Fig. 1: The bone marrow contains a major population of isotype-switched non-proliferating memory B cells.

a Quantification of NP-specific IgG2a/b⁺ spleen, peripheral lymph nodes, blood and bone marrow (BM) memory B cells. Female C57BL/6 mice immunized with NP-KLH/LPS SC. Numbers of NP-binding IgG2b⁺ cells in Spleen, BM, blood, and peripheral lymph nodes (pLN) determined by flow-cytometry on d421 or d426 post immunization; pooled from two independent experiments. OVA ctrl: staining controls from mice immunized with the irrelevant antigen ovalbumin (OVA). Gated for IgG2b⁺CD19⁺CD38⁺CD138⁻GL7⁻CD11c⁻IgM⁻IgD⁻PI⁻ small lymphocytes (cf. Supplementary Fig. 5). Lines connect samples from one individual, paired one-sided t-test for spleen and BM samples. b Flow-cytometric quantification of Ki-67 expression in IgG2b⁺ Bsm (IgG2b⁺CD19⁺CD38⁺CD138⁻GL7⁻CD11c⁻IgM⁻IgD⁻PI⁻ small lymphocytes) splenic naïve (IgM⁺IgD⁺IgG2b⁻CD19⁺CD38⁺CD138⁻GL7⁻CD11c⁻ PI⁻ small lymphocytes) and germinal center (GC) (CD19⁺CD38^loGL7⁺CD11c⁻PI⁻ lymphocytes) B cells. Frequencies of Ki-67⁺ cells within the subset, data in right graph from two independent experiments using pooled cells from 4 to 20 C57BL/6 mice, paired one-sided t-test. c Flow-cytometric quantification of CD19⁺ B cells and IgG2b⁺ memory B cells in female C57BL/6J mice treated with Cyclophosphamide (CyP) or untreated controls (PBS) after immunization with 3× NP-CGG/IFA. Analysis performed after 7 days of CyP. IgG2b⁺ B cells quantified as IgG2b⁺CD19⁺CD38⁺CD138⁻GL7⁻CD11c⁻IgM⁻IgD⁻PI⁻ small lymphocytes, CD19⁺ B cells as CD19⁺CD138⁻PI⁻ lymphocytes (Welch’s test, one-sided). Representative data for one of two independent experiments (n = 5 per group). Boxplot indicates median, first and third quartiles, whiskers: 1.5 IQR. d IgG2b⁺ B memory cells (Ki-67⁻ IgD⁻ Blimp1⁻GFP⁻) are dispersed as single cells throughout the BM. Arrows indicate IgG2b⁺DAPI⁺ cells. Scale bar: 20 µm. Micrograph representative of five slides from two female C57BL/6 mice. e Co-localization of IgG2b⁺GFP⁻IgD⁻IgG2b⁺ cells (arrows) with mesenchymal stromal cells. Arrows indicate IgG2b⁺DAPI⁺ cells. Representative micrograph. Scale bars: 10 µm. f Co-localization of IgG2b⁺ cells to mesenchymal stromal cells. Graph shows frequency of IgG2b⁺ cells in direct contact (black) or within 10 μm (gray) of a cell stained for the respective molecule. g Flow cytometric quantification of surface expression of the VLA-4 and VLA-6 components CD29, CD49d, CD49f in spleen and BM IgG2b⁺ Bsm. Gated for IgG2b⁺CD19⁺CD38⁺CD138⁻GL7⁻CD11c⁻IgM⁻IgD⁻PI⁻ small lymphocytes, histogram plots representative of three biological replicates from C57BL/6 females. Source data for Fig. 1a–c, f are provided as a Source Data file.