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Originally published online as doi:10.1189/jlb.0705402 on February 3, 2006

Published online before print February 3, 2006
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(Journal of Leukocyte Biology. 2006;79:852-859.)
© 2006 by Society for Leukocyte Biology

p53 regulates Btk-dependent B cell proliferation but not differentiation

Nathan W. Schmidt*, Lindsey D. Mayo{dagger}, David B. Donner* and Mark H. Kaplan*,1

* Departments of Pediatrics and Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, and Walther Cancer Institute, Indianapolis, Indiana; and
{dagger} Departments of Radiation Oncology and Pharmacology, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio

1Correspondence: Department of Pediatrics and Microbiology and Immunology, HB Wells Center for Pediatric Research, Indiana University School of Medicine, 702 Barnhill Dr., RI 2600, Room 302, Indianapolis, IN 46202. E-mail: mkaplan2{at}iupui.edu


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ABSTRACT
 
Btk is critical for B cell development and proliferation. Mice lacking Btk have a defect in B cell development, resulting in a loss of mature B cells and decreased proliferative responses following B cell receptor cross-linking. In contrast, mice deficient in the tumor suppressor p53 display increases in developing B cell populations in the bone marrow. To investigate the potential role of p53 in Btk-dependent B cell development and function, we generated mice doubly-deficient in p53 and Btk. Btk/p53-deficient mice showed an increase in splenic B220+ cell numbers compared with Btk-deficient mice, although there was no recovery in B cell subset differentiation. In contrast to the lack of recovery of B cell development, there was a recovery in lipopolysaccharide and anti-immunoglobulin M (IgM) plus interleukin-4-induced proliferation of Btk/p53-deficient B cells, although there was no recovery to anti-IgM stimulation alone. Thus, p53 promotes B cell expansion and proliferation, but p53 deficiency cannot compensate for Btk deficiency in the development of B cell subsets.

Key Words: B lymphocyte • immunoglobulin • tumor suppressor • development


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INTRODUCTION
 
Btk, a member of the Tec family of protein tyrosine kinases [1 ], is important for signaling downstream of the B cell receptor (BCR), interleukin-5 (IL-5) receptor, and Fc receptor for immunoglobulin E (IgE)I [2 3 4 5 6 7 8 ]. Ligation of the BCR results in tyrosine phosphorylation of Btk by other nonreceptor protein tyrosine kinases such as Lyn and Syk, which leads to the activation of Btk [9 ]. Btk is mutated in X-linked agammaglobulinemia (XLA) [2 3 4 ], and XLA patients display a severe loss of mature B cells (>95%) [10 , 11 ]. Btk function has also been assessed in CBA/N mice, which carry a spontaneous Btk mutation (xid), and by specific gene targeting [12 , 13 ]. The phenotype of Btk mutant mice is not as severe as the human condition, with a less dramatic loss of mature B cells. However, the decrease in mature B cells is still accompanied by a marked reduction of serum IgM and IgG3. Btk-deficient B cells also show a diminished capacity to proliferate upon stimulation with lipopolysaccharide (LPS) and BCR cross-linking. In addition, Btk may regulate the antiapoptotic proteins Bcl-2 and Bcl-xL [14 , 15 ]. In xid mice, there is a decrease in the amount of endogenous Bcl-2, and this correlated with an increase of B cell apoptosis in culture [14 ]. Following anti-Ig stimulation, xid B cells also fail to up-regulate Bcl-xL, correlating with an inability to complete the cell cycle and progression to apoptosis [15 ]. These data suggest that Btk has a cell-cycle regulatory role and may use several downstream effector proteins. Additional proteins that may be involved in Btk-dependent B cell development and function are still unidentified.

