Vol. 203, No. 11, October 30, 2006. Pages 2425–2431.
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We next assessed if these long-lived t(14;18)-bearing B cells in HI already transited through the GCs. Circulating naive and memory B cells can be distinguished on the basis of the CD27 cell surface marker and sIg isotypes (11–13). Fresh PBMCs from 10 healthy donors were collected, and IgD+/CD27− naive, IgD+/CD27+ memory, and IgD−/CD27+ switched memory B cell subsets isolated by cell sorting (Fig. 2 A). The occurrence of t(14;18) was then assessed in total PBMCs and in each fraction by the short-range BCL2/JH PCR assay (Fig. 2 B and Table II). As a first approach, we pooled data from the two CD27+ memory subsets and examined the overall contribution of naive (CD27−) and memory (CD27+) B cells to the total t(14;18)+ frequency calculated as a fraction of CD19+ B cells (Fig. 2 C). t(14;18) frequencies of CD19+ B cells ranged from <1/105 to the unexpectedly high rate of ∼1/3.500 B cells in some individuals (Fig. 2 C, squares). Strikingly, although the level of naive t(14;18)+ cells constantly remained at baseline (Fig. 2 C, circles), CD27+ B cells accounted in a large part for the amplitude of t(14;18) frequencies (Fig. 2 C, triangles). This clearly indicates that circulating t(14;18)+ clones in HI are indeed predominantly B cells which transited through the GCs. To determine if the presence of high levels of t(14;18) in some individuals was caused by a higher incidence of distinct translocations or to the clonal expansion of a given t(14;18)+ B cell, we cloned and sequenced 55 out of 61 BCL2/JH fragments from the CD27+ subset. One major BCL2/JH junction was observed in most individuals (Fig. 2 D, black bars), indicating that only one clone mainly accounted for t(14;18) frequencies. Remarkably, this data demonstrate that the wide modulation of t(14;18) frequency in HI is not caused by the accumulation of clonally unrelated t(14;18) naive B cells in some individuals, but rather to the clonal expansion in the GCs of t(14;18)+ B cells.
