CD40L expression in freshly purified CLL B cells. (A) Peripheral blood mononuclear cells from patients with CLL (all untreated for at least 4 months) were purified by Ficoll-hypaque centrifugation and depleted of T cells by rosetting with sheep red blood cells. Shown are the results of immunofluorescence flow cytometry in consecutive, unselected cases for which the cells could be stained immediately after purification. In each case, greater than 96% of the cells expressed high levels of CD19, and in the majority greater than 99% of the purified cells expressed CD19. As shown, each histogram except for the first represents the fluorescence intensity after incubation with an irrelevant, isotype-matched control antibody (murine IgG₁ antihuman TcRV_β13, shaded histograms) or with anti-CD40L (open histograms), followed by goat antimouse-FITC. In the first case, the shaded histogram indicates CD3 expression. (B) Peripheral blood mononuclear cells were washed briefly in neutral (pH 7.1) or acidic PBS (pH 4.1) to remove soluble CD40 from the surface39 before exposure to MoAbs for immunofluorescence cytometry. After washing in neutral PBS, cells from each sample were examined with CD19-FITC and CD3-FITC concomitantly with either CD40L-PE (clone 89-76) or with a negative, control PE-conjugated antibody. In each plot, the linear histogram reflects PE fluorescence intensity after exposure to CD40L-PE and the shaded histogram reflects fluorescence intensity after exposure to the control antibody, in each case analyzed among the gated, CD19-FITC–positive cells.