Article Title: Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow
Figure Lengend Snippet: 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 BM memory B cells. C57BL/6 mice were immunized with NP-KLH/LPS SC. Numbers of NP-binding+ IgG2b+ cells in Spleen, BM, blood, and peripheral lymph nodes (pLN) were determined by flow-cytometry on d421 or d426 post immunization; data pooled from 2 independent experiments. OVA ctrl indicates staining controls from mice immunized with the irrelevant antigen ovalbumin (OVA). Gated for IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes. Lines connect samples from one individual, p value of paired t test for spleen and BM samples is indicated. 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. Indicated are the frequencies of Ki-67+ cells within the subset indicated, data in right graph from 2 independent experiments using pooled cells from 4-20 C57BL/6 mice, p value (paired t test) as indicated. c) Flow-cytometric quantification of CD19+ B cells and IgG2b+ memory B cells in mice treated with Cyclophosphamide (CyP) or untreated controls (PBS) after immunization with 3x NP-CGG/IFA. Anaylsis was performed after 7 days of CyP. IgG2b+ B cells were quantified as IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes, CD19+ B cells as CD19+CD138-PI-lymphocytes, p value (Welch’s test). Representative data shown for one out of two independent experiments. d) IgG2b+ B memory cells (Ki-67-IgD-Blimp1-GFP-) are dispersed as single cells throughout the bone marrow. Arrows indicate IgG2b+DAPI+ cells. Scale bar: 20µm. e) Co-localization of IgG2b+GFP-IgD-IgG2b+ cells (arrows) with mesenchymal stromal cells. Arrows indicate IgG2b+DAPI+ cells. Representative micrograph. Scale bars: 10µm. f) Co-localization of IgG2b+ cells to mesenchymal stromal cells. Graph shows frequency of IgG2b+ cells in direct contact (black) or within 10mm (grey) of a cell stained for the molecule indicated. 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 are representative of three biological replicates.
Article Snippet: Antibodies against following murine antigens were used: Ki-67 (B56, BD Biosciences), CD11c (N418, DRFZ), CD19 (1D3, DRFZ and 6D5, BioLegend), CD38 (90, BioLegend), CD138 (281-2, BD Biosciences), GL7 (GL7, DRFZ), IgA (C10-3, BD Biosciences), IgD (11.26c, DRFZ), IgG1 (A85-1, BD Biosciences and X-56, Miltenyi), IgG2a/b (R2-40, BD Biosciences and X-57, Miltenyi), IgG2b (A95-1, BD Biosciences and MRG2b-85, BioLegend), IgM (M41, DRFZ and RMM-1, BioLegend), CD93 (AA4.1, BioLegend and DRFZ), CD21/35 (7G6, DRFZ and 7E9, BioLegend), CD23 (B3B4, BioLegend), CD29 (HMß1-1, Miltenyi), CD49d (R1-2, BioLegend), CD49f (GoH3, eBioscience), CD62L (MEL-14, DRFZ).
Techniques: Mouse Assay, Binding Assay, Flow Cytometry, Staining, Expressing, Immunofluorescence