IL-4 rescues sIgM on CLL cells. (A) IL-4 significantly rescues sIgM expression in CLL cells. U-CLL cells (n = 5; top) and M-CLL cells (n = 5; bottom) were cocultured in the presence (green line) or absence (purple line) of IL-4 (25 ng/mL) for 48 hours. The cocultured CLL cells were stained with mouse anti-human IgM antibody or isotype control mouse antibody (shaded area) and analyzed by flow cytometric assay. (B) IL-4 does not significantly change sIgD expression. CLL cells (n = 5) were cocultured in the presence (green line) or absence (purple line) of IL-4 (25 ng/mL) for 48 hours. The cocultured CLL cells were stained with mouse anti-human IgD antibody or isotype control mouse antibody (shaded area) and analyzed by flow cytometric assay. (C) Stromal cells do not affect IL-4–restored sIgM protein expression. CLL cells (n = 5) were cocultured in the presence (green line) or absence (purple line) of IL-4 (25 ng/mL), or cultured with IL-4 (25 ng/mL) alone (red line) for 48 hours. Viable CLL cells were stained with mouse anti-human IgM antibody or isotype control mouse antibody (shaded area) and analyzed by flow cytometric analysis. (D) Coculture-induced sIgM is IL-4 independent. CLL samples were noncultured (purple line), or cocultured in the absence (MED; green line) or presence of neutralizing anti-human IL-4 antibody (10 μg/mL) (NAb; red line). Viable CLL cells were stained with mouse anti-human IgM antibody or isotype control mouse antibody (shaded area) and analyzed by flow cytometric assay. (E) CLL samples were stained with mouse anti-human CD19 antibody, CXCR4 antibody, CD5 antibody, or isotype control mouse antibody (shaded area). sIgM expression in CXCR4^dimCD5^hi subpopulation (green line; “b” in the right panel) and CXCR4d^hiCD5^dim subpopulation (purple line, “a” in the left panel) was examined by flow cytometric analysis. (F) CLL samples were stained with mouse anti-human CD19 antibody, CXCR4 antibody, and CD5 antibody. CXCR4^dimCD5^hi subpopulation (“b”) and CXCR4d^hiCD5^dim subpopulation (“a”) were sorted, and CD79b protein was examined by immunoblot. Membranes were stripped and reprobed with anti-actin antibody as a loading control. In (A-F), the left panels represent 1 of 5 independent experiments and the right panels are the summarized results. Lines represent mean ± SEM of 5 samples. Each spot represents 1 sample. **P < .01; *P < .05. MFI, mean fluorescence intensity; NAb, neutralizing antibody; NC, noncultured; NS, not significant; SC, stromal cells.