Article Figures & Data
Figures
- FIGURE 1.
Splenic white pulp is disorganized in the absence of Nkx2-3. Spleen cryosections were stained with anti-B220 (red) and anti-CD3 (blue) (A and B), anti-IgM (red) and anti-IgD (blue) (C and D), anti-B220 (green) and anti-CD35 (red) (E and F), or FDC-M2 (red) (G and H). Cryosections of mesenteric lymph node were also stained with FDC-M2 (red) (I and J). MZs are indicated by arrows (C). Stainings shown are representative of at least five mice of each genotype.
- FIGURE 2.
Chemokine genes are expressed in the spleens of Nkx2-3−/− mice. Total RNA, extracted from pooled spleens of each genotype, was separated by electrophoresis, transferred to a nylon membrane, and probed with labeled cDNAs corresponding to the indicated chemokines. Membranes were stripped and rehybridized with GAPDH to assess RNA loading. Each lane is an independently prepared RNA sample from a unique tissue pool.
- FIGURE 3.
Differential blood cell counts. Peripheral blood from 20 control and 13 Nkx2-3−/− mice was examined. The number of all cell types was significantly elevated in knockouts compared with littermate controls (∗∗, p < 0.001), although the relative ratios remained normal.
- FIGURE 4.
B cell distribution is altered in Nkx2-3−/− mice. Two-color flow cytometric analysis of bone marrow (A), mesenteric lymph node (B), and peritoneal cavity (PerC) (C) of control and knockout mice. Abs used are indicated. D, Three-color analysis of B cell subsets in spleen. Splenocytes were partitioned according to CD23 expression and the distribution of CD21 and IgM assessed on the positive and negative subsets. Transitional 1 (T1) and MZ B cells are boxed with the percentages of gated cells given. Percentages are the average of three mice of each genotype. FACS data are depicted as probability plots with logarithmic scales. E, B cell subpopulation frequencies from control (filled symbols) and knockout (open symbols) mice. Each point is a single mouse. B cell frequencies in PerC are a percentage of recovered lymphocytes as defined by light scatter. Significant differences are indicated (∗, p < 0.01; ∗∗, p < 0.001).
- FIGURE 5.
B cell developmental defects in Nkx2-3−/− mice are host dependent. Flow cytometry was used to assess the frequency of B cell subsets in lymphoid tissues of bone marrow-reconstituted mice. A, Representative example of bone marrow is shown using the Abs indicated. FACS data are depicted as probability plots with logarithmic scales. B, The result of analyzing three mice of each donor-recipient pairing is shown. Only two data points are present for the spleens of Nkx2-3−/− recipients because one mouse in each group was asplenic. Architecture of the spleens of bone marrow-engrafted recipients was examined by staining cryosections with anti-B220 (red) and anti-CD3 (blue) (C–F), anti-IgM (red) and anti-IgD (blue) (G–J), and FDC-M2 (red) and anti-B220 (green) (K–N). G and H, MZ indicated by arrows.
- FIGURE 6.
Nkx2-3−/− mice show an abnormal response to TD but not TI Ags. A, Induction of Ag-specific serum IgM in knockout and control mice at the indicated time points after immunization with DNP-dextran. OD of 405 nM for the 1/10,000 dilution of serum is plotted for each sample at each time point. Filled and open symbols are from control and knockout mice, respectively. B, Serum titers taken at the indicated times after primary immunization with NP-KLH. Titers of total and high-affinity Ag-specific IgG1 are indicated in arbitrary units. Analysis of serum from pre-bleeds revealed no NP-binding IgG1 in all mice. Filled and open symbols are from control and knockout mice, respectively. C–J, Cryosections of spleens from immunized mutant and control mice, taken at day 7 (C–F) or day 14 (G–J) of the response, were stained for IgG1 AFCs with a polyclonal anti-IgG1 serum (C and D), GCs using anti-B220 (blue), PNA (green), and FDC-M2 (red) (E and F), and PNA (blue) and B220 (red) (G–J) with GCs indicated by arrows. Sections from at least three mice of each genotype were examined with identical results.
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