LEF-1 protein is specifically expressed by B-cell precursors and CLL cells but not mature peripheral B-cell subsets. (A) Representative intracellular flow cytometry for LEF-1 in B cells from healthy donor bone marrow, blood, tonsil, cord blood, and CLL B cells from a CLL patient. Isotype control histogram is shaded gray; LEF-1 histogram is black. (B) Detection of LEF1 and AICDA mRNA levels by quantitative PCR in B cells from a healthy donor at 24 and 72 hours after stimulation with CpG and cytokines or anti-immunoglobulin (anti-Ig) stimulation. Fold change was calculated relative to unstimulated donor B cells; an unstimulated CLL sample served as a positive control for LEF-1 expression. (C) Detection of LEF1 mRNA levels by quantitative PCR in 2 CLL patients before and after 2 weeks in culture in AIM-V media at a density of 10 × 106 cells/mL. (D) Western blot analysis of LEF-1 isoforms in B cells from 7 CLL samples; T cells from a healthy donor and Colo 320 cells served as a control for LEF-1 isoforms. β-Actin shown as a loading control of samples.
