Wip1 Phosphatase-Deficient Mice Exhibit Defective T Cell
Maturation Due To Sustained p53 Activation1
Marco L. Schito,2* Oleg N. Demidov,* Shin’ichi Saito,* Jonathan D. Ashwell,†and
The PP2C phosphatase Wip1 dephosphorylates p38 and blocks UV-induced p53 activation in cultured human cells. Although the
level of TCR-induced p38 MAPK activity is initially comparable between Wip1?/?and wild-type thymocytes, phosphatase-
deficient cells failed to down-regulate p38 MAPK activity after 6 h. Analysis of young Wip1-deficient mice showed that they had
fewer splenic T cells. Their thymi were smaller, contained significantly fewer cells, and failed to undergo age-dependent involution
compared with wild-type animals. Analysis of thymocyte subset numbers by flow cytometry suggested that cell numbers starting
at the double-negative (DN)4 stage are significantly reduced in Wip1-deficient mice, and p53 activity is elevated in cell-sorted DN4
and double-positive subpopulations. Although apoptosis and proliferation was normal in Wip1?/?DN4 cells, they appeared to be
in cell cycle arrest. In contrast, a significantly higher percentage of apoptotic cells were found in the double-positive population,
and down-regulation of thymocyte p38 MAPK activation by anti-CD3 was delayed. To examine the role of p38 MAPK in early
thymic subpopulations, fetal thymic organ cultures cultured in the presence/absence of a p38 MAPK inhibitor did not correct the
thymic phenotype. In contrast, the abnormal thymic phenotype of Wip1-deficient mice was reversed in the absence of p53. These
data suggest that Wip1 down-regulates p53 activation in the thymus and is required for normal ?? T cell development. The
Journal of Immunology, 2006, 176: 4818–4825.
the thymus as (CD4?CD8?) double-negative (DN)3T cell
CD44?CD25?(DN2), CD44?CD25?(DN3), and CD44?CD25?
(DN4) developmental stages (1). During the transition from the
DN3 to the DN4 stage, thymocytes must first pass ?-selection to
develop into ?? T cells (2–4). The transition from DN to
CD4?CD8?double positive (DP) depends on the expression of a
functional pre-TCR composed of the invariant pre-TCR? coupled
to the TCR?-chain (5). Thymocytes that express a pre-TCR are
rescued from apoptosis, differentiate (up-regulate CD4 and CD8
coreceptor expression and rearrange the TCR?-chain), and prolif-
erate (6–8). Thymocytes that successfully rearrange the TCR?-
chain gene undergo positive or negative selection during the tran-
sition between the DP to the CD4?CD8?or CD4?CD8?(single
positive; SP) stage. It is believed that a balance between proapo-
ptotic (Bim, Bad, Bax) and antiapoptotic (Bcl-2, Bcl-xL) Bcl-2
family members controls the fate of thymocytes. The particular
signaling pathways involved are diverse and depend on the nature
of the apoptotic signal and the stage of cell differentiation. Thus,
cell development is defined by a timely and ordered set
of phenotypic changes in the expression of multiple sur-
face markers and receptors. Hemopoietic precursors enter
maturation of thymocytes occurs through complex interactions that
involve TCR signaling (9), cell-cell communication (10, 11), and
soluble factors (12, 13).
The role of p38 MAPK is well established in the thymus. Using
a p38 dominant-negative transgene under control of the proximal
lck-promotor, it was demonstrated that activation of p38 MAPK
was required for the earliest stage of DN differentiation (14). Fur-
thermore, by using a constitutively active MAPK kinase (MKK)6
transgene that phosphorylates p38 in the absence of upstream stim-
ulation, Diehl et al. (14) found that activation of p38 MAPK
blocked thymocyte development at the DN3 stage. In addition,
more thymocytes from constitutively active MKK6 transgenic
mice were found to be cycling compared with nontransgenic lit-
termates. Therefore, p38 MAPK needs to be active for cells to
differentiate from the DN1 to DN3 stage, but kinase activity must
be turned off for cells to differentiate to the DN4 stage. Thus, initial
activation of p38 MAPK appears to be important for early thymo-
cyte development, but continuous activation blocks the generation
of T cells resulting in immunodeficiency.
The p53 tumor suppressor protein regulates cell cycle and ap-
optosis in response to DNA damaging events such as irradiation,
but p53 may also play a role under more physiological conditions
such as dsDNA breaks due to site-specific V(D)J recombination in
T cell precursors (15). Interestingly, T cell development can, to
some extent, be rescued by the simultaneous loss of p53 in mice,
where T cell development is otherwise blocked (RAG?/?, SCID,
CD3??/?) (16–19). Thus, down-regulation of p53 allows ?? T
cells to proceed from the DN to DP stage.
Previously, we identified wild-type (WT) p53-inducible phos-
phatase, Wip1, an irradiation inducible type 2C phosphatase
(PP2C), as a transcriptional target of p53 (20). PP2Cs have been
associated with stress responses, sexual differentiation, and cell
cycle control in a variety of organisms (21). We have generated
mice deficient for Wip1, and although they appear to develop nor-
mally, males have defects in reproductive organs, and embryonic
*Laboratory of Cell Biology and†Laboratory of Immune Cell Biology, National
Cancer Institute, National Institutes of Health, Bethesda, MD 20892
Received for publication June 23, 2005. Accepted for publication February 2, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the Intramural Research Program of the National In-
stitutes of Health, Center for Cancer Research, National Cancer Institute.
