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. 2016 Jun 14;7:11889. doi: 10.1038/ncomms11889

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(a) Murine Nkx2-3 expression measured by quantitative RT–PCR in haematopoietic cell subpopulations isolated from healthy C57BL/6 mice. Haematopoietic stem cells (HSC, lin^negSca-1⁺c-Kit⁺), pro-B (c-Kit⁺B220^low) and pre-B cells (CD25⁺B220^low) isolated from bone marrow; mature B cells (IgM⁺B220⁺) isolated from peripheral blood; B220⁺CD21^highCD23^low marginal-zone and B220⁺CD21^intCD23^high follicular B cells isolated from the spleen. Three to six mice were used in each experiment. Error bars represent standard deviation (s.d.). (b) Representative flow cytometry plots showing different B-cell subpopulations in 8-month-old WT and Nkx2-3^−/− mice (KO). (c) Sequential lymphocyte cell-count in peripheral blood from WT and TG mice at 4, 12 and 18 months (n=10 each). Error bars represent standard deviation (s.d.). Significance level of changes are indicated (two-tailed Student's t-test: *P<0.05, **P<0.001, ***P<0.0001). (d) Spleen size monitored by ultrasounds measured at 4, 12 and 18 months in WT and TG animals (WT, n=8; L1, n=10; L2, n=12). Spleen images from WT, TG (L1 and L2) and Nkx2-3-deficient (KO) mice. (e) Representative flow cytometry plots showing B- and T-cell subpopulations in PB and spleen from 18-month-old WT and 4-, 12- and 18-month-old Eμ-NKX2-3 mice. (f) Immunofluorescence analyses of WT and 12- and 18-month-old TG spleens. Left column: gradual expansion of MAdCAM-1-positive IgM and plasma cells with dissolution of the follicular architecture in ageing Eμ-NKX2-3 mice: in WT mice (top) marginal reticular cells (arrow, green) adjacent to MadCAM-1 sinus-lining cells (red) separate follicular (FoB) and MZ B cells (MZB, blue, IgM) mice. Middle column: Loss of MZ macrophages in ageing Eμ-NKX2-3 mice: in WT mice (top) two concentric layers of MZ macrophages consist of an expanded rim of MARCO-positive (red) MZ macrophages encircling sialoadhesin-positive metallophilic macrophages (green) intermingled with IgM-positive MZ B cells (blue; Fo). Right column: dissolution of follicular stromal architecture in ageing Eμ-NKX2-3 mice: in WT mice (top) CR1/2 receptors (green) mark FDC reticula (arrow) and outline several layers of marginal zone B cells (arrowhead) outside the MadCAM-1 rim of marginal sinus-lining cells (red; Fo, follicle; MZ, marginal zone). m, month.