Multicolor flow analysis of B-cell compartments in spleen, blood, BM, and gut. Splenocytes, blood, BM, or intestinal tissue cells (gut) were stained with flow panels 1 or 2 (supplemental Tables 2 and 3), respectively. (A) Gating strategy for 10-color panel “Stain 1” flow cytometric analysis of donor D215 splenocytes to identify CD27^– (referred to as “naive B cells”) and CD27⁺ (referred to as “MBC,” acknowledging the generally accepted but imprecise convention^43,44 ) and their isotype distribution. Transitional B cells were identified as CD10^hi CD38^hi B cells. AFCs of the plasmablast/plasma cell lineage were identified in a broader forward scatter/side scatter (FSC/SSC) gate as CD38^brightCD138⁺ in the right panel. The latter expressed CD27 and displayed a range of CD19 expression, with most cells being positive, consistent with a plasmablast identity rather than a fully differentiated plasma cell⁴⁵  (bottom right panel). MBCs showed clear surface IgM⁺ and IgG⁺ populations, which were less clear among these AFCs, likely due to reduced levels of surface immunoglobulin expression. Arrows indicate subsequent gating of populations, and numbers next to outlined areas indicate percentages of parent gate cells in gated populations. Key gated populations are labeled. (B) Distribution of B- cell compartments across human tissues (for gut data, after careful removal of mucosal epithelia and Peyer’s patches individually isolated cells from jejunum, ileum, and colon were analyzed and compiled, as these showed no significant differences among each other). The graphs show the percentages for CD27^–, CD27⁺, and transitional B cells of CD19⁺ live singlet lymphocytes (top panel), the percentages for total CD19⁺ lymphocytes and AFCs as percentage of all live cells cross indicated tissues (middle panel) and GC cells, which were gated as CD38^int or CD38^hi cells of all live CD19⁺ IgD^– CD10⁺ cells across tissues (for gating, see supplemental Figure 1). The blue numbers in the graphs represent the mean of the depicted population, also shown as purple lines in the graph. (C) Surface immunoglobulin isotype distribution of human organ donor samples. Upper panel: percentage of switched (IgG⁺; blue) and unswitched (IgM⁺ IgD⁺; red) CD27^– and CD27⁺ cells of CD19⁺ singlets with mean and standard deviation. Lower panel: graphing of data in upper panel with lines linking data from same samples. (D) “Inferred” IgA/IgE frequencies across tissues of CD27⁺ cells of CD19⁺ singlets with mean and standard deviation plotted as difference from 100% using the measured IgG and IgM/IgD frequencies depicted in panel C. (E) Correlation analysis between blood and spleen of CD27⁺, AFCs, and IgM⁺ IgD⁺ B-cell compartments within the same donors. P values were calculated as paired two-tailed Student t test (B-C) or linear regression (E).