The p53 protein is mutated in ~50% of all human cancers [16 ]. Several types of stress can induce p53 activity including DNA damage, telomere attrition, oncogene activation, and hypoxia [16 ]. Following activation, p53 is capable of inducing several responses including cell differentiation, cell senescence, DNA repair, inhibition of angiogenesis, apoptotic cell death, and cell-cycle arrest [16 , 17 ]. p53 functions as a transcription factor, which leads to the up-regulation of proteins that serve as the effector molecules associated with p53-dependent responses. One of the targets for p53 is Mdm2, which functions as a negative regulator of p53 [18 ]. Other transcriptional targets of p53 include p21Waf1/Cip1, 14-3-3 {sigma}, growth arrest and DNA damage 45 (GADD45), Fas/apolipoprotein 1, DR5/KILLER, and Fas ligand [19 ]. In addition, p53 can regulate members of the Bcl-2 family, such as Bcl-2, Bcl-xL, BID, and Bax [19 20 21 22 23 24 ].

The hyperproliferative capacity of cells during lymphopoeisis suggests a role for p53 in the regulation of B cell development. Several reports have supported a role for p53 in B cell development in the bone marrow, showing an increase in the number of pro-B cells [25 ] and pro-B cells/pre-B cells compared with wild-type controls [26 ]. In addition, when the proto-oncogene Mdm2, which regulates p53 activity and protein levels in vitro, was reduced to about half its wild-type level in vivo, there were defects throughout B cell development [27 ]. As Btk and p53 oppositely regulate antiapoptotic proteins, which are important for B cell survival and function, we hypothesized that p53 deficiency might complement the phenotype seen in Btk-deficient mice.

To determine if p53 functions as a suppressor of B cell development and functions in the absence of Btk signaling, we generated mice lacking p53 and Btk. We demonstrate that the loss of p53 on a Btk-deficient background was unable to restore B cell compartments in the spleen and bone marrow but did lead to an increase in total splenic B220+ cells. We also observed an increased proliferative capacity of Btk/p53-deficient B cells compared with Btk-deficient B cells following stimulation with B cell mitogens. These data support a role for p53 in B cell expansion but suggest that p53 cannot compensate for the absence of Btk in B cell development.


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MATERIALS AND METHODS
 
Mice
The generation of Btk-deficient and p53-deficient mice has been described previously [13 , 28 ]. Breeder pairs for single gene-deficient mice (Jackson Laboratories, Bar Harbor, ME) were mated to generate double-deficient mice (housed and bred in the Indiana University Laboratory Animal Research Center, Indianapolis). The Btk allele was genotyped by Southern blot using the same conditions described previously [13 ]. The p53 allele was genotyped using polymerase chain reaction to amplify a sequence from the 3' end of Exon 6 or the 3' end of Neo insert in the targeted allele through the 5' end of Exon 7: primers (p53-X6 5'-AGCGTGGTGGTACCTTATGAGC-3', p53-X7 5'-GGATGGTGGTATACTCAGAGCC-3', and Neo 5'-GCTATCAGGACATAGCGTTGGC-3'). This amplified an ~475 base-pair (bp) band from the wild-type allele and an ~700-bp band from the targeted allele. Control C57BL/6 mice were purchased from Harlan Bioproducts (Indianapolis, IN). For analysis of B cell development and in vitro Ig production, 6-week-old mice were used. Mice in other experiments were between 6 and 12 weeks of age.

Cellular phenotypic analysis by flow cytometry
Cells (1x106 per sample) were washed in phosphate-buffered saline with 2% bovine serum albumin and 0.1% NaN3 [fluorescein-activated cell sorter (FACS) buffer]. Cells were first incubated with anti-Fc receptor for IgG antibodies, Clone 2.4G2 (BD PharMingen, San Diego, CA), for 10 min. Cells were then stained with antibodies conjugated directly to fluorescein isothiocyanate, phycoerythrin, allophycocyanin, and peridinin chlorophyll protein-Cy5.5 (CD43, IgM, CD23, CD21, CD5, Annexin V, BD PharMingen; B220 and IgD, eBioscience, San Diego, CA) for 15 min. All staining was done at 4°C followed by one wash with FACS buffer. Cells were then fixed in FACS buffer, which contained 0.5% formaldehyde.

Isolation of peritoneal cavity cells
To isolate peritoneal cavity cells, 3 ml cold, supplemented RPMI was injected into the peritoneal cavity, which was then gently agitated to loosen the cells. Approximately, 2 ml media containing cells could be recovered from the peritoneal cavity of each mouse.

