American Society of Hematology

Fig. 5.

$Fig. 5. Kinetics of apoptosis induction by CH11 mAb and effects of IL-15 pretreatment. / (A) RPMI 8226 cells were either treated with IL-15 (10 ng/mL) or left untreated for 16 hours prior to addition of the agonistic anti-Fas mAb CH11 (50 ng/mL). The percentages of early and late apoptotic cells were determined 30 minutes to 48 hours after CH11 mAb addition. Results from 4 independent experiments ± SEM are given. Specificity control of IL-15 signaling via IL-2Rβ. Cells were pretreated with an anti–IL-2Rβ mAb (2.5 μg/mL; □) or an isotype-matched control mAb (Δ) 30 minutes prior to stimulation with IL-15 and CH11 mAb. The extent of apoptosis induction at 7, 24, and 48 hours was compared to that of cells treated with either CH11 mAb alone (▪) or the combination of IL-15 and CH11 mAbs (▴). Mean values ± SEM of 3 independent experiments are given. (B) Dose-dependence of IL-15 prestimulation to inhibit Fas-induced cell death. IM-9, MC-Car, and RPMI 8226 cells were stimulated with increasing concentrations of IL-15 (0-100 ng/mL) 16 hours prior to addition of CH11 mAb (50 ng/mL). The percentages of apoptotic cells were determined 7 hours after Fas-triggering by CH11 mAb (50 ng/mL). Results from 3 independent experiments ± SEM are given. (C) Dose-dependence of CH11 mAb-induced apoptosis in RPMI 8226 cells. Cells were treated for 24 hours with increasing concentrations of CH11 mAb, and the percentages of apoptotic cells were determined. Statistical analysis revealed a significant increase in the apoptotic cell fraction following CH11 mAb treatment compared with untreated cells (control versus 50 ng/mL:P = .02; control versus 100 ng/mL: P = .002; control versus 250 ng/mL: P = .001; control versus 500 ng/mL:P = .0007). No significant difference was observed when comparing the effects of 50 ng/mL with those at higher concentrations of CH11 mA: 50 ng/mL versus 100 ng/mL: P = .2; 50 ng/mL versus 250 ng/mL: P = .1; and 50 ng/mL versus 500 ng/mL:P = .07. (D) Interdependence of the IL-15 and Fas-pathways in RPMI 8226 cells. RPMI 8226 cells were pretreated with IL-15 (10 ng/mL) 16 hours prior to the addition of increasing concentrations of CH11 mAb (10-250 ng/mL). The percentages of apoptotic cells were determined 24 hours after CH11 mAb addition. Results of 3 independent experiments ± SEM are given, and P values were as follows: 10 ng ± IL-15: P = .0009; 20 ng ± IL-15:P = .0009; 50 ng ± IL-15: P = .001; 100 ng ± IL-15: P = .003; and 250 ng ± IL-15:P = .08. Apoptotic cells were detected by the annexinV/FITC/PI assay, and the sum of the percentages of early and late apoptotic cells was calculated as the percentage of the apoptotic cell fraction. Statistical analyses were done using a Fisher's PLSD test, and statistically significant different results are marked. *P < .05; **P < .01; ***P < .005; and **** P < .001.$

Kinetics of apoptosis induction by CH11 mAb and effects of IL-15 pretreatment.

(A) RPMI 8226 cells were either treated with IL-15 (10 ng/mL) or left untreated for 16 hours prior to addition of the agonistic anti-Fas mAb CH11 (50 ng/mL). The percentages of early and late apoptotic cells were determined 30 minutes to 48 hours after CH11 mAb addition. Results from 4 independent experiments ± SEM are given. Specificity control of IL-15 signaling via IL-2Rβ. Cells were pretreated with an anti–IL-2Rβ mAb (2.5 μg/mL; □) or an isotype-matched control mAb (Δ) 30 minutes prior to stimulation with IL-15 and CH11 mAb. The extent of apoptosis induction at 7, 24, and 48 hours was compared to that of cells treated with either CH11 mAb alone (▪) or the combination of IL-15 and CH11 mAbs (▴). Mean values ± SEM of 3 independent experiments are given. (B) Dose-dependence of IL-15 prestimulation to inhibit Fas-induced cell death. IM-9, MC-Car, and RPMI 8226 cells were stimulated with increasing concentrations of IL-15 (0-100 ng/mL) 16 hours prior to addition of CH11 mAb (50 ng/mL). The percentages of apoptotic cells were determined 7 hours after Fas-triggering by CH11 mAb (50 ng/mL). Results from 3 independent experiments ± SEM are given. (C) Dose-dependence of CH11 mAb-induced apoptosis in RPMI 8226 cells. Cells were treated for 24 hours with increasing concentrations of CH11 mAb, and the percentages of apoptotic cells were determined. Statistical analysis revealed a significant increase in the apoptotic cell fraction following CH11 mAb treatment compared with untreated cells (control versus 50 ng/mL:P = .02; control versus 100 ng/mL: P = .002; control versus 250 ng/mL: P = .001; control versus 500 ng/mL:P = .0007). No significant difference was observed when comparing the effects of 50 ng/mL with those at higher concentrations of CH11 mA: 50 ng/mL versus 100 ng/mL: P = .2; 50 ng/mL versus 250 ng/mL: P = .1; and 50 ng/mL versus 500 ng/mL:P = .07. (D) Interdependence of the IL-15 and Fas-pathways in RPMI 8226 cells. RPMI 8226 cells were pretreated with IL-15 (10 ng/mL) 16 hours prior to the addition of increasing concentrations of CH11 mAb (10-250 ng/mL). The percentages of apoptotic cells were determined 24 hours after CH11 mAb addition. Results of 3 independent experiments ± SEM are given, and P values were as follows: 10 ng ± IL-15: P = .0009; 20 ng ± IL-15:P = .0009; 50 ng ± IL-15: P = .001; 100 ng ± IL-15: P = .003; and 250 ng ± IL-15:P = .08. Apoptotic cells were detected by the annexinV/FITC/PI assay, and the sum of the percentages of early and late apoptotic cells was calculated as the percentage of the apoptotic cell fraction. Statistical analyses were done using a Fisher's PLSD test, and statistically significant different results are marked. *P < .05; **P < .01; ***P < .005; and **** P < .001.

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