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S. Delignat et al.
mice (565±170 AU, mean ± standard error of mean) (Figure 2D) were not statistically different from those in vehicle-treated mice (1133±376 AU, P=0.488). Similarly, inhibitory titers did not differ statistically between PF- 06250112-treated mice (123±88 AU) (Figure 2E) and con- trol mice (457±219 AU, P=0.572). The capacity of splenic T cells to proliferate in the presence of FVIII or con- canavalin A was similar in the two groups of mice (Figure 2F,G). Thus, inhibition of BTK at the time of FVIII injec- tion does not prevent the onset of a primary anti-FVIII immune response.
Bruton tyrosine kinase inhibition alters the memory factor VIII-specific B-cell response
We investigated the effect of BTK inhibition in the con- text of a memory anti-FVIII B-cell response, in which the FVIII-specific BCR in principle have a higher affinity for FVIII than BCR of naïve B cells. FVIII-deficient mice were injected with FVIII once a week for 4 weeks, and then left untouched for 90 days to allow the spontaneous elimina-
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tion of FVIII-specific short-lived plasmocytes.25 FVIII-sen- sitized mice were then fed with vehicle or PF-06250112 and treated with FVIII as described in Figure 3A. Anti- FVIII IgG titers measured prior to FVIII re-challenge were heterogeneous among mice (Figure 3B), as previously described.21,26,27 The levels of anti-FVIII IgG depicted in Figure 3C were, therefore, normalized with respect to the initial levels of anti-FVIII IgG measured for each individual mouse. Feeding mice with PF-06250112 significantly reduced the increase of the anti-FVIII IgG response, as compared to that in control mice (Figure 3C).
We then exploited an alternative model in order to study the effect of PF-06250112 in the absence of a poten- tial bias provided by the presence of plasma cells and cir- culating anti-FVIII IgG. Splenocytes from FVIII-treated mice or -naïve mice were depleted of CD138+ plasmocytes and adoptively transferred to FVIII-naïve FVIII-deficient mice as previously described.23 Feeding of the recipient mice with PF-06250112 or vehicle was initiated on the next day, and one injection of FVIII was given 2 h after the
BC
Figure 3. Treatment with PF-06250112 controls the recall response to factor VIII. (A) FVIII-deficient mice were injected with FVIII once a week for 4 weeks. After 90 days, mice were fed for 5 days a week, during 2 weeks, with PF-06250112 or vehicle, and injected with FVIII only once, 2 h after the second day feed. (B) Levels of anti-FVIII IgG were assessed by enzyme-linked immunosorbent assay using the mouse monoclonal anti-FVIII antibody mAb6 as a standard. Mice were then randomly attributed to either the PF-06250112- or the vehicle-fed groups. (C) Levels of anti-FVIII IgG were measured 7 and 14 days after the last injection of FVIII. IgG levels were normalized with respect to the initial levels of anti-FVIII IgG of each respective mouse measured at Bleed 0. Graphs depict means±SEM of three independent experiments with a total of 17 mice/group.
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haematologica | 2019; 104(5)