WM B cells fail to differentiate in response to the TLR7 agonist R848. (A) B cells derived from the peripheral blood of healthy donors were stimulated with 1 μg/mL R848 and F(ab′)2 anti-IgG/IgM. Each scatter plot displays the percentage of live cells expressing the stated CD marker, as determined at each time point via flow cytometry. Data from comparable intervals were grouped together for clarity. Each independent differentiation is represented by a different color. (B) The fold change in cell number from healthy donors (left panel) and WM samples (right panel) at each time point was determined by manual counts between day 0 and day 6 and by flow cytometry thereafter. The cell number at each point was normalized to the number of “input” cells for that specific donor obtained at day 0. WM samples with CXCR4 mutations are shown in red (WM7 and WM8). (C) B cells derived from the bone marrow of patients diagnosed with WM were stimulated with 1 μg/mL R848 and F(ab′)2 anti-IgG/IgM and profiled for markers as in panel A.