B Cell Receptor (BCR) Ligation Enhances Cell Survival in Progressive Chronic Lymphocytic Leukemia (CLL).

Abstract

Recent gene profiling studies demonstrated that signalisation through the BCR plays a central role during CLL disease progression. However, incidence of BCR engagement on cell survival remains unclear as both increased and decreased apoptosis have been reported after BCR ligation by anti-mu antibodies. We investigated the effect of anti-mu exposure on fresh cells from 32 untreated CLL cases. Cell viability was assessed both by MTT assay and Annexin V/ IP labelling. Culture conditions were set after time and dose experiments. A 25% increase of viability, obtained by MTT assay after 72h of culture, was considered as significant. When a coated rabbit anti-human IgH mu specific antibody was used, it significantly prevented apoptosis in 20/32 cases (named responders) and never increased it. On the same CLL cases, soluble goat F(ab')2 anti-human IgH mu specific antibody increased apoptosis in all but one case. However, coating of this latter antibody to the culture plate cancelled the pro-apoptotic effect and restored cell survival promoting effect in 13/14 cases tested. Unspecific effects were ruled out using control antibodies. Cytometry experiments and confocal microscopy demonstrated that uncoated antibody was rapidly internalized and degraded. In responders cases, enhanced survival was supported by increased expression of early cell cycle protein cyclin D2 and cdk4. There was no subsequent cell cycle progression as shown by IP staining and absence of pRB phosphorylation most likely related to the lack of p27 down regulation. There was a strong correlation between the extent of apoptosis prevention after anti-mu exposure and disease progression. Indeed, the 12 over 32 cases who were found to have no IgVH somatic hypermutations, happened to be all responders. Conversely among the 20 mutated cases, 12/20 were non responders and had a clinically stable disease. As for the 8 responders with somatic mutations, four had detectable levels of ZAP 70 protein and 2 others had a clinically progressive disease. In conclusion, coated anti-mu antibody is likely to mimic in vivo conditions of stimulation, suggesting that BCR ligation could play a major role in the pathophysiology of the progressive disease, giving a survival advantage to these CLL cells