Analysis of Ig class-switching
Serum was isolated by cardiac puncture. Spleens were disrupted into a cell suspension, which was treated with 8.3 g/L NH4Cl, 0.01 M Tris, pH 7.5 (red blood cell lysis solution), for 5 min. Cells were then washed in RPMI 1640 (BioWhittaker, Walkersville, MD), supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biologicals, Norcross, GA), 50 U/ml penicillin + 50 µg/ml streptomycin, 1 mM sodium pyruvate, 0.5x nonessential amino acids, 1 mM L-glutamine, 10 mM HEPES (all from BioWhittaker), and 50 µM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). Cells were plated at a density of 3 x 106/ml, stimulated or not with 5 µg/ml {alpha}CD40 (clone 3/23, BD PharMingen), 5 µg/ml {alpha}CD40 plus 5 ng/ml IL-4 (PeproTech, Rocky Hill, NJ), or 5 µg/ml {alpha}CD40 plus 50 ng/ml interferon-{gamma} (IFN-{gamma}; PeproTech) for 4 days at 37°C. At the end of the incubation period, supernatants were collected. Ig isotypes were analyzed by enzyme-linked immunosorbent assay (ELISA) using IgM, IgG2a, IgG3, IgE (BD PharMingen), and IgG1 (Southern Biotechnology Associates, Birmingham, AL) antibodies, according to the manufacturer’s recommendations.

Proliferation assay
Splenocytes (5x104/well) were pipetted in a 96-well plate and stimulated or not with 10 µg/ml {alpha}IgM F(ab')2 (Jackson ImmunoResearch Laboratories, West Grove, PA), 10 µg/ml {alpha}IgM F(ab')2 plus 5 ng/ml IL-4, 5 µg/ml {alpha}CD40, 5 µg/ml {alpha}CD40 plus 5 ng/ml IL-4, or 10 µg/ml LPS (Sigma-Aldrich). The cells were incubated at 37°C and pulsed with 1 µCi [3H]thymidine/well for the last 18 h of a 48-h incubation. Alternatively, the splenocytes were plated out in media at 1.5 x 106/ml and activated with 10 µg/ml {alpha}IgM F(ab')2 for 48 h at 37°C. Cells were then washed in media, pipetted (5x104/well) in a 96-well plate, stimulated with 10 ng/ml IL-4, and pulsed with 1 µCi [3H]thymidine/well for the last 18 h of a 48-h incubation at 37°C.

Statistical analysis
Statistical analysis was performed with SPSS software using the Tukey test or Student’s t-test, as indicated in figure legends.


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RESULTS
 
B cell development in Btk-deficient, p53-deficient B cells
To determine if p53 deficiency was able to restore B cell development on a Btk-deficient background, we analyzed B cell compartments in the bone marrow, spleen, peritoneal cavity, and lymph nodes of wild-type, Btk-deficient, Btk/p53-deficient, and p53-deficient mice. For analysis of IgM and IgD expression on more mature B cells, we gated on CD43–/B220+ bone marrow cells. There were similar increases in developing B cell subsets and significant decreases in recirculating, mature B cells in Btk/p53-deficient and Btk-deficient mice compared with wild-type (Fig. 1 ). Consistent with the B cell percentages (Fig. 1A) , there was no recovery in the number of recirculating, mature B cells in the Btk/p53-deficient mice compared with the Btk-deficient mice (Fig. 1B) . B cell development in wild-type and p53-deficient mice was similar with the exception of the significant decrease in recirculating, mature B cells in the p53-deficient mice compared with wild-type (Fig. 1) . This suggests that p53 deficiency does not affect Btk-dependent B cell development in the bone marrow.


Figure 1
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  		Figure 1. B cell populations in femur bone marrow. (A) Bone marrow cells from the femur of 6- to 7-week-old mice were extracted and incubated with antibodies that recognized CD43, B220, IgM, and IgD. Cells that were CD43–/B220+ were gated and analyzed for expression of IgM and IgD. Cells in gate A are late pre-B cells, gate B are immature B cells, and gate C are recirculating, mature B cells. B cell population percentages shown are averages from wild-type (Wt; n=10), Btk–/– (n=5), Btk/p53–/– (n=7), and p53–/– (n=5). (B) Cell numbers per femur (±SD) were calculated based on the percentage of B cells in each population. The results shown are the averages from the mice in A. *, Significant change from the wild-type mice using Tukey test (P<0.05).

