Coordinated events following TLR-9 and CD122 signaling promote NF-kB/AKT/STAT5-dependent B-CLL clonal expansion

Abstract

Abstract B-cell chronic lymphocytic leukemia (B-CLL) clones express BCR specific for microbes and/or apoptotic cells and elevated TLR-9. In patients, B-CLL clonal expansion occurs within lymphoid tissues. We recently reported that CpG DNA (ODN-2006) and IL-15, found within stromal cells of CLL-infiltrated lymphoid tissues, manifest synergy in promoting in vitro B-CLL growth (J. Immunol 195:901–923, 2015). This study explores the mechanism responsible by using purified B-CLL from peripheral blood to assess the temporal requirements for IL-15; effect of ODN on expression of IL-15 receptors (IL-15Rα and CD122 (IL-2/15Rβ)); and IL-15-induced activation of AKT and STAT5. Experiments with CFSE-labeled B-CLL show that IL-15 is critical during a 20–36 h window after ODN priming. Neutralizing mAbs to IL-15 or CD122 fully abrogate IL-15-driven growth when added during this interval. RT-PCR/immunofluorescence studies show that ODN triggers rapid increases in mRNA/protein for IL-15Rα and CD122 that are blocked by IkBα inhibitor. Furthermore, ODN-primed B-CLL exposed to IL-15 manifest elevated pAKT(Ser473) and pSTAT5(Tyr694) levels. Both PI-3K/AKT and STAT5 pathways are functionally relevant, as indicated by growth abrogation by pharmacologic inhibitors, LY294002, pimozide or STAT5 Inh II. Importantly, activated STAT5 levels are high in cycling CFSE-labeled B-CLL; furthermore, extended divisions were blocked by delayed (day 4) administration of STAT5 inhibitors or neutralizing mAbs to IL-15/CD122. Together, findings show that ODN + IL-15-driven growth represents coordinated early functions of TLR-9 and CD122 as well as later CD122 signaling during clonal expansion. This synergy may help drive in vivo growth of B-CLL clones in patients.