Gating strategies to identify peripheral blood B-cell subsets. Gating on lymphocytes was done according to forward scatter (FSC) vs side scatter area (SSC-A) features (upper left). Cell doublets were excluded by analyzing FSC area (FSC-A) vs FSC width (FSC-W) (upper right). In the next step, gating on CD20⁺ B cells was applied (middle left). CD27 vs CD23 staining was used to separate CD27⁺CD23⁻ memory B cells (A) from CD23⁺CD27⁻ naive, transitional, and mature CD5⁺ B cells (B), and from CD27⁻CD23⁻ memory B cells (middle right). CD27⁺CD23⁻ memory B cells were further subdivided into IgM-negative class-switched B cells (CSM) and IgM⁺ non–class-switched memory B cells (NCSM) (lower left). CD23⁺CD27⁻ B cells were further separated into CD5⁻CD38⁻ naive B cells, CD5⁺CD38⁺ transitional B cells, and CD5⁺CD38⁻ mature CD5⁺ B cells (lower right). MACS, magnetic-activated cell sorting.