2Address correspondence and reprint requests to Dr. Marco L. Schito, National Can-
cer Institute, National Institutes of Health, Building 37, Room 2140, Bethesda, MD
20892. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: DN, double negative; DP, double positive; SP,
single positive; WT, wild type; PI, propidium iodide; 7-AAD, 7-amino actinomycin
D; FTOC, fetal thymic organ culture.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00
fibroblasts derived from these mice have multiple defects in cell
cycle control (22). In addition, Wip1-deficient mice reportedly ex-
hibit immunological defects in the peripheral lymphoid organs that
result in an inability to control influenza infection, suggesting that
this phosphatase may be involved in immune responses (22).
p38 MAPK was the first Wip1 substrate identified. Takekawa et
al. (23) have shown that Wip1 inhibits p38 MAPK function by
dephosphorylating Thr180. It has been suggested that Wip1 may
mediate a negative feedback loop for p53, resulting in reduced p38
site-specific phosphorylation of p53 residues. More recently, Wip1
was found to regulate base excision repair through its dephosphor-
ylation of the nuclear isoform of uracil DNA glycosylase (UNG2)
in response to UV irradiation (24).
Our initial experiments determined that Wip1-deficient mice
had reduced numbers of peripheral T cells that could not be at-
tributed to reduced proliferation or enhanced apoptosis. Thus, we
examined T cell development and found that all populations of
cells in young Wip1?/?mice (?3 mo) were severely reduced in
number, with the exception of the DN thymocyte population. The
reduced number of cells appeared to be due to defects in the DN4
to DP transition during T cell development. Although p38 MAPK
did not appear to play a role, thymic development appeared normal
when Wip1-deficient mice were crossed onto a p53-null back-
ground. Our results suggest that Wip1 plays a role during the DN
to DP transition in ?? T cell development by down regulating the
p53 tumor suppressor.
Materials and Methods
The generation of Wip1?/?and p53?/?mice has been described previ-
ously (22, 25). All mice were backcrossed at least five times on to a
C57BL/6NCr background. Wip1?/?and p53?/?mice were mated to ob-
tain Wip1?/?p53?/?double knockout animals. The breeders used were
heterozygous for p53 to extend their survival, because the lack of this gene
predisposes mice (?6 mo) to thymic lymphomas and early death. DNA
(tail) samples were subjected to RT-PCR using the SuperScript preampli-
fication system (Invitrogen Life Technologies) according to the manufac-
turer’s protocol. All experiments used age- and gender-matched mice. WT
C57BL/6NCr control mice were purchased from Division of Cancer Treat-
ment/National Cancer Institute (Frederick, MD). All mice were bred and
housed in an Association for Assessment and Accreditation of Laboratory
Animal Care-accredited facility under an approved National Institutes of
Health animal study protocol and specific pathogen-free conditions.
Tissue culture and cell activation
Complete medium consisted of RPMI 1640 with glutamax (Invitrogen Life
Technologies) supplemented with 10% FCS (BioWhittaker), 5.5 ? 10?5M
2-ME (Invitrogen Life Technologies), 100 U/ml penicillin, and 100 ?g/ml
streptomycin (Invitrogen Life Technologies). Thymi were isolated from
mice, weighed, and pushed through a 70-micron membrane (BD Bio-
sciences) in complete medium. The thymocytes were washed and sus-
pended in complete medium at 10 ? 106cells/ml. Cells were cultured at
37°C with 5% CO2at a density of 5 ? 106cells/ml in the presence/absence
of plate-bound anti-CD3 (10 ?g/ml) and anti-CD28 (5 ?g/ml) (BD
Flow cytometry, cell cycle, and sorting analysis
Thymocytes (5 ? 105to 1 ? 106cells) were suspended in staining buffer
(PBS supplemented with 1% FCS) with the following Abs purchased from
BD Pharmingen (BD Biosciences): CD4; CD8; B220; pan-NK; CD11c;
CD3; ??-TCR; ?-TCR; CD5; CD44; CD25; and Bcl-xL. All Abs used for
flow cytometry were directly conjugated to FITC, PE, PerCP, or allophy-
cocyanin with the exception of CD25, which was biotinylated for some
experiments. Streptavidin conjugates were purchased from BD Bio-
sciences. For intracellular Bcl-xLstaining, cells were fixed and permeabil-
ized with BD Cytofix/Cytoperm (BD Biosciences) using the manufactur-
er’s directions. Flow cytometry was performed using a FACScan and
CellQuest software (BD Biosciences). For cell cycle analysis, thymocytes
(1 ? 106cells) were washed, stained for cell surface markers, suspended
in 70% ethanol, and fixed overnight at 4°C. The cells were then resus-
pended in staining solution containing either 50 ?g/ml propidium iodide
(PI) (Sigma-Aldrich) and 100 U/ml RNase A (Roche) or 7-amino actino-
mycin D (7-AAD). In experiments where cell cycle data was acquired from
four-color stained samples, cells were stained for cell surface markers,
fixed, and permeabilized with BD Cytofix/Cytoperm (BD Biosciences),
then stained with 4?,6?-diamidino-2-phenylindole (DAPI). Flow cytometry
data was acquired using the LSRII cytometer (BD Biosciences) and ana-
lyzed with FlowJo software (Tree Star). For DN cell sorting experiments,
thymocytes from five 4- to 6-wk-old Wip1?/?and WT mice were com-
plement-depleted of CD4 and CD8 cells (Cedarlane Laboratories). Dead
cells were removed by density gradient centrifugation using Histopaque-
1083 (Sigma-Aldrich). The remaining live cells were stained with CD4,
CD8, B220, CD3, pan-NK, and CD11c FITC-labeled Abs to gate these
cells out and stained with CD44-allophycocyanin and CD25-PE Abs to sort
the four DN cell populations using the FACSVantage SE sorter with DiVa
option (BD Biosciences). To sort out DP and SP cells, thymocytes isolated
from mice were stained with CD4-FITC and CD8-PE. Analysis was done
using FACS DiVa software (BD Biosciences).
