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S. Delignat et al.
RPMI-1640, with 10% fetal calf serum (Thermo Fisher), 100 U/mL penicillin, 100 mg/mL streptomycin, and 50 mM 2-mercap- toethanol without CD138 depletion for 6 days. At day 0, PF- 06250112 or vehicle was added at different concentrations. FVIII (1 mg/mL Advate®, Shire, Dublin, Ireland) was added to the culture 2 h later. Advate was used instead of Refacto to follow the seminal protocol established by Hausl et al. as closely as possible.23 Of note, Refacto and Advate present with the same immunogenicity in FVIII-deficient mice.21,24 After 6 days, the formation of FVIII-specif- ic antibody-secreting plasma cells was assessed by an enzyme- linked immunospot (ELISPOT) assay with FVIII-coated plates (0.5 mg Advate/well). After 2 h of blocking with 10% fetal calf serum in RPMI medium, cells were incubated on the membrane and cul- tured overnight at 37°C in 5% CO2. After washing with PBS, 0.1% Tween 20, antibodies were detected using a goat anti-mouse IgG alkaline phosphatase-conjugated antibody (Southern Biotech, Birmingham, Alabama, USA), and antibody-secreting plasma cells were revealed with Sigmafast 5-bromo-4-chloro-3-indolyl phos- phate/nitro blue tetrazolium (BCIP/NBT) tablets (Sigma). Plates were scanned on an ImmunoSpot Analyzer (CTL, Shaker Heights, OH, USA). Spots were automatically counted by ImmunoSpot software using the SmartCountTM and AutogateTM functions.
Statistical analysis
The statistical analysis was performed using the non-parametric two-tailed Mann-Whitney U test with a 95% confidence interval. P values ≤0.05 were considered statistically significant (ns: not sig- nificant). GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA) was used for the statistical analysis.
Results
PF-06250112 inhibits B-cell receptor signaling
BCR engagement initiates an intracellular signaling cas- cade that induces the BTK-dependent activation of B cells.14 PF-06250112 has recently been described to inhibit BCR-mediated B-cell signaling, activation and prolifera- tion.18 We confirmed the effect of PF-06250112 on splenic B cells by monitoring the upregulation of CD86 in response to B-cell triggering with anti-IgM or anti-IgD for in vitro and in vivo assays, respectively. Pre-treatment of purified splenic B cells by PF-06250112 prevented, in a dose-dependent manner, the induction of CD86 expres- sion on B cells upon stimulation with anti-IgM F(ab’)2 frag- ments (Figure 1A). It did, however, marginally affect CD86 expression upon stimulation of the cells with an anti-CD40 antibody. The calculated half maximal inhibitory concentration (IC50) was 1.1±0.6 nM (Figure 1A, inset), which is in agreement with previous observations.18 The in vivo validation of the effect of PF-06250112 was per- formed using an anti-IgD antiserum instead of the anti- IgM antibody in order to avoid quenching of the triggering antibody by endogenous circulating IgM. The treatment of FVIII-deficient mice with PF-06250112 2 h prior to the injection of the anti-IgD antiserum prevented anti-IgD- mediated CD86 induction, as measured ex vivo on splenic B cells (Figure 1B), and only marginally affected anti- CD40-mediated CD86 induction. The dose of 15 mg/kg of PF-06250112 was sufficient to prevent induction of CD86
AB
Figure 1. Inhibition of splenic B-cell activation by PF-06250112. (A) Splenic B cells from C57Bl/6 mice, purified by negative selection, were plated in RPMI, 10% fetal calf serum and treated with PF-06250112 or vehicle (dimethylsulfoxide) for 2 h. F(ab’)2 fragments of goat anti-mouse IgM (10 mg/mL) or a monoclonal hamster anti-mouse CD40 antibody (5 mg/mL) were added. After 24 h, the expression of CD86 by live B220+ cells was analyzed by flow cytometry. Results are representative of two independent experiments performed in triplicate (mean±SD). Treatment of B cells in vitro with PF-06250112 suppressed induction of CD86 expression with an IC50 of 1.1±0.6 nM (Inset). (B) C57Bl/6 mice were fed with PF-06250112 or vehicle. After 2 h, goat anti-IgD antiserum (400 mL, eBiosciences), or rat anti-mouse CD40 IgG (100 mg, Biolegend) was injected intraperitoneally. Eighteen hours later, mice were sacrificed and the expression of CD86 by live splenic B220+ B cells was analyzed by flow cytometry. Results are representative of two independent experiments performed with three mice per group (mean±SD).
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haematologica | 2019; 104(5)