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. 2008 Jan 8;111(9):4723–4730. doi: 10.1182/blood-2007-07-099531

Figure 1.

Figure 1

IL-21–induced cell death in B-CLL cells. (A) Dose kinetics. B-CLL cells isolated from 7 patients were left untreated in media or treated with IL-21 at 0.1, 1, 5, 10, 25, 50, 100, and 200 ng/mL for 72 hours. At 24, 48, and 72 hours, cells were stained with FITC-annexin V and PI and analyzed by flow cytometry. Direct cell death was evaluated by normalizing annexin V/PI–negative cells with the respective media control. The top panel shows a dot plot of one of the representative experiments. The bottom panel shows summarized average of results from the 7 independent experiments. The SD of the mean within the population is shown as error bars (P = .046, implying a slight decrease in the percentage of live cells with dose increases beyond 25 ng/mL). Number on plots are percentages of total cells. (B) Time kinetics. CD19+ B-CLL cells were treated with 50, 100, or 200 ng/mL of IL-21 in media, and the direct cell death caused by IL-21 at different time points were assessed by annexin V/PI staining. Direct cell death was evaluated by normalizing annexin V/PI cells with the respective media control. Error bars indicate SD of mean in 16 B-CLL patient cell samples. (C) IL-21–mediated direct cytotoxicity correlates with receptor expression. Primary cell samples from 14 B-CLL patients were stained with PE-labeled mouse mAb to human IL-21R or a nonbinding isotype control and analyzed by flow cytometry. The percentage of the CLL cells expressing IL-21R and percentage of viable CLL cells (annexin/PI compared with media) were analyzed by multiple regression (n = 16; P = .009; R = Pearson correlation). (D) IL-21–mediated direct cytotoxicity in responding patients-dose and time dependence. B-CLL cells were left untreated (media) or treated with IL-21 at 25, 50, 100, and 200 ng/mL for 72 hours. Cells were stained with FITC–annexin V and PI. Direct cell death was evaluated by normalizing annexin V/PI cells with the respective media control. Error bars represent SD of mean in 9 B-CLL patient cell samples (n = 9; P < .01 compared with media control).