RNA purification and real-time RT-PCR
Total RNA was isolated from sorted cells using RNeasy Micro kit per
manufacturer’s instructions (Qiagen). RNA concentration was determined
using a nanodrop (Grace Scientific). Primers for Wip1 (forward, 5?-GCTA
GAGGGAATATCCAGACTGTAGTGA-3? and reverse, 5?-AGTATTT
GTTGAATTGGTTGGAATGAGGC-3?) were designed using LightCycler
Probe Design 2 software (Roche). Primers for GAPDH (forward, 5?-AAT
GTGTCCGTCGTGGATCTGA-3? and reverse, 5?-GATGCCTGCTTCA
(PerkinElmer). One-step real-time RT-PCR was performed using the
LightCycler RNA Master SYBR Green I kit according to the manufactur-
er’s instructions (Roche). LightCycler conditions were as follows: each run
consists of reverse transcription at 61°C for 20 min, initial denaturation at
95°C for 30 s, followed by 45 cycles, denaturation; 15 s at 95°C, annealing;
12 s at 58°C, elongation; 30 s at 72°C, automatic measurement of the F2:F1
ratio. A melting curve was determined as another option by continuous
heating from 40 to 95°C and measurement of F2:F1ratio. Relative expres-
sion of the Wip1 gene was calculated using the delta-delta crossing point
method (2?ddCP) after normalization
Apoptosis and cell proliferation
To determine the percentage of cells undergoing apoptosis, thymocytes
were analyzed either directly after isolation or after incubation in complete
medium for 24 h. Cells were washed and stained using either the annexin
V-FITC apoptosis detection kit (Oncogene Research Products) with PI to
stain dead cells or the annexin V-PE apoptosis detection kit (BD Bio-
sciences) with 7-AAD to stain dead cells as directed by the manufacturer.
In some cases, FITC-labeled CD8 and allophycocyanin-labeled CD4 Abs
were also included with the annexin V-PE kit to examine thymocyte sub-
populations. To determine apoptosis in the DN subpopulations, thymocytes
were stained with CD4, CD8, B220, CD3, pan-NK, and CD11c FITC-
labeled Abs to gate these cells out and stained with annexin V-PE, CD25-
biotin, CD44-allophycocyanin, and streptavidin-PerCP. DN thymocyte
subset proliferation was determined by measuring BrdU incorporation by
flow cytometry. Six-week-old WT and Wip1?/?mice received injections
i.p. with BrdU (Zymed Laboratories) and sacrificed 1 h later. Thymocytes
were stained for surface markers (PE-CD3, -CD4, -CD8, -CD19, -CD11c;
biotin-CD25; allophycocyanin-CD44; streptavidin-PerCP) and fixed and
permeabilized with BD Cytofix/Cytoperm (BD Biosciences) using the
manufacturer’s directions. The cells were resuspended in permeabilization
wash buffer containing 300 ?g/ml DNase (Sigma-Aldrich) and incubated
for 60 min at 37°C, washed, and stained with anti-BrdU-FITC (BD Bio-
sciences). Flow cytometry was performed using a FACScan with CellQuest
software (BD Biosciences).
Protein sample preparation, immunoprecipitation, kinase
reactions, Western blotting, and immunodetection
p38 MAPK activity in the total thymocyte population was measured by
using a p38 MAPK Assay Kit (Cell Signaling Technology; catalog no.
9820) according to the manufacturer’s instructions. Cells were rinsed with
PBS then lysed with lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1
mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophos-
phate, 1 mM ?-glycerolphosphate, 1 mM NaVO4, 1 ?g/ml leupeptin, 1
mM PMSF). After centrifugation for 20 min at 15,000 ? g at 4°C, the
supernatant was stored at ?80°C. Protein content was measured using the
4819The Journal of Immunology
BCA Protein Assay (Pierce). Phosphorylated p38 MAPK was immunopre-
cipitated with immobilized anti-phospho-p38 mAb (Thr180/Tyr182) on aga-
rose hydrazide beads. Beads were washed twice with lysis buffer and twice
with kinase buffer (25 mM Tris (pH 7.5), 5 mM ?-glycerolphosphate, 2
mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2). Kinase reactions were per-
formed for 30 min at 30°C in 50 ?l of the kinase buffer supplemented with
200 ?M ATP and 2 ?g of activating transcription factor (ATF)-2 fusion
protein. Activity was detected using phospho-ATF2 (Thr71) (rabbit poly-
clonal; Cell Signaling Technology) and p38 MAPK (rabbit polyclonal; Cell
Signaling Technology) Abs. To detect cell signaling molecules in sorted
and unsorted thymocyte populations, 1 ? 106cells were prepared in sam-
ple buffer, and the proteins were separated by SDS-PAGE. Specific Abs
against p53 (CM-5; NovoCastra), phospho-p53Ser15(rabbit polyclonal;
Cell Signaling Technology), p21Waf1(Ab-4; Oncogene Research Prod-
ucts), and ?-tubulin (Ab-1; Oncogene Research Products) were used for
Western blotting and were detected with HRP-conjugated anti-mouse or
anti-rabbit (Jackson ImmunoResearch Laboratories) Abs.
