Figure 4
Figure 4. CpG-STAT3dODN augments immunogenicity while reducing immunosuppressive effects of patients’ AML cells. (A) CpG(D19)-STAT3dODNCy3 internalization by major compartments of AML. Patients’ leukemic blasts were incubated with CpG(D19)-STAT3dODNCy3 at 500 nM for 2 hours and analyzed by flow cytometry. Representative results from 1 of 14 analyzed samples from individual patients demonstrating gating for CD34+CD38− LSC, CD34+CD38+ LPC, and bulk leukemic cells (left) The uptake of the CpG(D19)-STAT3dODNCy3 by each AML compartment (top) and the corresponding intracellular TLR9 levels (bottom) with bar graphs summarizing results from 4 individual patients. (B) Correlation between the level of conjugate internalization and STAT3 inhibition. Shown are flow cytometric results of the internalization study (as a percentage of Cy3+ cells), assessment of pSTAT3 levels following CpG(D19)-STAT3dODN vs CpG(D19)-scrODN control treatment (compared with untreated control cells set as 100%) and pairwise analysis of correlation between levels of uptake and pSTAT3 reduction (n = 10). (C) CpG(D19)-STAT3dODN reduced Arginase-1 expression in primary AML cells. (Left) Intracellular levels of ARG1 were assessed using flow cytometry after treatment using indicated conjugates compared with untreated control set as 100%. (Right) Pairwise analysis of correlation between levels of ARG1 and pSTAT3 reduction in individual patients’ samples (n = 11). (D) Decrease in arginase activity in supernatants from cultured patients’ AML cells treated as indicated for 72 hours. Results from the analysis of 6 individual patients’ samples. (E) The surface expression of HLA-DR or costimulatory CD86, CD80, CD40 molecules was determined using flow cytometry. Shown are means ± SEM using samples from 5 individual patients. (F) Partial reduction of the immunosuppressive effect of primary AML blasts on T-cell proliferation. Primary AML cells pretreated in vitro using CpG-STAT3dODN were later cocultured with allogeneic T cells at 1:1 for 3 days. T-cell proliferation was assessed using CFSE dilution assay; percentages of dividing T cells past the parental generation are indicated. Shown are representative results from 1 of 5 independent experiments; means ± SEM (n = 5).

CpG-STAT3dODN augments immunogenicity while reducing immunosuppressive effects of patients’ AML cells. (A) CpG(D19)-STAT3dODNCy3 internalization by major compartments of AML. Patients’ leukemic blasts were incubated with CpG(D19)-STAT3dODNCy3 at 500 nM for 2 hours and analyzed by flow cytometry. Representative results from 1 of 14 analyzed samples from individual patients demonstrating gating for CD34+CD38− LSC, CD34+CD38+ LPC, and bulk leukemic cells (left) The uptake of the CpG(D19)-STAT3dODNCy3 by each AML compartment (top) and the corresponding intracellular TLR9 levels (bottom) with bar graphs summarizing results from 4 individual patients. (B) Correlation between the level of conjugate internalization and STAT3 inhibition. Shown are flow cytometric results of the internalization study (as a percentage of Cy3+ cells), assessment of pSTAT3 levels following CpG(D19)-STAT3dODN vs CpG(D19)-scrODN control treatment (compared with untreated control cells set as 100%) and pairwise analysis of correlation between levels of uptake and pSTAT3 reduction (n = 10). (C) CpG(D19)-STAT3dODN reduced Arginase-1 expression in primary AML cells. (Left) Intracellular levels of ARG1 were assessed using flow cytometry after treatment using indicated conjugates compared with untreated control set as 100%. (Right) Pairwise analysis of correlation between levels of ARG1 and pSTAT3 reduction in individual patients’ samples (n = 11). (D) Decrease in arginase activity in supernatants from cultured patients’ AML cells treated as indicated for 72 hours. Results from the analysis of 6 individual patients’ samples. (E) The surface expression of HLA-DR or costimulatory CD86, CD80, CD40 molecules was determined using flow cytometry. Shown are means ± SEM using samples from 5 individual patients. (F) Partial reduction of the immunosuppressive effect of primary AML blasts on T-cell proliferation. Primary AML cells pretreated in vitro using CpG-STAT3dODN were later cocultured with allogeneic T cells at 1:1 for 3 days. T-cell proliferation was assessed using CFSE dilution assay; percentages of dividing T cells past the parental generation are indicated. Shown are representative results from 1 of 5 independent experiments; means ± SEM (n = 5).

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