IL4 and CD40L Prevent Apoptosis of Chronic Lymphocytic Leukemia Cells Via Intracellular pSTAT6 and NFkB Signaling and Not Via Receptor Kinetics

Abstract 3893

Chronic lymphocytic leukemia (CLL) cells are highly dependent on microenvironmental input for their extended survival in vivo, but the underlying molecular mechanism is still unclear. Compared to non-malignant B-cells, CLL cells are more responsive to contact dependent complex stimuli like coculture on bone marrow derived stromal cell lines of both human (p<0.0001) and murine origin (p<0.01), but also to soluble factors (human conditioned medium p<0.0001, murine conditioned medium p<0.001, all student′s t-test). In order to understand the intrinsic difference of the anti-apoptotic phenotype of CLL cells, the signalling circuitry of the malignant cells was modelled. Compared to candidate ligands like SDF-1 (at concentrations between 10–1000ng/ml), BAFF (250–1000ng/ml), APRIL (250–1000ng/ml) and soluble anti-IgM (1–25μg/ml), the factors CD40L (10–2000ng/ml) and IL4 (0.1–10ng/ml) were the most efficient ligands in rescuing CLL cells from spontaneous death in vitro. The dose response of IL4 and CD40L displayed different saturation and cooperativity between CLL cells and non-malignant B-cells. Using IL4, saturation was reached both for CLL cells and B-cells at 0.2pM, but at 52% survival (+/− 8%) for CLL cells and 28% (+/−7%) for B-cells, and the estimated dissociation constant Kd was 0.01pM for both ligands. For CD40L, CLL cell survival reached saturation at 40nM, while no saturation was reached for B-cells. Intriguingly, B-cells showed cooperativity in their response to CD40L, with a cooperativity coefficient of 2.0 and a Kd of 70pM, while cooperativity for CD40L was lost in CLL cells (Kd of only 2.6pM). This pointed towards distinct differences in ligand-receptor interactions or in downstream signaling between CLL cells and non-malignant B-cells. However, high-throughput spatial analysis with a microscope-coupled cytometer did not show differences of receptor quantity or receptor distribution between malignant and non-malignant cells. In contrast, quantity and phosphorylation levels of downstream signalling nodes like STAT6 (measured by flow cytometry and validated by Western-blot) and the activity of NF-kB (p65 binding to DNA measured by oligonucleotide-coupled ELISA) were higher in CLL cells compared to B-cells from healthy donors. Therefore, the defect in IL4 and CD40L signalling that leads to an enhanced survival in CLL cells is likely caused by changes in the intracellular circuitry.