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. 2019 Apr 23;38(11):e100249. doi: 10.15252/embj.2018100249

Figure 7. Abnormal germinal center reaction in IgD‐deficient mice.

Figure 7

  1. Schematic overview of the procedure of mouse immunization with 2,4,6‐trinitrophenyl hapten conjugated to ovalbumin (TNP‐Ova) and analysis of germinal center (GC) reactions at different time intervals. Control animals were injected intraperitoneally (i.p.) with PBS and quantified as day 0 of immunization.
  2. Analysis of GC B cells in splenic B220+ B cells from wild‐type (WT, top) and IgD−/− (bottom) animals after 4, 7, and 10 days (left to right) postimmunization. Percentages of follicular B cells (Fo.B; CD38+/CD95−) and GC B cells (CD38−/CD95+) are depicted in the plots. GC B cells (CD38−/CD95+) were further analyzed for GL‐7 expression, compared to Fo.B cells and quantified.
  3. Quantification of Fo.B cells (top, percentages) and CD38−/CD95+/GL‐7+ GC cells (percentages and absolute numbers, bottom panels) in WT (black) and IgD−/− (green) animals after 4, 7, 10, and 14 days postimmunization, mean ± SD (numbers of animals indicated in the top plot). Statistical significance was analyzed by a two‐tailed unpaired t‐test.
  4. Analysis of GC in spleen sections from WT and IgD−/− animals after 4, 7, and 10 days postimmunization. Representative images of 2 × 2 mm sections stained with DAPI (nuclei blue) and peanut agglutinin (PNA red) are shown for the indicated time points. Scale bar represents 500 μm. Dashed white or yellow lines mark distinct GC foci in the merged images.
  5. Representative 800 × 800 μm regions of areas marked by yellow‐dashed lines within the images of D are shown as enlarged single GCs. As no distinct GC foci were observed in IgD−/− samples from days 4 and 7, an identical 800 × 800 μm region is shown in each case.
  6. Quantification of number of GCs per mm2 area of spleen sections from WT (black) and IgD−/− (green) animals after 4, 7, and 10 days postimmunization. Data represent mean ± SD of two sections (from three animals per group and time point) and were analyzed by two‐tailed unpaired t‐test.
  7. Total serum α‐TNP IgG. Sera from PBS‐ or TNP‐Ova‐immunized WT (left) and IgD−/− (middle) mice, collected at day 7 following immunization, were adjusted to an IgG concentration of 1 μg/ml and applied in duplicates and dilution steps of 1:3 to TNP‐coated plates, respectively, mean ± SEM. Right: Overlay of α‐TNP IgG titers from TNP‐Ova‐immunized WT and IgD−/− mice. Statistical significance was calculated by using the Mann–Whitney U‐test (see also Appendix Table S7), n.s. = not significant, *P ≤ 0.05, **P ≤ 0.01.