We next examined B cell compartments in the spleen, which can be separated into four populations based on the expression of B220, CD23, IgM, and CD21. B220+CD23– cells are further divided into marginal zone and T1 B cells, and the CD23+ population identifies follicular and T2 B cells. The B220+IgMdim cells observed in Figure 2A are B cells that are IgMlo/IgDhi or B cells that have undergone class-switching. Splenic B cells from Btk-deficient and Btk/p53-deficient mice showed similar profiles of B cell development with smaller percentages and numbers of mature B cells than wild-type mice (Fig. 2) . Although p53-deficient mice had increases in B cell compartments correlating with spleen size, Btk-deficient and Btk/p53-deficient mice had a severe loss of follicular B cells compared with wild-type mice (Fig. 2) . Splenic B220+ cell numbers were increased 150% in Btk/p53-deficient mice compared with Btk-deficient mice (Fig. 2B) .


Figure 2
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  		Figure 2. B cell populations in the spleen. (A) Spleens from 6- to 7-week-old mice were isolated, and B cell populations were identified using FACS analysis based on expression of B220, CD23, CD21, and IgM. Population percentages shown are the averages from wild-type (n=10), Btk–/– (n=5), Btk/p53–/– (n=7), and p53–/– (n=5). (B) Average number (±SD) of cell numbers in the spleen from 6- to 7-week-old mice. Based on the percentages of B cell populations in the spleen, the number of B cell populations was then calculated. *, Significant change from the wild-type mice using Student’s t-test (P<0.05); {dagger}, significant change between Btk- and Btk/p53-deficient mice using Student’s t-test (P<0.05). MZ, Marginal zone; FO, follicular; T1, transitional; T2, transitional type 2.

B cells were also decreased in other peripheral sites. Peripheral B220+ cell numbers in lymph nodes were also decreased in Btk- and Btk/p53-deficient lymph nodes compared with those from wild-type and p53-deficient mice (data not shown). Btk deficiency also results in dramatic decreases in peritoneal numbers [13 ]. Although total cell recoveries from peritoneal lavage were similar among the four genotypes analyzed (data not shown), there was a significant decrease in total B cell numbers from Btk-deficient and Btk/p53-deficient peritonea (Fig. 3A ). B1 cell numbers were decreased greatly in the Btk-deficient and Btk/p53-deficient peritoneum from levels in wild-type or p53-deficient peritonea (Fig. 3B) .


Figure 3
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  		Figure 3. Peritoneal B cell populations. (A) Total peritoneal lavage B cells (±SD) from WT (n=6), Btk–/– (n=5), Btk/p53–/– (n=7), and p53–/– (n=4) mice. (B) B cell numbers (±SD) were calculated based on B cell percentages and total cell recovery from peritoneal lavage. B cell population percentages were identified using FACS analysis based on expression of indicated cell-surface markers. *, Significant change from wild-type mice using Tukey t-test (P<0.05).

Functional analysis of B cells
Btk-deficient B cells have decreased serum IgM and IgG3 [13 ]. We next addressed whether the loss of p53 on the Btk-deficient background would restore any of these functions. We first analyzed serum Ig levels in wild-type, Btk-deficient, Btk/p53-deficient, and p53-deficient mice. Figure 4 shows that the loss of p53 did not restore the ability of Btk-deficient B cells to produce IgM or IgG3 compared with wild-type. These data correlate with there being fewer follicular B cells (Fig. 2) and B1 B cells (Fig. 3) producing Igs in Btk-deficient and Btk/p53-deficient mice rather than in wild-type mice. It is surprising that there was a significant increase in the amount of serum IgE in the Btk-deficient and Btk/p53-deficient serum compared with wild-type, although the mechanism for this is unknown. p53-deficient serum Ig levels are not different from wild-type levels (Fig. 4) .


