peptide-specific Th1 responses by IL-2 ELISA (A), IFN-γ ELISPOT (B), and CTL assay (C). To induce OVA_257-264 peptide-specific IFN-γ–producing cells and IL-2 secretion, the splenocytes were restimulated with OVA, OVA_257-264 peptide, Con A, or unrelated control peptide VSV_324-332. For CTL assay, the splenocytes were restimulated with irradiated E.G7-OVA cells and then used as effector cells (E) in an OVA-specific lysis test by standard 4-hour ⁵¹Cr-release assay. EL4 cells pulsed with OVA_257-264 peptide and E.G7-OVA cells were used as OVA-specific targets (T); control targets were EL4 cells. Various E/T ratios were tested as indicated. IL-2 concentration is expressed in pg/mL, with ELISPOT results expressed as the number of IFN-γ–positive spot-forming cells (SFCs)/10⁶ splenocytes. Data are mean ± SEM of 3 independent experiments.