Figure 3. AIRE Inhibits CSR and SHM in B cells.

(A) The generation and immunization of Aire^+/+ and Aire^−/− BM and B cell chimeric mice.

(B) The percentage of CD45.1⁺ Aire^+/+ and CD45.2⁺ Aire^−/− B cells in the splenic GC B cells of the secondary μMT recipient mice (n = 6) after the immunizations. ***P < 0.001, by 2-tailed paired t-test.

(C and D) Flow cytometric and statistical analyses of the ratio of NP₈- to NP₃₆-binding (C) and NP₈- to total NP-binding (D) GC B cells after immunizations of the secondary μMT recipient mice (n = 6). **P < 0.05, **P < 0.01, by 2-tailed paired t-test.

(E) The generation and immunization of Aire^+/+ and Aire^−/− B cell chimeric mice.

(F) Flow cytometric analysis of surface IgD and IgM on NP₃₆-binding B cells in μMT recipients of Aire^+/+ or Aire^−/− B cells immunized with NP₃₂-KLH. The result represents 3 age- and sex-matched μMT recipients each of B cells from 3–5 age- and sex-matched littermate donor Aire^+/+ or Aire^−/− mice.

(G) The ratios of the titers of circulating NP₄-binding to NP₂₉-binding IgM, IgG1, IgG2b and IgG3 in immunized μMT recipient mice of Aire^+/+ or Aire^−/− B cells. The results represent 4 experiments, each consisting of B cells from 3–5 age- and sex-matched littermate donor mice and 6–8 age- and sex-matched littermate μMT recipient mice. *P < 0.05, **P < 0.01, by 2-tailed unpaired t-test.

(H) The SHM landscape across IgHV, including FR2, CDR2, FR3, CDR3 and FR4, of NP₃₆-binding IgM⁻IgD⁻ or IgM⁺IgD⁺ Aire^+/+ and Aire^−/− donor B cells in μMT recipients after immunizations with NP₃₂-KLH. The result represents 3 μMT recipients of Aire^+/+ donor B cells and 3 μMT recipients of Aire^−/− donor B cells.

(I) Frequencies of C-to-T transitions in SHMs in IgHV of NP-specific IgG⁺, IgA⁺ or IgE⁺ splenic B cells from μMT recipient mice of Aire^+/+ or Aire^−/− B cells after immunizations with NP₃₂-KLH. Data are represented as median ± upper/lower quartile. *P < 0.05, **P < 0.01, by 1-tailed unpaired t-test.

(J) qRT-PCR analysis of the fold induction of Iμ-Cγ1 and Iμ-Cγ3 post-switch transcript levels in peripheral blood IgD⁺CD27⁻ naïve B cells from healthy subjects (n = 5) or APS-1 patients (n = 5) stimulated for 3 d with CD40L and IL-4 or IFN-γ over the respective unstimulated control B cells. *P < 0.05, by 1-tailed unpaired t-test (upper panel) or 1-tailed Mann-Whitney U test (lower panel).