We analyzed the effect of CD40 triggering on the fludarabine-induced apoptosis of B chronic lymphocytic leukemia (B-CLL) cells. Peripheral blood samples obtained from 15 patients were incubated with fludarabine in the absence or the presence of the anti-CD40 monoclonal antibody (MoAb) G28-5. In 12 patients a significant proportion of apoptotic cells, ranging from 22% to 38% (mean +/- SE: 28.5 +/- 1.6), were detected after 3 days of culture. In 9 of these samples, the addition of G28-5 reduced apoptosis by at least 30.1% and by 57.1% +/- 7.8% on average (P = .0077). Because the CD40 antigen activates NF-kappaB/Rel transcription factors in B cells, and NF-kappaB/Rel complexes can inhibit cell apoptosis, we investigated whether the antiapoptotic effect of G28-5, in our system, could be related to modulation of NF-kappaB/Rel activity. As expected, B-CLL cells displayed significant levels of nuclear NF-kappaB/Rel activity; p50, RelA, and c-Rel components of the NF-kappaB/Rel protein family were identified in these complexes. After exposure to fludarabine, NF-kappaB/Rel complexes were decreased in the nuclei. The addition of G28-5 upregulated the NF-kappaB/Rel levels. To determine the involvement of NF-kappaB/Rel activity in the G28-5-mediated inhibition of apoptosis, we blocked the transcription factor with a decoy oligonucleotide, corresponding to the NF-kappaB/Rel consensus sequence. Cells incubated with the anti-CD40 MoAb in the presence of the decoy oligonucleotide but not a control oligonucleotide displayed a complete impairment of the G28-5 antiapoptotic effect, indicating that NF-kappaB/Rel activity was required for the inhibition of apoptosis. These results suggest that CD40 triggering in vivo could counteract the apoptotic effect of fludarabine on B-CLL cells and that its neutralization, or the use of NF-kappaB/Rel inhibitors, could improve the therapeutic effect of fludarabine.