RB expression correlates with increased signs of activation and selection and is highest among late-stage and/or terminally differentiated peripheral B-cell subsets. (A) RB versus RO dot plots were generated for naive, pre-GC, GC, Pblast, and memory B-cell subsets that collectively represent the spectrum of peripheral B-cell development, ranging from early (naive) to late (memory). The “TB45^BO” system was used to assign RB^−/lo, RB⁺, RO⁻, and RO⁺ (combined RO^+/− and RO⁺) gate limits. The percentage of cells in each fraction is indicated. (B) Mean fluorescence intensities (MFIs) for several additional markers were quantified among RB^−/lo and RB⁺ fractions from each B-cell subset. Markers were partially indicative of selection (CD77, IgM), late-stage development or terminal differentiation (CD27), potential interactions with accessory cells (CD25, CD40), and activation/proliferation (CD69, Ki67). (C) The total GC (IgD⁻CD38⁺) pool was subdivided into RB^−/lo and RB⁺ fractions. Each RB fraction was then applied to a CD38 histogram to characterize the distribution of cells across the CD38 spectrum. RB^−/lo GC B cells exhibited a bell-curve distribution across the CD38 spectrum, showing a strong central tendency (indicated by the bisecting dotted line). In contrast, RB⁺ GC B cells produced a “V-shaped” distribution across the CD38 spectrum and were skewed toward either the CD38⁻ or CD38⁺⁺ gate limits. (D) RB⁺ GC B cells were further split into CD38^LO and CD38^HI fractions in accordance with the dividing gate depicted in panel C. In separate analyses, the percentage of IgM⁺, RO⁺, and CD27⁺ cells were calculated for each fraction. RB⁺CD38^HI GC B cells were relatively more similar to Pblast B cells than memory cells (↑RO, ↑CD27). Conversely, the RB⁺CD38^LO fraction was more consistent with memory B cells than Pblast. (E) Isotype control antibodies for FITC (RB), Alexa Fluor 750 (A750; CD27), and PE analyses (RO).