Figure 1.

CpG/TLR9 stimulation results in regression of Cbfb/Myh11/Mpl leukemia in mice only when combined with STAT3 inhibition

C57BL/6 mice were intravenously injected with 1 × 10⁶Cbfb/Myh11/Mpl (CMM) leukemia cells. After tumors were established (1%–2% of AML cells in blood), mice were treated i.v. using CpG-STAT3d, control CpG-scr oligonucleotides (5 mg/kg), or PBS every other day for six times (days 6–16). (A) CpG-STAT3d, but not control CpG-scr treatment, resulted in survival of the majority of mice. The presented result represents one of two independent experiments with similar outcome (n = 7–8 mice/group). (B) CpG-STAT3d-treated mice that rejected CMM leukemia were rechallenged with CMM cells or with an unrelated FBL3 leukemia. Shown are survival curves for rechallenged mice as well as naive mice engrafted with both AML types in parallel (n = 5–10 mice/group). (C) Mice were euthanized 1 day after the last treatment to assess leukemia burden, cell morphology, and immune infiltration. The percentages of c-Kit⁺ leukemic cells, CD8⁺ T cells, F4/80⁺ macrophages, and Ly6B.2⁺ neutrophils were assessed using immunohistochemical staining in spleens from mice treated as indicated above; scale bar, 200 μm. Shown are data representative of one of three independent experiments. (D–F) Treatment with CpG-STAT3d triggers AML cell differentiation to the antigen-presenting phenotype. CMM leukemia-bearing mice were treated three times every other day using CpG-STAT3d, control CpG-scr (5 mg/kg), or PBS and euthanized 1 day later. Data are representative of 1 of 2 independent experiments. (D) Reduction of leukemia burden in spleen after CpG-STAT3d treatment is inversely correlated with AML cell differentiation. The percentages of GFP⁺c-Kit⁺ AML cells and differentiated GFP⁺CD11b⁺/MHC-II⁺/CD86⁺ AML cells were assessed using flow cytometry; shown are means ± SEM (n = 4 mice/group). (E and F) The cytofluorimetric analysis of intracellular staining for proliferation marker Ki-67 (E) and phagocytosis assay using pHrodo-E.coli-BioParticles (F) on freshly isolated GFP⁺CD11b⁺ vs. GFP⁺CD11b⁻ leukemic cells after in vivo treatments as indicated earlier; shown as means ± SEM (n = 3–6 mice/group). (G) CpG-STAT3d induces morphological changes in ultrastructure of the differentiating AML cell subset. Representative TEM images of splenic AML cell cytomorphology from two independent experiments; scale bar, 2 μm. Statistically significant p values were indicated as follows: ∗∗∗, p < 0.001; ∗∗, p < 0.01; and ∗, p < 0.05