Effect of rituximab on freshly isolated B-CLL cells.
(A) First, 2 × 106/mL freshly isolated B-CLL cells were cultured for 24 hours in the presence (right panel) or absence (left panel) of 4 μg/mL rituximab and 20 μg/mL mouse anti–human IgG1 F(ab)2 fragment. Cytospin slides were prepared from 2 × 105 cells and stained with May-Grünwald-Giemsa (top panels). Apoptotic cells are indicated by arrows. Original magnification × 100. For quantification of the degree of apoptosis, isolated nuclei from 5 × 105cells from the same experiment were lysed, stained with PI, and analyzed for DNA content on a FACSort flow cytometer. Several condensed and fragmented apoptotic nuclei appear in the cells cultured in the presence of rituximab (top, right panel); concomitantly, apoptotic nuclei appear within the hypodiploid region (M1) of the DNA histogram (bottom, right panel). (B) Freshly isolated B-CLL cells from 7 patients were cultured for 24 hours in triplicate with the indicated concentrations of rituximab and secondary antibody in 5-fold excess. Following harvest and washing, the cells were subsequently analyzed for DNA content as above. Dots and squares indicate the mean percentages of apoptotic cells at the indicated concentrations; error bars indicate the estimated 95% confidence intervals of the means. (C) Freshly isolated B-CLL cells were cultured as described in panel A, for 24 or 48 hours. After harvest, cells were incubated with FITC-conjugated annexin V and counterstained with PI in order to detect early apoptotic and late apoptotic/necrotic cells by flow cytometry. The lower left quadrants of each panel show the viable cells, which exclude PI and are negative for annexin V–FITC binding. The lower right quadrants represent the early apoptotic cells, annexin V–FITC+ and PI−, demonstrating cytoplasmic membrane integrity. The upper right quadrants contain the nonviable, late apoptotic/necrotic cells, positive for annexin V–FITC binding and for PI uptake.
