Expression of cytokine receptors on LSCs and effects of cytokine ligands. (A) Expression of surface antigens on CD34⁺/CD38⁻ cells and CD34⁺/CD38⁺ cells in patients with AML, CML (at diagnosis and relapse), and normal donors (nBM). Cells were stained with antibodies against CD114 (G-CSFR), CD116 (GM-CSFR), CD123 (IL-3RA), and CD221 (IGF-1R), and marker expression on CD34⁺/CD38⁻ and CD34⁺/CD38⁺ cells was determined by multicolor flow cytometry. The dotted line represents clear expression (staining index > 3). The range of each box represents the 25th to 75th percentiles. The middle line inside the boxes represents the median expression level. Red asterisk (*): P < .05 vs normal CD34⁺/CD38⁻ BM cells; gray asterisk: P < .05 vs normal CD34⁺/CD38⁺ BM cells. Pairwise Wilcoxon test and P values were adjusted for multiple comparisons using the Benjamini-Hochberg procedure. (B) Highly purified CD34⁺/CD38⁻ AML LSCs (from patients at diagnosis or relapse) were incubated in control medium (n = 5) or in G-CSF (n = 4), IL-3 (n = 4), SCF (n = 4), or a combination of all cytokines (n = 5) (100 ng/mL each) for 48 hours at 37°C. Then, uptake of ³H-thymidine was measured. Results are expressed as percentage of control (100%) and show the mean ± standard deviation (SD) of 4 or 5 experiments. *P < .05 vs control. The table shows the expression of cytokine receptors CD114 (G-CSFR), CD117 (SCFR), and CD123 (IL-3RA) on CD34⁺/CD38⁻ LSCs used in these experiments. Results are expressed as staining index (SI), as described in supplemental Patients and methods, and were graded using the following score: −, SI = 0 to 1.3; +/−, SI = 1.31 to 3; +, SI = 3.01 to 10; ++, SI > 10. Patient numbers (#) refer to those defined in supplemental Table 3. (C) AML MNCs were incubated in control medium or in G-CSF, IL-3, SCF, or a combination (100 ng/mL each) at 37°C for 48 hours. Then, apoptosis was determined in CD34⁺/CD38⁻ and CD34⁺/CD38⁺ cells by annexin V/4′,6-diamidino-2-phenylindole (DAPI) staining by multicolor flow cytometry, as described in supplemental Patients and methods. D, diagnosis; n.t., not tested.