Fetal thymic organ cultures (FTOC)
Timed pregnancies were initiated for WT and Wip1?/?mice, and, at day
15, fetal thymi were isolated and cultured on 0.8 ?M nucleopore mem-
branes (Whatman) in 12-well plates with complete medium supplemented
with 100 ?M nonessential amino acids (Invitrogen Life Technologies), 1
mM sodium pyruvate (Invitrogen Life Technologies), and 10 mM HEPES
(Invitrogen Life Technologies). In some cultures, the p38 MAPK inhibitor,
SB203580 (Calbiochem), was added for a final concentration of 10 ?M.
FTOC were cultured for 3 days, and then analyzed by flow cytometry as
Thymic tissues were fixed in 10% buffered formaldehyde, sectioned, and
stained with H&E (American HistoLabs).
Young Wip1?/?mice have significantly lower thymic cell
Wip1-deficient mice have small thymi, immunological defects in
the peripheral lymphoid organs, and are more susceptible to influ-
enza infection (22). Although the number of peripheral T cells in
Wip1?/?mice was reported to be normal (22), that study was
performed in old animals (?8 mo). We have repeatedly observed
substantially lower splenic T cell numbers in younger animals (Ta-
ble I). However, T cell numbers in the periphery approaches that
of WT animals as the mice age, suggesting that homeostatic pro-
liferation may eventually reconstitute normal T cell numbers.
To determine whether the lower number of T cells in the spleen
is due to abnormal T cell development, we examined thymocyte
ontogeny. The lack of Wip1 expression resulted in a 50–75% re-
duction in thymocyte numbers (Table II). Although the thymi of
8-wk-old Wip1-deficient mice appeared structurally normal, the
medulla was small, suggesting that there are fewer mature T cells
(Fig. 1, A and B). In older Wip1?/?animals (?3 mo of age), the
thymus appeared normal in size and was not histologically differ-
ent from thymi from age-matched WT mice (Fig. 1, C and D).
Thus, there appears to be a defect in early T cell development in
young Wip1?/?mice that is not evident in older animals.
Young Wip1-deficient mice have lower numbers of DP and SP
cells and are unable to down-regulate TCR-induced p38 MAPK
To clarify the mechanism for the reduced thymic cellularity of
Wip1-deficient mice, thymocytes were analyzed for the expression
of CD4 and CD8 surface markers by flow cytometry. The percent-
age of CD4 and CD8 SP as well as DP cells were not affected.
However, we consistently observed that Wip1-deficient mice ex-
pressed higher percentages of DN cells compared with their WT
counterparts (Fig. 2A). When the number of cells were taken into
account, Wip1-deficient mice had significantly lower numbers of
DP and SP thymocytes, whereas DN numbers were near normal
(Fig. 2B). Therefore, Wip1-deficient mice either have a defect in
the DN to DP thymocyte transition and/or more DP cells undergo
To determine whether all thymocytes were affected, CD3?cells
were analyzed for the expression of the ??- or ??-TCR. The num-
ber of ?-TCR?cells was significantly reduced in the Wip1-defi-
cient animals, whereas the number of ??-TCR?cells was not sig-
nificantly different (Fig. 2B) when compared with WT mice. This
result indicates that Wip1 plays a role in ?? but not ?? T cell
development in the thymus.
dulla compared with WT animals. Thymic sections of eight (A and B) and
24 (C and D)-wk-old WT (A and C) and Wip1?/?(B and D) mice were
stained with H&E. Results are representative fields observed in sections of
thymus from three individual WT and Wip1?/?mice examined. Images were
examined at ?100 total magnification and digitally captured using a SPOT
camera (Diagnostic Instruments). M, Medulla; Cx, cortex; bar, 100 ?m.
Thymi from young Wip1-deficient mice have smaller me-
Table I. Reduced numbers of splenic T cells in young Wip1-deficient
aNumbers in the table (?106) represent the mean number of splenic T cells
(CD3?B220?) of at least three mice with SD in parentheses.
Table II. Reduction of thymic cellularity during the first 6 mo of age in
aNumbers in the table (?106) represent the mean number of thymocytes of at
least three mice with SD in parentheses.
? The p values were calculated between mouse strains with Slidewrite Pro soft-
ware using the homoscedastic t test.
