Table 1.
Patient cohort characteristics.
Figure 1.
CpG/c stimulation induces proliferation and plasmacytoid morphology in CLL cells.
A. Detection of proliferation in leukemic B cells from 6 CLL patients by [3H]-thymidine incorporation. (*p = .02; error bars represent standard error of the mean) B. Morphologic analysis of leukemic B cells on days 0, 3, and 5 after CpG/c stimulation as revealed by Wright Giemsa staining.
Figure 2.
CpG/c stimulated CLL B cells secrete light chain restricted Ig.
A. Representative propidium iodide and immunofluorescence staining for cytoplasmic Ig in CpG/c stimulated leukemic B cells. B. ELISA detection of IgM and IgG levels in cell free culture supernatants from 14 patients in unstimulated or CpG/c treated CLL cultures after 5 days. (*p = .02; error bars represent standard error of the mean) C. ELISA detection of kappa and lambda light chain Ig levels in cell free culture supernatants from 7 patients in nil or CpG/c treated CLL cultures after 5 days.
Figure 3.
CpG/c stimulated CLL B cells decrease LEF-1 expression and increase PRDM1 expression.
A. Detection of PRDM1 (left) and LEF1 (right) mRNA levels by qPCR in 3 CLL patients in nil or CpG/c treated CLL cultures after 5 days. Transcript levels were normalized to 18 s rRNA and relative fold change was calculated using the 2∧−ΔΔCT method. B. Representative western blot analysis of LEF-1 full length and ΔN LEF-1 isoforms in B cells from 3 CLL samples in nil or CpG/c treated CLL cultures after 5 days of culture; β-actin shown as a loading control of samples. C. Representative intracellular FACS analysis for LEF-1 in B cells from 2 CLL samples in nil or CpG/c treated CLL cultures after 5 days; Isotype control histogram is shaded grey; LEF-1 histogram is black. D. Inverse correlation between LEF-1 Δ mean fluorescence intensity (ΔMFI) and IgM secretion in 14 CpG/c treated CLL samples (best fit line equation y = −0.3606Ln(x)+4.4207; r2 = 0.505).
Figure 4.
CpG/c stimulated CLL B cells exhibit decreased survival and decreased activation of the Wnt pathway.
A. TCF/LEF dual luciferase reporter assay relative fold change was measured in samples from 3 CLL patients in nil or CpG/c treated cultures after 5 days of stimulation and then 2 days in media alone (**p = .0098; error bars represent standard error of the mean). B. CLL percent survival 48 hours post-ficoll isolation of 5 day CpG/c stimulated or untreated samples from 3 CLL patients (*p = .0039; error bars represent the standard error of the mean).
Figure 5.
Multiple agents are able to induce CLL B cell differentiation and subsequent decrease in LEF-1 expression.
A. ELISA detection of IgM levels in cell free culture supernatants from culture of CLL cells from 6 patients. The CLL cells were either unstimulated, or stimulated with CpG, CpG/c, TPA, interferon-alpha, or activated T cells for 5 days of culture (error bars represent standard error of the mean). B. Intracellular FACS LEF-1 Δ mean fluorescence intensity (ΔMFI) was measured in the same samples (error bars represent standard error of the mean, dashed line represents no increase in anti-LEF-1 staining over isotype control).