Antigen-specific CD8⁺T cells contribute to antileukemic effects of CpG-Stat3 siRNA. (A) CD8⁺ T-cell depletion prevents the antitumor effect of CpG-Stat3 siRNA in vivo. C57BL/6 mice were injected intraperitoneally with CD8-specific control immunoglobulin G (IgG) antibodies or left untreated and then injected intravenously with 1 × 10⁶CMM⁺ AML cells. After tumors were established, both antibody-treated groups of mice were injected with CpG-Stat3 siRNA every other day 6 times. Percentages of AML cells in blood, spleen, and bone marrow were determined by flow cytometry. Shown are combined results from 1 experiment on 6 mice per group; means ± SEM (n = 6). (B) STAT3 targeting using CpG-siRNA results in tumor antigen–specific IFN-γ production. Splenocytes from untreated, CpG-Luc siRNA–, or CpG-Stat3 siRNA–treated mice were incubated overnight with irradiated (100 Gy) CMM⁺ AML cells. Production of IFN-γ was assessed by ELISPOT assay. Shown are representative images (top) and results combined from the group of 4 to 6 individual mice. (C) CpG-Stat3 siRNA augments recall response to SIY model tumor antigen. C57BL/6 mice were injected with 1 × 10⁶ C1498.SIY cells intravenously and treated as described above. IFN-γ ELISPOT assay was performed by using splenocytes from 4 to 6 individual mice in each group after overnight restimulation with SIY peptide. Shown are the representative results from 2 independent experiments; means ± SEM. Statistically significant differences were indicated by asterisks: ***P < .001; **P < .01; *P < .05.