4820Wip1 REGULATION OF THYMOCYTE DEVELOPMENT
Wip1 acts as a phosphatase that regulates p38 MAPK activity in
vitro. To determine whether Wip1 alters p38 MAPK signaling in
mature TCR-expressing thymocytes, total thymocytes from Wip1-
deficient and WT mice were stimulated with plate-bound anti-
CD3/CD28, and MAPK activity was followed over time. TCR-
stimulation induced p38 MAPK activity within the first 30 min.
Increased MAPK activity was sustained for over 3 h in both WT
and Wip1?/?thymocytes. However, whereas WT cells began to
down-regulate p38 MAPK activity at 6 h and had little or no ac-
tivity by 18 h, Wip1-deficient thymocytes still expressed high lev-
els of p38 MAPK activity even up to 18 h after TCR-stimulation
(Fig. 2C). This indicates that the TCR-induced activation of
Wip1?/?thymocytes are unable to down-regulate p38 MAPK
once activated, consistent with it being a substrate for Wip1.
DP thymocytes from Wip1?/?mice undergo spontaneous
apoptosis and have cell cycle abnormalities
We can directly compare both DP and SP thymocytes from
Wip1?/?and WT mice because these populations constitute the
majority of the cells and are in similar proportions in both WT and
progression and undergo a higher rate of apoptosis. A, Thymocyte size and
granularity from 6- to 8-wk-old Wip1-deficient mice were compared with
that of age-matched WT control cells as determined by flow cytometric
analysis of forward and side scatter. Values indicate the percentage of
larger thymocytes in the boxed gate. B, Total thymocytes, as well as the DP
and DN subset, were subjected to cell cycle analysis using flow cytometry.
Values indicate the percentage thymocytes in cell cycle. C, Thymocytes
were stained after incubation in medium for 24 h to determine apoptosis
(AnnexinV?) and necrosis (7-AAD?or PI?). The increase in apoptosis
was confined to the CD4?CD8?population, which had lower numbers of
cells that expressed Bcl-xLby intracellular staining. f represent WT, and
? represent Wip1?/?mice. Each bar represents the mean with SD whis-
kers of three individual animals. The data are representative of two or more
separate experiments. The ? indicate significant differences (p ? 0.05)
between WT and Wip1-deficient cells.
DP thymocytes from Wip1?/?mice have altered cell cycle
lower numbers of DP and SP cells and fail to regulate p38 MAPK. A,
Thymocytes from 4-wk-old animals were analyzed for CD4 and CD8 sur-
face expression using flow cytometry, and the percent positive of each
subpopulation is depicted. Similar results were obtained for mice at 6, 8,
and 12 wk of age. B, The total number of each thymocyte population from
4- to 6-wk-old WT and Wip1?/?mice was calculated from the percentage
of cells that was determined by flow cytometry. The numbers of CD3?
cells expressing the ?-TCR vs the ??-TCR were also determined. Similar
results were obtained with 8-wk-old mice. C, To determine the level of p38
MAPK activation, thymocytes were cultured with plate-bound anti-CD3
and anti-CD28 Abs for the indicated times. Cells were lysed, protein levels
were determined, and kinase activity was assessed. Numbers within FACS
panels represent the percentage of cells in the respective quadrants. Each
bar represents the mean with SD whiskers of three individual animals. The
data are representative of three or more separate experiments. The ? indi-
cate significant differences (p ? 0.05) between WT and Wip1-deficient
Thymocytes from Wip1-deficient mice have significantly
4821 The Journal of Immunology
knockout mice. However, compared with WT mice, Wip1?/?
mice contain a higher percentage of larger thymocytes, suggesting
that more cells were in cycle (Fig. 3A). To evaluate this more
closely, thymocytes were subjected to cell cycle analysis by flow
cytometry to determine whether the reduced cellularity of the thy-
mus could be accounted for by alterations in cell cycle progression.
Freshly isolated total thymocytes, as well as the DP and DN sub-
populations, from Wip1?/?mice consistently had more cells in
S/G2/M than WT animals (Fig. 3B) despite the fact that DP cells
do not proliferate.
To determine whether Wip1?/?thymocytes were more prone to
undergo apoptosis, thymocytes were stained for annexin V and PI
or 7-AAD, and examined by flow cytometry. On average, the per-
centage of cells undergoing spontaneous apoptosis was 2-fold
higher in freshly isolated Wip1?/?thymocytes (4.3 ? 0.1%) com-
pared with WT (2.4 ? 0.4%) control cells. The difference in ap-
optosis in Wip1-deficient compared with WT thymocytes was in
the DP population, whereas the DN thymocytes of WT and
Wip1?/?mice had similar levels of apoptosis (annexin positive,
7-AAD negative) (Fig. 3C). The increase in the number of
Wip1?/?DP thymocytes undergoing apoptosis correlated with a
reduced number of DP cells expressing the antiapoptotic factor
A block from the DN to DP transition is due to a block in
thymocyte maturation at the DN4 stage of development
To determine which subpopulations within the DN compartment
expresses Wip1, thymocytes from WT mice were sorted by flow
cytometry and isolated based on surface marker expression. In WT
mice, Wip1 mRNA expression was dramatically increased at the
DN3 and DN4 developmental stages with lower levels present in
DP and SP cells (Fig. 4A). Further analyses by flow cytometry of
the DN subpopulations identified consistent defects in the absence
of Wip1. A greater percentage of Wip1?/?thymocytes were ob-
served at the DN3 stage and a lower percentage at the DN4 stage
when compared with age-matched WT animals (Fig. 4B). When
cell numbers were taken into account, there were no differences in
the number of DN3 cells, but there were lower numbers of DN4
cells (Fig. 4C). This suggests that thymic ?? T cell development
has a partial defect at the DN4 stage in Wip1-deficient mice.