Figure 4
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  		Figure 4. Serum Ig levels. Serum was obtained from 12 wild-type, Btk–/–, or Btk/p53–/– mice and four p53–/– mice (6–12 weeks old). Serum Ig levels were analyzed by ELISA for detection of IgM, IgG1, IgG2a, IgG3, and IgE. Each data point represents the serum from one mouse. *, Significant change from wild-type mice using Tukey t-test (P<0.05).

To further address the ability of B cells to produce Igs and undergo class-switching, we stimulated the splenocytes from wild-type, Btk-deficient, and Btk/p53-deficient mice for 4 days with {alpha}CD40, {alpha}CD40 plus IL-4, or {alpha}CD40 plus IFN-{gamma}. After 4 days, the supernatant from each incubate was analyzed by ELISA for IgM, IgG1, IgG3, and IgE. The presence of IL-4 induces class-switching from IgM to IgG1 or IgE, and IFN-{gamma} induces class-switching from IgM to IgG2a. Btk-deficient and Btk/p53-deficient B cells do not produce IgM, IgG3, or IgG2a in vitro following CD40 stimulation (Fig. 5 and data not shown) compared with wild-type B cells. Analysis of Ig production from purified, splenic B220+ cells showed Ig production similar to total splenocytes (data not shown). Addition of IL-4 to the cultures augmented the ability of Btk-deficient and Btk/p53-deficient B cells to produce IgM, IgG1, and IgE, although levels were still diminished compared with wild-type cells (Fig. 5) . p53-deficient B cells had no defect in Ig production or class-switching (data not shown).


Figure 5
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  		Figure 5. In vitro Ig production and class-switching. Splenocytes from 6-week-old mice of the indicated genotypes were stimulated with 5 µg/ml {alpha}CD40, 5 µg/ml {alpha}CD40 plus 5 ng/ml IL-4, or 5 µg/ml {alpha}CD40 plus 50 ng/ml IFN-{gamma} for 4 days. After 4 days, supernatant was collected and assayed by ELISA for the levels of IgM, IgG1, IgG3, and IgE (mean±SEM). Results are representative of three experiments.

Analysis of B cell survival and proliferation
As p53 plays an important role in regulating apoptosis, we wanted to determine whether the modest expansion of B cell populations in double-deficient mice was a result of decreased cell death or increased cell expansion. A previous report demonstrated that low levels of Annexin V can bind to B cells that are not undergoing apoptosis; we analyzed the percentages of cells that were Annexin V bright [29 ]. The largest difference between Btk-deficient or Btk/p53-deficient mice from wild-type mice is the increase in the number of apoptotic cells in the T2 and follicular B cell population (Fig. 6 ). These data are consistent with there being fewer T2 and follicular B cells in the Btk-deficient and Btk/p53-deficient mice compared with wild-type and the requirement for BCR/Btk signaling at these stages of development [30 ]. The lack of increased apoptosis between Btk-deficient and Btk/p53-deficient B cells suggests that the modest increase in B cell numbers in double-deficient mice is not a result of altered levels of apoptosis.


Figure 6
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  		Figure 6. Ex vivo apoptosis analysis of B cell populations. Femur bone marrow cells and splenocytes were isolated from 6- to 7-week-old mice. Cells were incubated with antibodies that recognized CD43, B220, IgM, and CD23. The B cell populations were segregated as follows: bone marrow pro/pre-B cells (B220+/CD43+/IgM–), bone marrow late pre-B cells (B220+/CD43–/IgM–), splenic marginal zone and T1 B cells (B220+/CD23–), and splenic T2 and follicular B cells (B220+/CD23+). Apoptosis in the different populations was then determined by high binding of Annexin V to the cell. The values are averages (±SD) from wild-type (n=6), Btk–/– (n=6), and Btk/p53–/– (n=10). *, Significant change from the wild-type mice using Student’s t-test (P<0.05).

