CD38 Expression in B-CLL Is Dynamic and Changes Due to Contact with Activating Stimuli Found within the Leukaemic Microenvironment.

Abstract

High levels of CD38 expression in B-cell chronic lymphocytic leukaemia (B-CLL) confer a poor prognosis. Although its role in B-CLL is unknown, signalling through CD38 has been implicated in cell survival, trafficking and proliferation. Since proliferation in B-CLL is thought to take place within both bone marrow (BM) and secondary lymphoid tissue, we investigated whether CD38 expression might vary in response to stimuli that occur in these tissue compartments.Firstly, we compared the percentage CD38 expression of CD5/19 cells on 35 paired PB and BM aspirate B-CLL samples. The mean CD38% was significantly higher in BM than PB in all samples (27% vs 19%, p=0.009) including samples with a PB CD38 of 7% or more (33% vs 42%, p=0.047), indicating that factors present in the BM up regulate CD38 expression. Next, CD38 expression and cell division of B-CLL peripheral blood mononuclear cells (PBMCs) were examined in an in vitro system aimed at mimicking the proliferation centre microenvironment where leukaemic cells are situated in close proximity to activated T lymphocytes. Positively selected T cells from 15 B-CLL patients were activated overnight with CD3/28 beads and subsequently cultured with autologous B-CLL PBMCs. Both the percentage of CD19+ CD38+ cells (29.9% vs 59.9%, p=0.003) and CD38 mean fluorescence intensity (75.1 vs 830.8, p=0.005) increased over the 6 day culture period. B-CLL cell division was assessed using the dye carboxyfluorescein diacetate succinimidyl ester (CFSE) in the same co-culture system. This showed that co-culture with autologous activated T-cells can result in B-CLL cell division, and is preceded by CD38 up regulation. In addition, significantly more B-CLL cells underwent at least one division from patients with an initial CD38 level of 7% or more, as compared to under 7% (24.6% vs 10.9%, p=0.031). To further investigate the relationship between B-CLL cell proliferation, CD38 expression and the role of T-cells we examined tissue sections known to contain paraimmunoblasts and other proliferating B-CLL cells. Four colour confocal microscopy using CD3, Ki67, CD38 and CD23 to label frozen B-CLL lymph nodes was employed. Large Ki67+ CD23+ cells were present in close proximity to CD3+ T-cells and these large B-CLL cells had higher CD38 expression than the surrounding small B-CLL lymphocytes.These results support the proposal that CD38 expression in B-CLL is dynamic and may reflect exposure to T-cell derived stimuli which contribute to proliferation in the BM or LN microenvironment. A possible explanation for the poorer prognosis of patients with higher CD38 expression may be that their disease has more proliferative potential.