TLR9 signaling by CpG-B ODN induces apoptotic death of B-CLL cells. (A) B-CLL cells were cultured in media with or without CpG 2216 or CpG 2006 for 9 days. Annexin V/PI-positive, TMRE-negative apoptotic (R1 gate) and annexin V/PI-negative, TMRE-positive viable (R2 gate) B-CLL cells in cultures were determined at the indicated time points. Flow cytometry data shown are representative for day 7 culture with indicated number (%) of viable B-CLL cells. Kinetic changes of viable B-CLL cell number in cultures are summarized in the adjacent bar graph. Data are the mean ± SD from 5 independent experiments with B-CLL cells from different CLL patients. *P < .01, viable B-CLL cells in media with versus without CpG ODN at each time point. (B[b]) B-CLL cells were cultured in media with or without the indicated CpG ODNs. Kinetic changes of annexin V/PI-negative, TMRE-positive B-CLL cells in cultures were determined. Data are representative results from 1 of 5 independent experiments with B-CLL cells from different CLL patients. The number (%) of viable B-CLL cells is indicated. (C) Representative results showing the addition of CpG-B ODNs to unpurified patient PBMCs containing different percentages (96%, 79%, or 45%) of B-CLL cells. (D) Data are results of day 5 cultures from independent experiments with purified B-CLL cells from the 23 CLL patients listed in Table 1. Each dot represents an individual patient, and the horizontal bar represents the median level. *P < .01, B-CLL cell numbers in cultures with versus without CpG ODNs. Annexin V/PI staining and TMRE staining were concurrently performed to determine the number of viable B-CLL cells in cultures with B-CLL cells from 10 CLL patients, and the results were comparable (data not shown).