Given that Btk-deficient B cells have decreased proliferative responses to {alpha}IgM [13 , 31 32 33 ], we next assayed the proliferative capacity of the B cells to determine if there was a recovery upon the loss of p53 on the Btk-deficient background. Splenocytes from each genotype were stimulated with LPS, {alpha}CD40, or {alpha}IgM, with or without IL-4. There was no defect in the proliferative capacity in Btk-deficient or Btk/p53-deficient cells following {alpha}CD40 ± IL-4 stimulation, consistent with previous results [13 , 34 ], and p53-deficient B cells showed increased responses to these stimuli (Fig. 7A ). When cells were stimulated with LPS, Btk/p53-deficient and wild-type cells showed similar proliferative responses, whereas Btk-deficient cells showed a decreased proliferative response (Fig. 7B) . Following 48 h of {alpha}IgM stimulation, the ability of B cells to proliferate was decreased in the Btk-deficient and Btk/p53-deficient cultures compared with wild-type cells (Fig. 7C) . However, in the presence of {alpha}IgM plus IL-4, there was a similar level of proliferation in wild-type and Btk/p53-deficient B cells compared with the diminished proliferative response in Btk-deficient B cells and a striking hyperproliferation of p53-deficient B cells in response to these stimuli (Fig. 7C) . The proliferative response of splenic B220+ cells was also examined and showed results similar to those seen with total splenocytes (data not shown). To determine if p53 deficiency affects apoptosis in mitogen-stimulated cultures, we analyzed B cell survival following stimulation. We observed only marginal differences in the survival of B cells from each of the four genotypes following stimulation with {alpha}IgM ± IL-4 for 24 h and 48 h (data not shown). These data suggest that p53 suppresses proliferation in B cells, and deficiency can increase proliferation, even in the absence of Btk-mediated signals. Conversely, these data demonstrate that p53-deficient B cell hyperproliferation is dependent on Btk activity.


Figure 7
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  		Figure 7. Proliferative response following stimulation with B cell mitogens. (A) Splenocytes from mice of the indicated genotypes (6–12 weeks old) were stimulated with 5 µg/ml {alpha}CD40 and 5 µg/ml {alpha}CD40 plus 5 ng/ml IL-4. (B) Splenocytes from each genotype were stimulated with 10 µg/ml LPS. (C) Cells from each genotype were stimulated with 10 µg/ml {alpha}IgM or 10 µg/ml {alpha}IgM plus 5 ng/ml IL-4. Cells from each stimulation were pulsed with [3H]thymidine for the last 18 h of a 48-h stimulation. Results are shown as mean ± SD. Results are representative of more than three experiments. *, Significant change from wild-type mice using Student’s t-test (P<0.05); {dagger}, significant change between Btk- and Btk/p53-deficient mice using Student’s t-test (P<0.05). CPM, Counts per minute.


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DISCUSSION
 
To investigate a role for p53 in B cell development, we mated p53-deficient mice with Btk-deficient mice, which have multiple defects in B cell development and function. p53 deficiency led to an increase in splenic B220+ cells but did not recover B cell development or Ig production in double-deficient mice. Proliferative responses to LPS and anti-IgM plus IL-4 were recovered to wild-type levels in double-deficient mice, and there was a hyperproliferative response in p53-deficient B cells. Therefore, p53 plays only a modest role in B cell development and function in the presence or absence of Btk signaling.

Several reports have supported a role for p53 in B cell development in the bone marrow, showing an increase in the number of pro-B cells [25 ] and pro-B/pre-B cells compared with wild-type controls [26 ]. In one report, it was shown that the increase in pro-B cells correlated with a decrease in the level of apoptosis in the p53-deficient cells compared with wild-type [25 ]. In contrast to these reports, we observed no difference in the numbers and frequency of p53-deficient bone marrow B cell populations (Fig. 1) . One possible explanation for the difference between our results and the previous reports is the genetic background of the p53-deficient mice; previous analyses were done with 129 background mice, and the mice in this report were backcrossed 10 generations to C57BL/6. However, the hyperproliferative response of p53-deficient B cells upon stimulation with anti-IgM or LPS was similar in our report and a previous study (Fig. 7) [26 ]. Although our data suggest that p53 does not play an important role in regulating Btk-dependent B cell development, when Mdm2 levels are reduced, there is an increase in the apoptotic activity of p53, resulting in multiple defects in B cell development [27 ]. These defects include a 63% decrease in white blood cells, an 85% decrease in splenic B cells, a 40% decrease in bone marrow B220+ CD43+ cells, and an 87% decrease in bone marrow B220+ IgM+ cells [27 ]. The decreased number of bone marrow B cells correlated with an almost twofold increase in the level of apoptosis in these cells [27 ]. However, deficiency in Mdm2 increases p53 expression to supra-physiological levels, suggesting that this is not a normal role for p53 in B cell development. Moreover, Mdm2 reduction could also affect the function of other molecules.

