CpG ODN uses autocrine IL-10 to provoke STAT1 tyrosine phosphorylation and apoptosis in B-CLL cells. (A) IL-10 and TNF-α production at 72 hours by CpG 2006-stimulated B-CLL cells with or without CAPE or AG490 pretreatment. Data are the mean ± SD from 5 independent experiments. *P < .01, IL-10 production by CpG 2006-stimulated B-CLL cells with versus without CAPE or AG 490 pretreatments. (B) Viable B-CLL cell number in day 5 cultures with or without CpG 2006 and in the presence or absence of anti–IL-10 or anti–TNF-α Abs. Data are the mean ± SD from 5 independent experiments. *P < .01, the viable B-CLL cell number of anti–IL-10 Ab group versus the CpG 2006 only group. (C) Western blot time course of tyrosine-phosphorylated or serine-phosphorylated STAT1 expression in CpG 2006-stimulated B-CLL cells in the presence or absence of anti–IL-10 or anti–TNF-α Abs. The data shown are representative results from 1 of 2 experiments. (D) Dose effect of IL-10 on B-CLL cell apoptosis in culture at day 5. Data are the mean ± SD from 7 independent experiments with B-CLL cells from 7 individual CLL patients (left panel). The number (%) of viable B-CLL cells in IL-10 (5 ng/mL) cultures with or without anti–IL-10 or anti–TNF-α Abs was determined at day 5. Data are the mean ± SD from 5 independent experiments with B-CLL cells from different CLL patients (right panel). *P < .01, B-CLL cell numbers in IL-10 cultures with versus anti–IL-10 or anti–TNF-α Abs. (E) Western blot of tyrosine-phosphorylated or serine-phosphorylated STAT1 expression in B-CLL cells cultured with or without IL-10 in the presence or absence of anti–IL-10 Abs was determined at 24 hours of culture. The data shown are representative results from 3 of 5 independent experiments. Data from the 5 CLL patients are densitometrically assessed and presented as the mean ± SD in the adjacent bar diagrams. *P < .01, STAT1 expression in B-CLL cells in IL-10 cultures with versus without anti–IL-10 Abs to the media only group. (F) Expression of the indicated surface markers on B-CLL cells cultured with or without CpG 2006 and/or IL-10 was analyzed at day 3, indicated with MFI number, and overlaid with isotype control. Data are representative results from 1 of 3 independent experiments with B-CLL cells derived from different CLL patients. (G) Schematic representation of TLR9-CpG ODN ligation induced apoptotic pathway in B-CLL cells. CpG ODN is recognized by TLR9 and ligated with it in the endosome engaging an intracellular pathway and leading to NF-κB activation and translocation. Binding of activated NF-κB fragments to DNA induces the production of cytokines (eg, IL-10, TNF-α). Extracellular binding of IL-10 with its receptor activates JAK/STAT pathway-dependent tyrosine phosphorylation of STAT proteins, leading to the activation and cleavage of caspase-9, caspase-3, and PARP, and consequent apoptosis of B-CLL cells. CAPE specifically inhibits NF-κB activation. AG490 blocks JAK phosphorylation, thus blocking the JAK/STAT signaling pathway. STAT1 antisense ODN blocks STAT monomer production from mRNA.