CD40 triggering enhances fludarabine-induced apoptosis of chronic lymphocytic leukemia B-cells through autocrine release of tumor necrosis factor-α and interferon-γ and tumor necrosis factor receptor-I-II upregulation

Background and Objectives. In chronic lymphocytic leukemia (CLL) B-cells are refractory to activation signals and to apoptosis. CD40 triggering, however, rescues CLL B-cells from their anergic state and upregulates the FAS receptor. We therefore studied whether CD40 triggering enhances CLL B-cell sensitivity to fludarabine, and receptors or cytokines potentially involved in apoptosis. Design and Methods. CD40-activation of CLL B-cells was carried out by co-culture with CD40L-transfected cells. After fludarabine treatment, apoptosis was evaluated by propidium iodide (PI), annexin-V/PI or Di0C6 staining and flow cytometry analysis. Modulation of Bcl-2, of tumor necrosis factor receptor (TNFRI/II) and release of tumor necrosis factor (TNF)α/interferon (IFN)γ was also analyzed. Furthermore, addition of caspaseinhibitors or anti-TNFα/-IFNγ monoclonal antibodies to fludarabine-treated cells allowed us to determine the mediators of apoptosis. Student's t tests or ANOVA variance statistical analysis were performed to evaluate whether any differences observed might be considered significant. Results. CD40 triggering enhanced fludarabine sensitivity of CLL B-cells, downmodulated Bcl-2 and upregulated TNFRI/II. Caspases 1 and 6 were the major caspases involved in fludarabine apoptosis induction in resting B cells, while only anti-TNFα/-IFNγ monoclonal antibodies reduced apoptosis in activated cells. In agreement with this observation, autocrine production of TNFα and IFNγ by CD40-activated CLL B cells was found. Interpretation and Conclusions. B-cells from a considerable proportion of CLL cases studied (11/20) are more prone to fludarabine-induced apoptosis after CD40 triggering; accordingly Bcl-2 expression was lower in activated cells. Moreover, upregulation of TNFRI/II, release of TNFα and IFNγ, and inhibition of apoptosis by anti-TNFα/-IFNγ monoclonal antibodies in CD40-activated cells strongly suggest that these cytokines may play a role in sensitizing B-cells to fludarabine treatment.