As there is not a change in the level of apoptosis in B cells deficient in Btk and p53 compared with the Btk-deficient B cells (Fig. 6) , we would predict that p53 is regulating B cell expansion/proliferation through regulation of cell-cycle regulatory proteins as opposed to regulation of apoptosis. p53 controls cell-cycle progression through the transcriptional regulation of several proteins including p21WAF1/CIP1, 14-3-3 {sigma}, GADD45, and Reprimo [11 , 19 ]. As the anti-IgM plus IL-4-stimulated decrease in p21WAF1/CIP1 expression was similar between wild-type and Btk- or Btk/p53-deficient B cells (data not shown), p53 must regulate additional genes important for B cell mitogen responses.

Our results suggest the partial recovery in cell numbers and function, resulting from p53 deficiency of Btk-deficient B cells, results from the hyperproliferative response observed in p53 deficiency. However, there are also similarities between the Btk/p53-deficient mice, which we have described here, and the xid-bcl-2 and xid-bcl-xL transgenic mice, which rescue Btk-dependent responses by increasing cell survival [14 , 15 ]. All three models showed a partial recovery in splenic B cell numbers compared with xid or Btk-deficient mice (Fig. 2) [14 , 15 ]. However, neither the xid-bcl-xL transgenic mice nor the Btk/p53-deficient mice demonstrate a recovery in serum IgM or IgG3, in contrast to the recovery of IgM levels in xid-bcl-2 transgenic mice (Fig. 5) [14 , 15 ]. This correlated with a recovery in follicular B cells in the xid-bcl-2 transgenic mice, not observed in the other models. These models are distinguished further by the response of B cells to BCR stimulation. There was no recovery in anti-Ig-induced proliferation in xid-bcl-2 transgenic B cells [14 ]. In contrast, the xid-bcl-xL transgenic B cells show a recovery in anti-IgM-induced proliferation [15 ], and Btk/p53-deficient B cells display normal proliferation in response to anti-IgM plus IL-4 (Fig. 7) . These results suggest that p53 and Bcl-2 family proteins have distinct but overlapping roles in facilitating B cell responses to BCR signaling.

Although there was no recovery in serum IgM or IgG3 in Btk/p53-deficient mice, we observed a significant increase in the level of serum IgE in Btk- and Btk/p53-deficient mice (Fig. 4) . In contrast, there was a defect in the ability of Btk-deficient B cells to produce IgE in the in vitro assay, consistent with the inability of Btk-deficient B cells to undergo class-switching to other Igs (Fig. 5) . The reason for increased IgE in vivo is unclear but could represent an altered in vivo cytokine environment or a requirement for Btk in suppressing IgE class-switching. The decrease in IgE production from Btk-deficient cells in vitro would support an altered in vivo environment hypothesis.

Btk is an essential protein involved in B cell biologic activity. In this report, we found that the loss of p53 on a Btk-deficient background led to an expansion of splenic B220+ cells in vivo and B cell proliferation in vitro. Despite the recovery in B cell proliferation, there was no recovery in splenic B cell subsets. Further investigation is required for understanding how Btk and p53 regulate B cell survival and proliferation and how the balance of these two is necessary for proper B cell development.


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ACKNOWLEDGEMENTS
 
This work was supported by U.S. Public Health Service Awards (to M. H. K.) from the National Institutes of Health. N. W. S. was a predoctoral fellow of the American Heart Association. We thank J. Blum for critical review of this manuscript and members of the Kaplan laboratory for their assistance and support.

Received July 21, 2005; revised November 29, 2005; accepted December 6, 2005.


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N. W. Schmidt, V. T. Thieu, B. A. Mann, A.-N. N. Ahyi, and M. H. Kaplan
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