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BTK inhibition prevents anti-FVIII memory response
expression in the mice and was used for further experi- ments.
Bruton tyrosine kinase inhibition does not prevent a primary anti-factor VIII immune response
We then investigated the potential of PF-06250112 to prevent the development of a primary anti-FVIII immune response in FVIII-deficient mice (Figure 2A). The effect of 4 weeks of treatment with PF-06250112 was first evaluat- ed on the splenic B- and T-cell compartments of FVIII-
A
injected mice. Analysis by flow cytometry revealed that chronic inhibition of BTK with PF-06250112 resulted in an increase in CD11b+ cells (P=0.040) (Figure 2B), which include monocytes, macrophages and natural killer cells, and in a significant decrease in CD4+ T cells (P=0.031) and in different B-cell subsets, including follicular and MZ B cells (P=0.038) (Figure 2C). Although statistically signifi- cant, changes in percentages of the cell populations were biologically marginal. The anti-FVIII IgG titers measured after the fourth injection of FVIII in PF-06250112-treated
BCD
EFG
Figure 2. Treatment with PF-06250112 does not prevent the onset of a primary anti-factor VIII immune response. (A) Experimental scheme for the preventive treat- ment of FVIII-deficient mice. Mice were fed with PF-06250112 (15 mg/kg) or vehicle for 5 days a week, and injected with FVIII once a week, 2 h after the second feed of the week. Mice were sacrificed 5 days after the fourth injection of FVIII (day 27). Spleens and sera were recovered. (B and C) At sacrifice, the isolated spleno- cytes were labeled with anti-CD45, anti-CD11b, anti-CD19, anti-CD3 and anti-CD4 antibodies (% of live CD45+ cells) (B). Follicular B cells (Fo) were identified as CD19+CD23highCD21low, marginal zone B cells (MZ) as CD19+CD21highCD23low, and germinal center B cells (GC) as CD19+GL7+ (% of live cells) (C). (D and E) Anti-FVIII IgG titers (D) and inhibitory titers (E) were measured in the sera of mice by ELISA with the mouse monoclonal anti-FVIII IgG mAb6 as a standard (expressed in arbitrary units, AU), and chromogenic assay, respectively. (F and G) Splenocytes were incubated for 72 h with FVIII (F) or with concanavalin A (ConA) (G). The incorporation of tritiated thymidine is depicted as counts per minute (CPM) after an additional 18 h of incubation (mean±SEM). Data are representative of two independent experi- ments with nine mice per group.
haematologica | 2019; 104(5)
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