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BTK inhibition prevents anti-FVIII memory response
first feed (Figure 4A). The treatment of mice with PF- 06250112 prevented the production of anti-FVIII IgG as compared to that in control mice (0.9±0.3 versus 124.0±115.7, P=0.011) (Figure 4B). Anti-FVIII IgG titers in PF-06250112-treated mice injected with FVIII were similar to those of control mice adoptively transferred with splenocytes from FVIII-sensitized mice injected with PBS and to those of control mice adoptively transferred with splenocytes from FVIII-naïve and FVIII-injected mice. We further performed re-stimulation of FVIII-specific memory B cells ex vivo. Splenocytes from FVIII-sensitized FVIII- deficient mice having developed a humoral response to FVIII were isolated and pre-treated with PF-06250112 or vehicle before stimulation with FVIII. After 6 days of cul- ture, FVIII-specific antibody-secreting plasma cells were
A
measured by ELISPOT. A statistically significant dose- dependent reduction of antibody-secreting plasma cell for- mation was observed when splenocytes were pre-incubat- ed with PF-06250112 as compared to splenocytes pre- incubated with vehicle, prior to stimulation with FVIII (P≤0.003) (Figure 5). Thus, inhibition of BTK prevents the activation of the FVIII-specific memory B-cell response.
Discussion
The occurrence of FVIII inhibitors following FVIII replacement therapy remains a major clinical and societal challenge in hemophilia A. B cells are a key effector of the anti-FVIII immune response. Depending on their subtype
B
Figure 4. Treatment with PF-06250112 inhibits the anti-factor VIII memory B-cell response. (A) FVIII-deficient mice were injected with phosphate-buffered saline (PBS) or FVIII once a week for 4 weeks. Seven days after the last injection, mice were sacrificed and spleens were collected and pooled. CD138-depleted splenocytes from FVIII-treated or naïve (PBS- treated) FVIII-deficient mice were adoptively transferred to naïve FVIII-deficient mice. From the next day onwards, host FVIII-deficient mice were fed during 2 weeks with 15 mg/kg of PF- 061250112 (18 mice) or vehicle (20 mice), daily for 5 days. Two control groups were inject- ed with PBS or were adoptively transferred with splenocytes from naïve mice (7 mice per group). Mice were injected with FVIII or PBS only once 2 h after the first feed. (B) Levels of anti-FVIII IgG were measured by enzyme-linked immunosor- bent assay 14 days after FVIII challenge. Means±SEM are depicted on the graph.
haematologica | 2019; 104(5)
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