IL-1B potently stimulates primitive CP CML cells. (A-C) Cells from CML BM (n = 3) or normal BM (n = 3) were sorted based on CD34 and CD38 expression and counted after 7 days culture in serum-free medium supplemented with indicated cytokines. The dotted lines represent seeded cell numbers. Cell numbers from seeded CD34⁺ CML cells (A), CD34⁺CD38⁻ CML cells (B), and CD34⁺CD38⁻ normal BM cells (C). (D) Flow cytometric analysis of cell surface markers following the 7-day culture of CD34⁺CD38⁻ CML cells in the presence or absence of IL-1B. (E-F) Colony formation of CD34⁺CD38⁻ CML cells plated in methylcellulose with or without the addition of IL-1B. Primary colony numbers (E) and colony numbers following replating (F). (G-H) CD34⁺ CML cells were stimulated with IL-1B for 15 minutes and analyzed for phosphorylation of NF-κB within the CD34⁺CD38⁺ and CD34⁺CD38⁻ cell populations by phospho-flow cytometry. The histogram (G) shows the phosphorylation levels in the CD34⁺CD38⁻ cells from a representative sample, and the diagram (H) summarizes data from 4 patients. (I-J) CD34⁺CD38⁻ CML cells from 5 patients were incubated in the presence or absence of IL-1B for 3 or 24 hours and analyzed by RNA-sequencing and GSEA. NF-κB target genes (I) and cell cycle–associated genes (J). For complete gene list, see supplemental Tables 1-5. BFU-E, burst forming unit–erythroid; CFU-G, colony forming unit–granulocyte; CFU-GEMM, colony forming unit–granulocyte, erythrocyte, monocyte, megakaryocyte; CFU-M, colony forming unit–macrophage.