WM B cells generate PCs in vitro following culture with TD stimuli. (A) Representative flow cytometric plots depicting phenotypic characterization of differentiating B cells derived from the peripheral blood of healthy donors (left panels) or WM patients (right panels) stimulated with CD40L and F(ab′)2 anti-IgG/IgM. The day of culture is indicated on the left. (B) The proportion of the IgH repertoire occupied by each clone was plotted for healthy donor (upper panel) or WM (lower panel) samples stimulated with CD40L and F(ab′)2 anti-IgG/IgM, with each unique clone represented by a colored band. The top 250 clones are shown. (C) Reads from RNA-seq from representative day-6 samples taken from healthy donors and WM differentiations, highlighting the L265P somatic variant (T>C). (D) Phenotypic profiles for B cells derived from the peripheral blood of healthy donors (D1-D12). 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. (E) Phenotypic profiles for B cells derived from the bone marrow of WM patients stimulated with CD40L and F(ab′)2 anti-IgG/IgM. Each independent differentiation is represented by a different color, and samples with CXCR4 mutations are shown in red (WM4 and WM7). (F) CXCR4 mutations detected among WM samples. (G) Representative plots of CD38 and CD138 expression on day-13 cells from 1 healthy donor and 6 WM samples. (H) The fold change in cell number from healthy donors (upper panel) and WM samples (lower 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. Samples with CXCR4 mutations are indicated by red font.