A r t i c l e Figure 6. Restoration of B cell homeo- stasis in BMKO mice by genetic ablation of p53. (A) Lysates of untreated Mulefl/y (control) and Muledel/y (Mule-deficient) MEFs; control and BMKO B cells; and Mulef/y (control E14K) and Muledel/y (Mule deficient) ES cells were immunoblotted to detect total p53 pro- tein. p53/ MEFs, Ab specificity control. -Tub, -tubulin (loading control). MEF and B cell results are representative of two to three independent experiments. ES cell data are from a single experiment involving one con- trol and three Mule-deficient clones. (B) The mRNA expression of the indicated p53 target genes was determined by quantitative real- time PCR in lysates of purified splenic B cells from mice of the indicated genotypes. The relative change in gene expression was calcu- lated using the comparative threshold cycle method (2Ct). Results are the mean ± SD after normalization to 18S or actin mRNA and are representative of two to five independent experiments involving one to five mice per genotype. P-values (*, P < 0.05; **, P < 0.005) are from the Student’s t test of control versus BMKO or BDKO. (C, left) The development of pro–B cells (B220+IgMcKit+; top row) and pre–B cells (B220+IgMCD25+; middle row) in the BM of mice of the indicated genotypes was monitored by flow cytometry. The bottom row shows BrdU uptake by total splenic B cells as determined by flow cytometry. Num- bers are the percentage of gated IgM lym- phocytes (top and middle), or of gated B220+ cells (bottom). (C, right) Absolute numbers of pro–B and pre–B cell cells in the BM of mice of the indicated genotypes were determined as in Fig. 2 C. Results are the mean ± SD (*, P = 0.018, Student’s t test of control versus BMKO) and are representative of two to three independent experiments involving one to two mice per genotype. (D, left) purified splenic B cells from mice of the indicated genotypes were treated with 20 µg/ml anti-IgM or 20 µg/ml anti-IgM plus 1 µg/ml anti-CD40 and [3H]-thymidine incorporation was measured at 24, 48, and 72 h after stimulation. Results are the mean± SD and are representative of two independent experiments involving one to two mice per genotype. (D, right) Splenic cellularity of mice of the indicated genotypes was determined as in Fig. 2 F. Results are the combined mean ± SD (*, P = 0.0073, Student’s t test of control versus BMKO) of four independent experiments each involving one to three mice per genotype. in splenic B cell proliferation disappeared upon genetic ablation of p53 (Fig. 6 C). B cell proliferation in response to anti-IgM or anti-IgM plus anti-CD40 was also restored upon loss of p53 (Fig. 6 D, left), and total B cell numbers in BDKO spleens returned to normal levels (Fig. 6 D, right). Our stud- ies therefore suggest that Mule controls p53 activity essen- tial for B cell development, proliferation, and homeostasis under steady-state conditions. We noted that our BDKO mice still showed signifi- cantly lower levels of serum IgM and IgG1 compared with control mice and B cell–specific p53 KO mice (Fig. 7 A). Consistent with the reduced serum IgG1 in BDKO mice, the impaired LPS-plus-IL-4–induced CSR to IgG1 observed in BMKO B cells was not rescued by genetic ablation of JEM Vol. 209, No. 1 p53 (Fig. 7 B). Indeed, the percentage of class-switched IgG1-producing cells was reduced even further in BDKO B cells, suggesting that both Mule and p53 are indispens- able for CSR to IgG1. In addition, the B1a cells missing from the peritoneal cavity of BMKO mice were not re- stored by loss of p53 (Fig. 7 C). Because B1a cells are a major producer of IgM (Herzenberg, 2000), this finding may account for the reduced serum IgM levels in BMKO and BDKO mice (Fig. 4 A and Fig. 7 A). Notably, deletion of p53 alone in B cells affected neither LPS-plus-IL-4– induced CSR nor B1a cell numbers (Fig. 7, A–C). Thus, the defective CSR, impaired serum Ig production, and lack of B1a cells in BMKO mice are not attributable to increased p53 transactivation. 179