Btk and PLC-γ2 phosphorylation inhibition by imatinib, dasatinib, and nilotinib. (A) To assess the impact of TKI on normal B cells, PBMCs from healthy controls were isolated and cultured in the presence or absence of increasing concentrations of TKIs, namely, 1 to 50 μM of imatinib (LC Laboratories), 1 to 50 μM of nilotinib (LC Laboratories), or 1 to 100 nM of dasatinib (LC Laboratories) for 2 hours. PBMCs were then stimulated with goat anti-human IgG and IgM F(ab')2 (0.5 mg/mL solution) at a final concentration of 10 μg/mL for 20 minutes, and cells were stained with pBtk-PE or pPLC-γ2-PE, APC-conjugated anti-CD19 (BD Biosciences, San Jose, CA), PerCP-conjugated anti-human IgM (BD Biosciences, Oxford, United Kingdom), and FITC-conjugated anti-CD27 (DakoCytomation). Cells were gated on lymphocytes: the panels on the top depict the unstimulated negative control, and on the bottom anti-IgG and IgM-induced phosphorylation of Btk (left) and PLC-γ2 (right). (B) PBMCs were coincubated with imatinib (10 μM), nilotinib (10 μM), and dasatinib (100 nM) for 14 hours, and the viability of CD19+ B cells was assessed by staining with FITC-conjugated annexin and APC-conjugated anti-CD19 (both BD Biosciences, San Jose, CA). (C) Curve fit (linear regression) of TKI doses plotted against the percentage of Btk phosphorylation inhibition induced by each of the 3 TKIs, imatinib, nilotinib, and dasatinib (each experiment was performed a minimum of 3 times). The Y bar represents the percentage of gated population in which phosphorylated Btk or PLC-γ2 are detected.
