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

Figure 3. IgD BCR expression requires Pten.

Figure 3

  • A, B
    Splenocytes from mice of the indicated genotypes, (A) Pten f/f, Pten f/f × mb1‐cre, and Pten f/f × mb1‐cre 3‐83 ki mice on the respective backgrounds, (B) Pten f/f × MD4 tg and Pten f/f × mb1‐cre × MD4 tg mice in presence and absence of antigen (ML5 tg), were analyzed by flow cytometry. B cells were pre‐gated by B220 and CD19 expression, and the IgM/IgD surface expression (dot plots, left) was determined. Histograms (right) show a comparison of intracellular IgD expression (Ig‐δHC ic) between the splenic populations of B cells (orange, identified by B220 and CD19 expression) and non‐B cells (gray). Numbers in the histograms indicate the percentages of the positive populations. Data are representative of 11–35 individual mice per genotype.
  • C
    Quantification of surface IgD mean fluorescence intensity (MFI) in Pten f/f × MD4‐transgenic mice in the presence (mb1‐cre +/+) and absence (mb1‐cre +/ki) of Pten. Single dots represent the average MFI of four independent measurements per mouse (n), mean ± SD. Statistical significance was calculated by using an unpaired two‐tailed t‐test.
  • D
    Serum anti‐HEL IgM titers. Sera from MD4 tg mice of the indicated genotypes were adjusted to an IgM concentration of 500 μg/ml and applied in triplicates to HEL‐coated plates in dilution steps of 1:3, mean ± SEM. Statistical significance was calculated by using the Kruskal–Wallis test (see also Appendix Table S4). Statements of significance in the figure refer to the comparison between Pten f/f × MD4 tg (filled red squares) and Pten f/f × MD4 tg × MD5 tg sera (filled orange triangles), **P ≤ 0.01; ***P ≤ 0.001.
  • E
    Immunohistochemistry of sections from spleens of Pten f/f, Pten f/ × mb1‐cre, Pten f/f × mb1‐cre × 3‐83 ki , and Pten f/f × mb1‐cre × MD4 tg mice for CD169 (green), CD5 (red), and IgM (cyan) at 10× magnification. Pictures in the second row show enlarged areas indicated by the white squares, respectively. Double arrows indicate the distance between T cell (red) and marginal zone (green). Shown pictures are representative of 2–3 mice per genotype (see also Appendix Fig S2).