Identification of 2 subsets of human transitional B cells. (A-B) Peripheral blood mononuclear cells (PBMCs) were stained with mAbs against CD20, CD24, CD38, and CD10. (A) Left panel: Expression of CD24 and CD38 on CD20⁺ B cells; transitional B cells were initially defined as CD24^hiCD38^hi cells. Right panel: Expression of CD10 on CD24^hiCD38^hi B cells. (B) PBMCs were stained with mAbs specific for CD20, CD27, and CD10 to identify transitional (CD20⁺CD27⁻CD10⁺), naive (CD20⁺CD27⁻CD10⁻), and memory (CD20⁺CD27⁺CD10⁻) B cells (left panel). The transitional B-cell population was further divided into CD21^lo and CD21^hi subsets (right panel). (C) MNCs from CB, PB, and spleen were stained with mAbs against CD10, CD20, CD27, and CD21. Expression of BAFF-R, CD23 CD44, IgD, and CD10 on CD21^lo (gray line) and CD21^hi (black line) transitional B-cell subsets was determined by flow cytometry. The corresponding isotype control is represented by the light gray filled and dark gray filled histograms for CD21^lo and CD21^hi transitional B cells, respectively. Note that, for the depiction of the CD10 histograms, the solid gray overlay represents the fluorescence of all lymphocytes after labeling with an isotype control mAb. The plots are representative of more than 5 experiments using cells from different donors. (D) The frequencies of total transitional B cells as well as CD21^lo and CD21^hi subsets in PB (n = 35), spleen (n = 7), and CB (n = 6). The results are expressed (left to right) as percentage of CD20⁺ B cells, and percentage of CD20⁺CD27⁻CD10⁺ transitional B cells. (E) PB B cells were labeled with mAbs against CD10, CD21, and CD27, together with R123, and then incubated at 37°C. Samples were collected at various times and analyzed for extrusion of R123. Left panel: Representative data for R123 extrusion after 60 minutes. Right panel: Kinetics of extrusion of R123 (expressed as the percentage of R123⁻ cells; mean ± SEM, n = 4). (F) The replication history of each subset was determined by KREC analysis.^30,31  This was performed on B-cell subsets isolated from 4 unrelated donors; each symbol (●, ■, ★, ▲) represents an individual donor, whereas the horizontal line represents the mean. **P < .01. ***P < .001.