Activation and Proliferation of B Cell Chronic Lymphocytic Leukaemia (B-CLL) Cells through Anti-CD180, Anti-CD40 and IL-4.

Introduction: We have previously shown that Toll-like receptor RP105 (CD180) is heterogeneously expressed on B-CLL cells and that the ligation of CD180 by monoclonal antibodies (mAb) on CD180+ B-CLL cells resulted in delineation of responder and non-responder B-CLL clones [1]. In this study we have examined the role of IL-4 together with CD180 and CD40 as activation signals.

Methods: Blood mononuclear cells were separated from 7 responder B-CLL patients with both mutated and unmutated Ig Vh genes and 11 controls and were cultured for 72 hours in optimum concentrations of anti-CD180 (G28-8) or anti-CD40 mAb or both in presence and absence of 15 ng/ml of IL-4. CD19+ B cells were stained with mAb to the activation marker CD86 or cell cycle protein Ki-67, measured by flow cytometry and expressed as Mean Fluorescence Intensity (MFI) or % of Ki-67+ cells.

Results: B-CLL cells and normal control B cells responded to CD180-ligation by activation and proliferation (Table). Higher levels of CD86 and Ki67 were detected when both anti CD40 mAb and anti CD180 mAb were added (p<0.05) compared with either alone. IL4 alone induced both activation and proliferation of control cells and this was even higher with the leukemic cells (p<0.01) confirming that IL-4 also provides a strong survival/activatory stimulus for B-CLL cells. Addition of IL-4 had no significant enhancing effect on normal B-cells stimulated with both anti-CD180 and anti-CD40, although IL-4 synergised with anti-CD40 in B cell activation (p=0.026) and with CD180 in B cell proliferation (p=0.044).

Conclusion: CD180 had an additive effect with CD40 ligation in activation and proliferation of both B-CLL cells and normal control B cells. IL-4 provides a strong additional stimulus for B-CLL cells.

CD86 and Ki-67 expression by CD19+ cells