To determine why fewer DN4 cells were present in mice that
lack Wip1, we assessed the levels of p53-related biochemical
markers (Fig. 4D). Thymocyte DN4 and DP subsets from both WT
and Wip1-deficient mice were purified by FACS sorting. Higher
levels of total p53, phospho-p53Ser15, and p21Waf1were found in
Wip1?/?DN4 and DP population. This also correlates with an
increase in the number of Wip1?/?DN4 cells that appear to be in
cell cycle (Fig. 4E), suggesting that lower numbers of DN4 cells in
Wip1?/?mice may be the result of increased p53 activity, which
blocks the cell cycle, without affecting cell proliferation or apo-
ptosis (Fig. 4E).
To determine whether elevated levels of p38 activity within the
DN subpopulations are responsible for the reduced numbers of DP
and SP cells, FTOC were used to determine whether blocking p38
MAPK can correct the thymic phenotype. Thymi from day 15
fetuses were cultured for 3 days in the presence (10 ?M) and
absence of the p38 MAPK inhibitor SB203580 (Fig. 4F). The
ment. A, FACS-sorted DN thymocytes from WT mice were analyzed for
Wip1 expression by real-time RT-PCR and evaluated in reference to
GAPDH. B, Thymocytes from 4- to 6-wk-old animals were used to deter-
mine the phenotype of the DN population with respect to CD44 and CD25
expression. Numbers within FACS panels represent the percentage of cells
in the respective quadrants. C, The number of thymocytes in each of the
DN subpopulation was calculated based on the number of DN cells. D,
Levels of p53, phospho-p53Ser15, p21Waf1, and ?-tubulin were detected in
FACS-sorted DN4 and DP thymocyte subsets from WT and Wip1-deficient
mice by Western blotting. E, The number of DN4 apoptotic cells (annexin
V), the number of cells (DN3 and DN4) in cycle (DAPI) and proliferating
(BrdU) were determined by flow cytometry. F, DN3 to DP thymic devel-
opment of day 15 fetal thymi cultured for 3 days in the presence and
absence of the p38 MAPK inhibitor SB203580 (10 ?M). Cell subset num-
bers were based on the percentage of cells, as determined by flow cytom-
Lack of Wip1 expression affects early thymocyte develop-
etry, and number of cells isolated after culture. f represent WT, and ?
represent Wip1?/?mice. Each bar represents the mean with SD whiskers
of three individual animals. The data are representative of two or more
separate experiments. The ? indicate significant differences (p ? 0.05)
between WT and Wip1-deficient cells.
4822 Wip1 REGULATION OF THYMOCYTE DEVELOPMENT
numbers of DN3 and DN4 cells were not affected, regardless of
whether the inhibitor was present. Although treatment with
SB203580 did reduce DP cell numbers from WT thymi, it had no
effect on Wip1-deficient thymi. Thus, p38 MAPK is not respon-
sible for the phenotype observed in Wip1?/?mice.
Thymocyte abnormalities of Wip1?/?mice are not observed for
mice that are also deficient for p53
Knowing that p53 needs to be suppressed for T cells to proceed
from the DN to DP stage (16–19) and that p53 levels are higher in
the absence of Wip1, we attempted to correct the T cell develop-
mental defects in Wip1?/?mice by removing p53. The thymic
abnormalities observed in mice that lack Wip1 were reversed when
the animals were bred onto a p53-null background. Not only did
the thymic cellularity (T cell numbers, percentage of DN thymo-
cytes, and their phenotype (CD44 and CD25 expression)) from
Wip1-deficient mice return to WT levels, but WT levels of apo-
ptosis (primarily in the DP population) and cell cycle progression
were restored as well (Fig. 5). This suggests that the block in T cell
development from DN to DP in Wip1-deficient thymocytes results
primarily from the inability to down-regulate p53.
In this study, we have defined the role of Wip1 phosphatase in the
control of p53 activity during murine thymic ?? T cell develop-
ment, and p38 MAPK regulation after TCR stimulation. Wip1
mRNA is induced at the DN3 and DN4 stage with lower levels
present in the DP and SP stage of thymocyte development. Al-
though Wip1 can dephosphorylate p38 MAPK, the kinase activity
was not different between WT and Wip1?/?thymocytes when the
cells were cultured in medium alone. It remains possible that Wip1
phosphatase may be induced, as part of a feed-back mechanism
due to p38 MAPK activation, because there was a delay in turning
off kinase activity in TCR-stimulated Wip1-deficient thymocytes
(Fig. 2C). Regardless, these data suggest that TCR activation may
use Wip1 to down-regulate T cell activation and supports p38
MAPK as a substrate for Wip1 in TCR-stimulated thymocytes.
