Dynamic variation of CD5 surface expression levels within individual chronic lymphocytic leukemia clones

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonally derived mature CD5 high B cells; however, the cellular origin of CLL is still unknown. Patients with CLL also harbor variable numbers of CD5 low B cells, but the clonal relationship of these cells to the bulk disease is unknown and can have important implications for monitoring, treating, and understanding the biology of CLL. Here, we use B-cell receptors (BCRs) as molecular barcodes to first show by single-cell BCR sequencing that the great majority of CD5 low B cells in the blood of CLL patients are clonally related to CD5 high CLL B cells. We investigate whether CD5 state switching was likely to occur continuously as a common event or as a rare event in CLL by tracking somatic BCR mutations in bulk CLL B cells and using them to reconstruct the phylogenetic relationships and evolutionary history of the CLL in four patients. Using statistical methods, we show that there is no parsimonious route from a single or low number of CD5 low switch events to the CD5 high population, but rather, large-scale and/or dynamic switching between these CD5 states is the most likely explanation. The overlapping BCR repertoires between CD5 high and CD5 low cells from CLL patient peripheral blood reveal that CLL exists in a continuum of CD5 expression. The major proportion of CD5 low B cells in patients are leukemic, thus identifying CD5 low B cells as an important component of CLL, with implications for CLL pathogenesis, clinical monitoring, and the development of anti-CD5-directed therapies.