IL-7 induces rapid IL-7Rα surface down-regulation resulting from receptor internalization. (A) HPB-ALL cells were cultured in the presence or absence of IL-7 (50 ng/mL) in culture medium for the indicated time points and subsequently analyzed by flow cytometry for surface IL-7Rα expression, as described in “Flow cytometric analysis.” Relative IL-7Rα expression was calculated as the geometric mean intensity of fluorescence normalized to the time 0 control. Data represent mean ± SEM from at least 3 independent experiments. *P < .05, ***P < .001 (1-way analysis of variance, with Bonferroni post test). (B) Representative flow cytometric histogram overlay of IL-7Rα surface expression in HPB-ALL cells stimulated for 30 minutes with IL-7 (gray line) or left unstimulated (black line). (C) IL-7Rα internalization visualized by time-lapse microscopy of HPB-ALL cells. IL-7Rα initially expressed at the cell surface was stained with α-human IL-7Rα unconjugated antibody, subsequently reincubated with a secondary α-mouse IgG-Alexa 488–conjugated antibody, and imaged for the indicated time points, with or without addition of IL-7 (50 ng/mL), as described in “Antibody ‘chase’ for assessment of receptor colocalization.” See supplemental data for full time-lapse microscopy video. Representative cells of each condition from 2 independent experiments are shown.