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+CD38loGL7+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.