Immunostimulatory ORNs trigger the production of antigen-specific, IFN-γ–producing CD8 cells. Mice were immunized as described in Figure 5 with 20 μg OVA together with ORN 9.2dr PTO, PolyA PTO, or CpG ODN twice at a 14-day interval. One week after the second immunization, spleen cells were isolated. (A,C) The generation of OVA-specific CTLs was assessed by flow cytometry using H-2k^b-OVA_257-264 peptide pentamers and an anti-CD8 mAb. (B,D) Splenocytes were restimulated with the OVA_257-264 peptide for 4 hours and cytoplasmic expression of IFN-γ in CD8⁺ T cells was examined by flow cytometry. (A-B) Representative data from one of 3 experiments are gated on CD8⁺ cells. Numbers indicate the percent of CD8⁺ cells that are OVA-pentamer positive or IFN-γ⁺. (C-D) Data show the mean values of individual mice (n = 4) ± SEM. Results are representative of 3 independent experiments. *P < .05; **P < .02; ***P < .001; ns indicates not significant. The asterisk without brackets indicates comparison to OVA group.