IL-1 signaling in primary CD34⁺ CD38⁻ CP CML cells is intact despite BCR/ABL1 inhibition but suppressed by IL1RAP antibodies. (A-D) CD34⁺CD38⁻ CML cells (n = 2-3 as presented) were incubated in the presence or absence of 5 μM imatinib (IM) for 4 hours. Cell surface expression of IL1RAP (gray, isotype control [no IM]; red, without IM; blue, with IM) (A). NF-κB phosphorylation following 15-minute IL-1B stimulation. The histogram (B) shows the NF-κB phosphorylation in a representative sample, and the diagram (C) summarizes data from 3 patients. Level of tyrosine phosphorylation following 15 minutes of IL-1B stimulation (D). (E) CD34⁺CD38⁻ CML cells were seeded in serum-free medium supplemented with IL-1B. Total cell numbers were determined following 7 days culture in the presence of the IL1RAP antibodies mAb81.2 and mAb3F8 or an isotype control antibody. Data are presented as fold change in cell numbers relative to conditions without IL-1B (n = 3). (F-G) CD34⁺CD38⁻ CML BM cells (n = 3) were incubated with the IL-1–blocking mAb3F8 or an isotype control antibody, stimulated with IL-1B, and analyzed for phosphorylation of NF-κB and AKT using phospho-flow. The levels of phosphorylated NF-κB (F) and AKT (G) are presented.