Antileukemic effect of CpG-STAT3dODN in vivo is immune-mediated and depends on the induction of AML immunogenicity. (A-B) NOD/SCID/IL-2RγKO (A) or Tlr9^−/− mice (B) were challenged IV using CMM cells and treated using CpG(1668)-STAT3dODN, CpG(1668)-scrODN, or PBS as in Figure 5A. The percentages of GFP⁺c-Kit⁺ AML cells in different organs were determined by flow cytometry. Shown are means ± SEM from 2 independent experiments; n = 6 per group for each experiment. Statistically significant differences were indicated by asterisks. (C-D) CMM-bearing C57BL/6 mice were treated using CpG-STAT3dODN, CpG-scrODN, or PBS siRNA as in panel A. The surface expression of MHC class II (C) and costimulatory molecules CD40, CD80, CD86 (D) on splenic AML cells was assessed by flow cytometry. Shown are representative histogram overlays and bar graphs summarizing results from each group of mice (n = 6); mean ± SEM. (E-G) Systemic administration of CpG(B)-STAT3dODN treatment induces infiltration of CD8⁺ T cells while reducing percentages of Tregs and MDSCs in spleens of AML-bearing mice. Mice were treated as described in Figure 5A. Percentages of immune cells were assessed using flow cytometry in cell suspensions prepared from spleens of treated mice. Shown are representative histograms and combined results in bar graphs; means ± SEM (n = 6). Statistically significant differences were indicated by asterisks. (H) CD8⁺ and CD4⁺ T-cell populations are required for the antitumor effect of CpG-STAT3dODN. C57BL/6 mice were injected intraperitoneally with neutralizing antibodies specific to CD8 (2.43), CD4 (GK1.5), control immunoglobulin G (IgG) or left untreated and then challenged IV with CMM leukemia as in Figure 5A. Mice with established leukemia were injected 6 times every other day using CpG-STAT3dODN or left untreated. Percentages of AML cells in spleens were determined by flow cytometry; shown are representative dot plots and the combined results from 1 experiment using 6 mice per each experimental group; means ± SEM.