Figure 5. Impaired Ig CSR in Mule-deficient B cells. (A) Purified splenic B cells from mice of the indicated genotypes were stimulated with either LPS (left) or LPS plus IL-4 (right) for 3–5 d and analyzed by flow cytometry to detect IgG3+ class-switched B cells (left) and IgG1+ class-switched B cells (right). Numbers are the percentage of class-switched B cells among gated B cells. Results are representative of three independent experiments involving two to five mice per genotype. (B) Purified splenic B cells were labeled with CFSE and stimulated for 4 d with LPS plus IL-4. Cell divisions tracked by CFSE were ana- lyzed by flow cytometry, with each peak indicating one cell division. Results depicted are representative cell division profiles of littermate control B cells (left), BMKO B cells (middle), and the overlay of control cells over BMKO cells (right). Results from A and B are representative of two independent experiments involving two to three mice per genotype. a Ti-neutralizing IgM response by days 2–4, followed by a Td IgG response by days 4–6 (Hangartner et al., 2006). In control mice, total VSV-neutralizing Abs (IgM + IgG) ap- peared by day 2 after infection, peaked on day 4, and re- mained high until day 12 (Fig. 4 D, left). In contrast, total anti-VSV Abs in BMKO mice were undetectable on day 2 and were only 1/8 of control titers on day 4 after infec- tion. However, by day 6, levels of total anti-VSV Abs in the mutant had caught up to the control, indicating that Mule- deficient B cells can mount an IgM response but with delayed kinetics. When we specifically examined VSV-neutralizing IgG, control mice first exhibited these Abs on day 6 after infection, and their levels continually increased until day 12 after infection (Fig. 4 D, right). However, in BMKO mice, anti-VSV IgG levels were only 1/5 of those in the control on day 6 after infection and failed to achieve control levels even by day 12. In vitro, B cells undergo class switch recombination (CSR) and differentiate into either IgG3+ plasmablasts in response to LPS or into IgG1+ plasmablasts in response to LPS plus IL-4 (Stavnezer et al., 2008). To determine whether Mule affects CSR, we cultured naive BMKO and control B cells with LPS, or LPS plus IL-4, and measured the percentages of B cells producing IgG3 or IgG1. In both cases, Mule-deficient B cells produced 50% less of the expected isotype than controls (Fig. 5 A). CSR re- quires that a minimum of two rounds of B cell division be completed to allow IgG production (Hodgkin et al., 1996). To assess whether the CSR defect in BMKO B cells was associated with impaired proliferation, we labeled BMKO and control B cells with CFSE, activated them with LPS plus IL-4, and followed their cell division by flow cytometry. Compared with control B cells, BMKO B cells underwent one cell division less (Fig. 5 B). Thus, Mule is required for proper B cell proliferation and CSR in vitro, as well as normal Ti and Td humoral responses in vivo. 178 Mule maintains cellular homeostasis under steady-state conditions by controlling p53 activity We next examined the effect of Mule deletion on the func- tion of p53, a known Mule substrate and a key proapoptotic molecule involved in maintaining homeostasis (Chen et al., 2005; Murray-Zmijewski et al., 2008).To aid in these analyses and examine additional cell types, we created Mule-deficient embryonic stem (ES) cell lines and E1A/Ras-transformed Mule-deficient MEFs via Cre-mediated deletion of Mulefl/y (Peitz et al., 2002). Immunoblotting showed that, compared with controls, p53 protein was clearly elevated in both Mule-deficient ES cells and MEFs at steady state (Fig. 6 A). Although p53 protein was not detectable by immunoblotting in either BMKO or control B cells at steady state (Fig. 6 A), quantitative real-time PCR analysis showed that mRNA levels of its proapoptotic target genes Bax, Mdm-2, Noxa, p21, and Puma (Villunger et al., 2003; Green and Kroemer, 2009), including p53, were all significantly higher in resting Mule- deficient B cells than in control B cells (Fig. 6 B).These data are consistent with our earlier observation of increased apop- tosis in B cells isolated from unstressed BMKO mice (Fig. 2 D). Thus, loss of Mule has profound effects on p53 transcriptional activity at steady state. To assess whether the increased p53 transactivation in BMKO B cells accounted for their defects, we crossed our BMKO mice to floxed p53 (p53fl/fl ) mice (Jonkers et al., 2001) to generate compound mutants lacking both Mule and p53 specifically in B cells (B cell–specific double KO [BDKO] mice). Quantitative real-time PCR analysis showed that the abnormal elevations of Bax, Mdm-2, Noxa, and Puma mRNAs (but not p21 mRNA) in BMKO B cells were abrogated upon genetic ablation of p53 (Fig. 6 B). These results suggest that the increase in Bax, Mdm-2, Noxa, and Puma mRNAs in BMKO B cells is p53 dependent. Flow cytometric analysis showed that both the block in BM B cell development at the pre–B cell stage and the defect E3 ligase Mule is essential for B cell homeostasis | Hao et al.