Proliferating CLL cells express high levels of CXCR4 and CD5

Chronic lymphocytic leukemia (CLL) is an incurable progressive malignancy of CD5⁺ B cells with a birth rate between 0.1% and 1% of the entire clone per day. However, the phenotype and functional characteristics of proliferating CLL cells remain incompletely understood. Here, we stained peripheral blood CLL cells for ki67 and DNA content and found that CLL cells in G1-phase have a CXCR4^loCD5^hi phenotype, while CLL cells in S/G2/M-phase express high levels of both CXCR4 and CD5. Induction of proliferation in vitro using CD40L stimulation results in high ki67 levels in CXCR4^loCD5^hi cells with CXCR4 expression increasing as CLL cells progress through S and G2/M-phases, while CXCR4^hiCD5^lo CLL cells remained quiescent. Dye dilution experiments revealed an accumulation of Ki67^hi-divided cells in the CXCR4^hiCD5^hi fraction. In Eµ-TCL1 transgenic mice, the CXCR4^hiCD5^hi fraction expressed high levels of ki67 and was expanded in enlarged spleens of diseased animals. Human peripheral blood CXCR4^hiCD5^hi CLL cells express increased levels of IgM and the chemokine receptors CCR7 and CXCR5 and migrate efficiently toward CCL21. We found higher levels of CXCR4 in patients with progressive disease and the CXCR4^hiCD5^hi fraction was expanded upon clinical relapse. Thus, this study defines the phenotype and functional characteristics of dividing CLL cells identifying a novel subclonal population that underlies CLL pathogenesis and may drive clinical outcomes.