B cell developmental arrest in Pax5 ^R31Q/− and Pax5 ^R31Q/E242 * mice. (A) Generation of the Pax5^R31Q allele by introducing the R31Q mutation into the Pax5 locus by CRISPR/Cas9-mediated mutagenesis in injected wild-type zygotes (see Materials and methods). The introduced mutation was verified by PCR amplification, cloning, and Sanger sequencing of genomic DNA from a Pax5^R31Q/+ mouse. In addition to the R31Q mutation (red), three silent mutations (blue) were introduced to prevent Cas9 cleavage and to generate an HhaI restriction site (GCGC) for genotyping. (B) Generation of the Pax5^E242* by introducing the E242* mutation into the Pax5 locus, as described in A. In addition to the E242* mutation (red), two silent mutations (blue) were introduced to prevent Cas9 cleavage and to generate an XbaI restriction site (TCTAGA) for genotyping. (C) Flow-cytometric analysis of the indicated B cell types in the bone marrow of 3–4-wk-old Pax5^+/+, Pax5^+/−, Pax5^R31Q/−, and Pax5^R31Q/E242* mice. Numbers refer to the percentage of cells in the indicated gate. Pre-B cells (B220⁺CD19⁺Kit⁻CD2⁺IgM⁻IgD⁻) are defined by the expression of CD2, which is, however, not expressed on Pax5^R31Q/− and Pax5^R31Q/E242* pre-B cells, as Cd2 is a directly activated Pax5 target gene (Revilla-i-Domingo et al., 2012). The B220⁺CD19⁺Kit⁻CD2⁻IgM⁻IgD⁻ B cells were identified as pre-B cells (red), as they expressed the in-frame rearranged Igμ protein in contrast to pro-B cells (blue), as shown to the right. Immature, imm; Rec, recirculating. (D) Loss of all B cells (B220⁺CD19⁺) in the bone marrow of Pax5^E242*^/E242* mice in contrast to Pax5^R31Q/R31Q mice, as shown by flow cytometry. (E) Flow-cytometric analysis of long-lived plasma cells (PCs) in the bone marrow of nonimmunized mice of the indicated genotypes. The different B cell types were defined as shown in C and E and Materials and methods. (F) PTEN expression and AKT phosphorylation at Ser473 in EBV-immortalized B cells of the patient and a representative control, as revealed by immunoblot analysis of whole-cell extracts with the respective antibodies. The expression of β-actin (ACTB) and total AKT protein served as loading controls. One of two experiments is shown. Similar results were obtained with a second independently generated EBV-immortalized B cell culture of the patient and EBV-immortalized B cells of two additional controls. (G) Impaired PI3K-AKT signaling in B cells of the patient. B cells from the blood of the patient and a healthy control were left untreated (0 min) or stimulated for 30 min with anti-IgM before intracellular analysis of AKT phosphorylation at Ser473. One of two experiments is shown. (H) Immunohistological analysis of spleen sections from Pax5^+/+ and Pax5^R31Q/− mice 14 d after NP-KLH immunization. The sections were stained with PNA (red) and antibodies detecting IgD (green) and CD3 (blue). A higher magnification of the region indicated by a white box (left) is shown to the right. FO B, GC B, and T cell zones are indicated. One of three experiments is shown. The scale bar represents 200 μm.