Figure 2
Figure 2. Partial B-cell depletion using IgG1 anti-CD20 promotes tolerance to FVIII during an ITI protocol. (A) Outline of the experimental protocol. B-cell depletion using a single dose of anti-CD20 antibody treatment in the FVIII primed E16 mice was followed by a daily high-dose FVIII intravenous injection to model clinical ITI therapy. The mice were bled at various time points for evaluation of anti-FVIII antibody levels and inhibitor formation. (B) Inhibitor formation during the course of an ITI protocol in IgG1 anti-CD20 mAb or control IgG1-treated mice. The FVIII-primed mice (n = 7-11) received IgG1 anti-CD20 mAb (right panel) or control IgG1 (left panel) were further treated with daily intravenous injections of high-dose FVIII (2 μg per mouse) to mimic the clinical ITI therapy. The mice were bled before (day 56), during (day 69), and one week after the end of an ITI protocol (day 83). Inhibitor titers were evaluated by Bethesda assay. A single dose of IgG1 anti-CD20 pretreatment prevented the increase in inhibitor formation in the majority of treated mice. (C) Inhibitor formation on delayed challenge after the IgG1 anti-CD20 treatment. Three months after the IgG1 anti-CD20 or control IgG1 treatment followed by an ITI protocol, the surviving mice were boosted again by intraperitoneal injection of 2 μg of FVIII. The inhibitor titer remained significantly lower in the IgG1 anti-CD20 group than in the control group. (D) The effect of B-cell depletion with IgG2a anti-CD20 on inhibitor formation during the course of an ITI protocol. In an independent experiment, FVIII-primed mice (n = 15) received either IgG2a anti-CD20 mAb (right panel) or control IgG2a (left panel) and were further treated with daily intravenous injection of high-dose FVIII (2 μg per mouse) to mimic the clinical ITI therapy. The mice were bled before (day 56), during (day 69), and one week after the end of an ITI protocol (day 83). Inhibitor titer was evaluated by Bethesda assay. n.s. indicates not significant. *P < .05, compared with control IgG treatment in terms of increase for inhibitor titers at day 69 and day 83, respectively (B,D). *P < .05 compared with control (C) (Mann-Whitney U test, 1-tailed).

Partial B-cell depletion using IgG1 anti-CD20 promotes tolerance to FVIII during an ITI protocol. (A) Outline of the experimental protocol. B-cell depletion using a single dose of anti-CD20 antibody treatment in the FVIII primed E16 mice was followed by a daily high-dose FVIII intravenous injection to model clinical ITI therapy. The mice were bled at various time points for evaluation of anti-FVIII antibody levels and inhibitor formation. (B) Inhibitor formation during the course of an ITI protocol in IgG1 anti-CD20 mAb or control IgG1-treated mice. The FVIII-primed mice (n = 7-11) received IgG1 anti-CD20 mAb (right panel) or control IgG1 (left panel) were further treated with daily intravenous injections of high-dose FVIII (2 μg per mouse) to mimic the clinical ITI therapy. The mice were bled before (day 56), during (day 69), and one week after the end of an ITI protocol (day 83). Inhibitor titers were evaluated by Bethesda assay. A single dose of IgG1 anti-CD20 pretreatment prevented the increase in inhibitor formation in the majority of treated mice. (C) Inhibitor formation on delayed challenge after the IgG1 anti-CD20 treatment. Three months after the IgG1 anti-CD20 or control IgG1 treatment followed by an ITI protocol, the surviving mice were boosted again by intraperitoneal injection of 2 μg of FVIII. The inhibitor titer remained significantly lower in the IgG1 anti-CD20 group than in the control group. (D) The effect of B-cell depletion with IgG2a anti-CD20 on inhibitor formation during the course of an ITI protocol. In an independent experiment, FVIII-primed mice (n = 15) received either IgG2a anti-CD20 mAb (right panel) or control IgG2a (left panel) and were further treated with daily intravenous injection of high-dose FVIII (2 μg per mouse) to mimic the clinical ITI therapy. The mice were bled before (day 56), during (day 69), and one week after the end of an ITI protocol (day 83). Inhibitor titer was evaluated by Bethesda assay. n.s. indicates not significant. *P < .05, compared with control IgG treatment in terms of increase for inhibitor titers at day 69 and day 83, respectively (B,D). *P < .05 compared with control (C) (Mann-Whitney U test, 1-tailed).

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