AML patients with high CD123 show downregulation of CXCR4, which can be rescued by blocking IL-3 signaling. (A) AML samples were cultured in the absence (no IL-3) or presence of 10 ng/mL human IL-3 for 24 hours. RT-PCR was used to analyze CXCR4 gene expression. Data are normalized to β-actin and each line represents a paired measurement in an individual patient. Significance was calculated by performing Wilcoxon matched-pairs signed rank test. (B) AML samples were cultured in the absence (no IL-3) or presence of 10 ng/mL human IL-3 and ± 1 μM of the IL-3 blocking mAb 7G3 or a control IgG2a for 24 hours. RT-PCR was used to analyze CXCR4 gene expression. Data are normalized to β-actin and are presented as foldchange in response to IL-3 and antibody. Significance was calculated using a 2-tailed paired t test. (C) CXCR4 function was compared between CD123-high and CD123-low AML BM cells after 4 hours of migration to SDF-1. Cells were cultured overnight in 10 ng/mL human IL-3 and plated into 96-well transwell plates with SDF-1 (or chemotaxis buffer as a control) in the lower chamber. Migration of specific populations was analyzed using flow cytometric staining for CD34, CD38, and CD123 postmigration. Data are normalized to beads. Statistical analysis: #nonsignificant difference, *P < .05, **P < .01, ***P < .001.