Early T cell development is, in part, controlled by the activity of
p38 MAPK (26, 27). At the earliest DN stages, DN1 to DN3, p38
MAPK needs to be active, but is down-regulated as the cells
progress to the DN4 stage (14). The lower number of DN4 cells
isolated from Wip1?/?mice suggests that some cells are able to
down-regulate p38 MAPK, likely by alternative phosphatases that
may be less efficient. To determine whether p38 MAPK overex-
pression is playing a role in thymocyte development in Wip1?/?
mice, FTOC from both WT and Wip1-deficient mice were assayed
for thymocyte development in the presence and absence of
SB203580, a specific p38 MAPK inhibitor. The inhibitor did not
affect T cell development nor did it restore DP cell numbers, sug-
gesting that Wip1 does not play a role in early T cell development
through p38 MAPK.
p53 activity has been shown to affect T cell development in the
thymus. Several studies have shown that thymocyte differentiation
in some T cell-deficient mice (SCID, RAG1/2?/?, CD3??/?, and,
to some degree, CD3??/?) can be rescued in the absence of p53
(16–19). Normally, these cells are blocked at the DN3 stage, but
thymocytes will progress from a DN to a DP phenotype if the mice
are bred to a p53-deficient background. Furthermore, the presence
of a functional pre-TCR was shown to down-regulate phospho-p38
MAPK and phospho-p53Ser15and that cytoplasmic deletion mu-
tants of the pre-TCR?-chain failed to suppress this activity (28).
Previous observations have shown that p53 activity is elevated in
embryo fibroblasts derived from Wip1-deficient mice (29). Our
results show that p53 activity is increased in DN4 and DP thymo-
absence of p53. A, Representative thymi from 4- to 6-wk-old WT,
Wip1?/?, and Wip1?/?p53?/?double knockout mice were isolated and
photographed to show size differences. Thymocytes from all mice were
subjected to cell cycle analysis using PI (B), counted using trypan blue
exclusion (C), and analyzed by flow cytometry to determine the following:
the number of CD3?cells that express the ??-TCR vs the ??-TCR (D); the
percentage of cells that were CD4?CD8?(E); the percentage of cells un-
dergoing apoptosis after culturing in medium for 18–24 h (F); and the
phenotype of the DN population with respect to CD44 and CD25 expres-
sion (G). Each bar represents the mean with SD whiskers of three individ-
ual animals. The data are representative of three separate experiments.
Thymic abnormalities of Wip1?/?mice are rescued in the
4823The Journal of Immunology
cyte subsets, which suggests that the defect in thymic development
of Wip1?/?mice arose from the inability to down-regulate p53.
Although the signaling pathways required to inactivate p53 from
the pre-TCR are currently not known (18), our data suggests that
Wip1 may play a role because its expression is highly up-regulated
at the DN3 and DN4 stage.
p53 can control the fate of cells either through apoptosis or cell
cycle arrest. Although the numbers of apoptotic DN4 cells were
not significantly different in either WT or Wip1-deficient mice,
there was a significant increase in the number of Wip1?/?DN4
thymocytes in cell cycle (Fig. 4E), suggesting that Wip1?/?DN4
thymocytes were proliferating more than WT cells. However,
BrdU incorporation in the DN4 subset was not different in the
absence of Wip1 (Fig. 4E), demonstrating that the higher p53 ac-
tivity in the DN4 cells negatively affects the transition to DP via
cell cycle arrest. However, cells that express both CD4 and CD8
are more susceptible to apoptosis, presumably due to higher p53
levels and/or the lower number of Bcl-xLDP thymocytes, suggest-
ing that Wip1 plays different roles depending on the maturation
state of the thymocyte.
To determine whether p53 overexpression is responsible for the
phenotype described for Wip1-deficient mice, we bred the mice
onto a p53-deficient background. Interestingly, the thymic devel-
opmental defects in mice that lack Wip1 were corrected in the
absence of p53 (Fig. 5). This strongly supports the hypothesis that
p53-induced Wip1 acts as a negative feedback loop that down-
regulates p53 activity (23, 30). The importance of p53 in T cell
development has been largely ignored because p53-deficient mice
do not appear to have any early thymic defects. Although T cell
development has not been addressed in p53-transgenic mice, it
would be interesting to see whether the abnormalities observed in
Wip1-deficient mice are also recapitulated in mice that overex-
press p53. Nevertheless, our results suggest that Wip1 down-reg-
ulates p53 activity, which plays a role in the DN to DP transition
during ?? T cell development.
p53 has been shown to regulate other down-stream cell signal-
ing pathways that are known to affect thymic development. Re-
cently, p53 was found to negatively regulate thymic Notch1 acti-
vation (31), which was shown to be required for ?-selection (32)
and the DN to DP transition (33) of thymocytes. If the above
observations are true, then Notch1 activation and the expression of
downstream targets such as HES1 in Wip1?/?thymocyte DN4
subpopulations and the percentage of CD8 SP cells should be sig-
nificantly reduced. Although we have not determined HES1 pro-
tein levels in thymocyte subpopulations, we did not see reduced
percentages restricted to the CD8 SP population in Wip1-deficient
mice, suggesting that Notch signaling is not significantly compro-
mised. Interestingly, Wnt signaling through ?-catenin was also
shown to be negatively regulated by p53 (34). Thus, the possibility
exists that the Wnt pathway may be altered in Wip1-deficient mice.
