Expression of BAFF mRNA and protein. (A) Quantitative real-time RT-PCR was performed on RNA samples isolated from the blood mononuclear cells of individual patients with CLL before (left) and after (right) depletion of CD2⁺ and CD14⁺ cells. The lines connect the preisolation and postisolation levels of BAFF mRNA detected in each sample. The amount of BAFF mRNA detected is indicated in arbitrary units. The amount of BAFF mRNA detected in an equivalent number of U937 cells is 1000 units (data not shown). (B) Quantitative real-time RT-PCR measurement of the average amount of BAFF mRNA detected in CD14⁺ cells (n = 4), NLCs (n = 12), purified CLL B cells (n = 12), and isolated CD19⁺ blood B cells of healthy donors (n = 2), as indicated at the bottom of the panel, ± SD (^**the level of BAFF mRNA detected in NLCs was significantly greater than that found in isolated CLL B cells, P < .001). (C) Reconstitution experiments in which small numbers of CD14⁺ blood mononuclear cells are added to 5 × 10⁶ isolated CLL B cells that subsequently were evaluated for BAFF mRNA in 2 representative patients. On the x-axis is the percent of CD14⁺ cells detected by FACS in the reconstituted cell population prior to extraction of RNA. The y-axis indicates the level of BAFF mRNA detected in units as defined in the description for panel A. (D) Representative histograms depicting surface BAFF detected by flow cytometry on CD14⁺ cells, NLCs, CD19⁺ CLL B cells, or CD19⁺ blood B cells of healthy donors, as indicated at the top of each graph. Shaded histograms represent the fluorescence of cells stained with a fluorochrome-labeled anti-BAFF mAb, whereas the open histograms depict the fluorescence of cells stained with an isotype control mAb. (E) An immunofluorescence picture of NLCs and CLL cells stained with fluorescein-labeled anti-CD19 mAb (green) and a PE-labeled anti-BAFF mAb (red). The nuclei are labeled blue with Hoechst 33342.