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J.R.B. IgG, suggestive of CSR failure (Fig. 1a). Defective CSR was confirmed in immunisation experiments in which serum concentrations of antigen-specific IgG1 in mice were ~10-fold lower than in control mice, while IgM reactions were similar between organizations (Fig. 1b). Similarly, REV7-deletion severely jeopardized (by up to 90%) the production of class switched B splenocytes upon activation in Gepotidacin tradition (Fig. 1c and Extended Data Fig. 1c), without impacting cell proliferation (Fig. 1d). Comparative CSR frequencies in and double knockout cells furthermore confirmed REV7-53BP1 cooperation is essential for CSR (Fig. 1e). Open in a separate window Number 1 REV7 and 53BP1 cooperate during CSR, yet are functionally uncoupled in V(D)J recombination.(A) Serum immunoglobulin in and mouse cohorts; mice per genotype; ideals, unpaired two-tailed t-test. Mean 95% CI. (B) NP-specific serum IgM (left) and IgG (ideal) at indicated instances after NP-CGG immunisation; Representative data, Gepotidacin independent experiments, each with 4 mice. Mean 95% CI. (C) Cell trace violet (CTV)-labelled splenic B cells were stimulated as indicated and stained for surface IgG1 or IgE on Gepotidacin day time 4. Muc1 Representative data, mice. (D) CTV dilution in purified B cells cultured in the presence of LPS and IL-4 for 96 hours. Representative data, mice. (E) Splenic B cells cultured with the indicated stimuli (96 h) and stained for surface IgG1, IgE, IgG2, IgG3. mice per genotype. 100% CSR, imply Ig isotype switch frequency of 2 control animals in each experiment. ideals, two-way ANOVA with Tukeys correction. Mean 95% CI. (F) Complete numbers of B220+ Gepotidacin B cells in the BM (1 femur plus 1 tibia) and spleen. mice per genotype, except ideals, unpaired two-tailed t-test, Mean 95% CI. (G) Complete numbers of B cell precursors (Hardy27 fractions A, B220+CD43+BP-1-CD24-; B, B220+CD43+BP-1-CD24+; C, B220+CD43+BP-1+CD24+; D, B220+CD43-IgM-IgD-; E, B220+CD43-IgM+IgD- ; and F, B220+CD43-IgM+IgD+) in the BM (one femur and one tibia) from mice. ideals, unpaired two-tailed t-test. Mean 95% CI. (H) Top, schematic of the and loci and FISH probes. Bottom, representative metaphase images showing normal and irregular and loci. C, centromere-proximal; T, telomere-proximal. (I) and locus breakage in splenic B cells of indicated mice upon activation (anti-CD40 + IL-4) for 96 h. mice per genotype, between 98 and 151 metaphases were analysed from each mouse and mice (Fig. 1f). While animals (Fig. 1f), leading us to query whether 53BP1-dependent DNA repair activities during B cell development require REV7. Detailed BM analysis showed that Gepotidacin B lymphocytes of mice became gradually depleted, with ~70% deficits in total lymphocytes from the late small pre-B and immature B cell phases (Hardy fractions D and E, respectively; Fig. 1g and Extended Data Fig. 2a). Losses were accompanied by improved apoptosis in BM and follicular (Fo) splenic B cell fractions (Extended Data Fig. 2b-c). In contrast, mice showed normal B cell counts and apoptotic indices (Fig. 1g and Extended Data Fig. 2a-c), despite the complete absence of REV7 protein in B220+ CD43+ pro-B progenitors (Extended data Fig. 2d). To exclude the possibility that developmental problems in mice could be masked by compensatory changes, we generated combined BM chimeric mice. Equal mixes of CD45.1 WT and CD45.2 or CD45.1 WT and CD45.2 whole BM cells were injected intravenously into lethally irradiated CD45.1 WT recipient mice (Extended data Fig. 2e). 8 weeks after BM transfer, the reconstitution of pro, pre, immature.