Gating schemes for the analysis of human B cells. (A) Cell aggregates and dead cells were further removed from the lymphocyte population, and the resulting live CD19+CD3− cells were selected for analysis. The customarily used IgD/CD27 scheme classifies peripheral blood B cells into four core subsets: naïve and transitional (N + T) IgD+CD27− B cells, unswitched memory (UM) IgD+CD27+ B cells, switched memory (SM) IgD−CD27+ B cells, and double-negative (DN) IgD−CD27− B cells. Rightmost panel: autoreactive 9G4+ B cells concentrate within the naïve compartment. (B) With the additional memory panel specific markers, SM and DN cells both exhibit heterogeneous subpopulations. A great majority of DN cells downregulate the expression of CD24 and CD21, while CD95+ and CXCR3+ cells are more frequently observed in SM cells. (C) MitoTracker Green (MTG) in the transitional panel separates IgD+CD27− N + T cells into MTG− resting naïve cells (rN) and MTG+ fraction. The latter can be further subset into early (T1/T2) transitional B cells, late (T3) transitional B cells and activated naïve (aN) B cells based on the CD24/CD38 expression pattern. A sizable IgM-only memory cells can be identified in the SM subset as well as in the DN subset. (D) Plasma cell panel illustrates that IgD−CD27++CD38++ cells include CD138− plasmablasts (PB) and CD138+ plasma cells (PC); both subsets are highly proliferative in the peripheral blood. The IgD−CD27−/+CD38++ region contains a CD24− fraction that is also highly proliferative and is considered to be a pre-plasmablast subset (Pre-PB). 9G4+ plasmablasts are readily identified from systemic lupus erythematosus patients. FSC, forward scatter; SSC, side scatter.