It has been observed that p53 down-regulates ?-catenin at the level
of transcription (35) and through a proteosome-mediated mecha-
nism involving glycogen synthase kinase-3? activity (34, 36, 37).
Interestingly, ?-catenin-deficient mice were also found to have de-
fective T cell development at the ?-selection checkpoint (38), sug-
gesting that the thymic phenotype of Wip1?/?mice may be due to
altered Wnt signaling resulting from elevated p53 activity. Cur-
rently, we are pursuing both of these options to determine whether
the expressionlevels correlate
Although we cannot rule out the involvement of thymic epithe-
lial cells in the Wip1 phenotype, the biochemical and in vitro bi-
ological differences observed support the premise that Wip1 di-
rectly affects thymic T cells. The finding that Wip1-deficient mice
are relatively resistant to the development of certain types of breast
cancer (29) have far-reaching implications. Because Wip1 is am-
plified in a number of human cancers, including breast tumors
(30), the phosphatase could be a new target for treating breast
cancer patients (39). Wip1 is also overexpressed in neuroblastomas
and could also be a potential target for therapy (40). Neuroblas-
tomas are one of the most common cancers in children accounting
for ?15% of all cancer-related pediatric deaths in the United
States (41) and about half of all cases result in death. Because most
neuroblastoma patients are young infants with a developing im-
mune system, treatments that would inhibit Wip1 may have im-
munological complications. The severity and extent that Wip1 in-
hibition could affect thymic development in these young patients
would likely be dependent on patient age and length of treatment.
Therefore, appropriate precautions must be taken into account to
identify any long-term immunological risks that may be associated
with inhibiting Wip1 in pediatric patients.
We are indebted to C. Sommers and B. J. Taylor for their help with flow
cytometry, sorting, and insightful discussion of the data. We are grateful to
A. Cheever for evaluating histological samples and M. Vacchio for setting
up the FTOC.
The authors have no financial conflict of interest.
1. Godfrey, D. I., J. Kennedy, T. Suda, and A. Zlotnik. 1993. A developmental
pathway involving four phenotypically and functionally distinct subsets of
CD3?CD4?CD8?triple-negative adult mouse thymocytes defined by CD44 and
CD25 expression. J. Immunol. 150: 4244–4252.
2. Dudley, E. C., H. T. Petrie, L. M. Shah, M. J. Owen, and A. C. Hayday. 1994.
T cell receptor ? chain gene rearrangement and selection during thymocyte de-
velopment in adult mice. Immunity 1: 83–93.
3. Fehling, H. J., A. Krotkova, C. Saint-Ruf, and H. von Boehmer. 1995. Crucial
role of the pre-T-cell receptor ? gene in development of ?? but not ?? T cells.
Nature 375: 795–798.
4. Mombaerts, P., A. R. Clarke, M. A. Rudnicki, J. Iacomini, S. Itohara,
J. J. Lafaille, L. Wang, Y. Ichikawa, R. Jaenisch, M. L. Hooper, et al. 1992.
Mutations in T-cell antigen receptor genes ? and ? block thymocyte development
at different stages. Nature 360: 225–231.
5. von Boehmer, H. 1999. T-cell development: what does Notch do for T cells?
Curr. Biol. 9: R186–R188.
6. Falk, I., G. Nerz, I. Haidl, A. Krotkova, and K. Eichmann. 2001. Immature thy-
mocytes that fail to express TCR? and/or TCR?? proteins die by apoptotic cell
death in the CD44?CD25?(DN4) subset. Eur. J. Immunol. 31: 3308–3317.
7. Groettrup, M., K. Ungewiss, O. Azogui, R. Palacios, M. J. Owen, A. C. Hayday,
and H. von Boehmer. 1993. A novel disulfide-linked heterodimer on pre-T cells
consists of the T cell receptor ? chain and a 33 kd glycoprotein. Cell 75: 283–294.
8. Hoffman, E. S., L. Passoni, T. Crompton, T. M. Leu, D. G. Schatz, A. Koff,
M. J. Owen, and A. C. Hayday. 1996. Productive T-cell receptor ?-chain gene
rearrangement: coincident regulation of cell cycle and clonality during develop-
ment in vivo. Genes Dev. 10: 948–962.
9. Werlen, G., B. Hausmann, D. Naeher, and E. Palmer. 2003. Signaling life and
death in the thymus: timing is everything. Science 299: 1859–1863.
10. Lehar, S. M., and M. J. Bevan. 2002. T cell development in culture. Immunity 17:
11. Petrie, H. T. 2002. Role of thymic organ structure and stromal composition in
steady-state postnatal T-cell production. Immunol. Rev. 189: 8–19.
12. Porter, B. O., and T. R. Malek. 2000. Thymic and intestinal intraepithelial T
lymphocyte development are each regulated by the ?c-dependent cytokines IL-2,
IL-7, and IL-15. Semin. Immunol. 12: 465–474.
13. Rossi, D., and A. Zlotnik. 2000. The biology of chemokines and their receptors.
Annu. Rev. Immunol. 18: 217–242.
14. Diehl, N. L., H. Enslen, K. A. Fortner, C. Merritt, N. Stetson, C. Charland,
R. A. Flavell, R. J. Davis, and M. Rincon. 2000. Activation of the p38 mitogen-
activated protein kinase pathway arrests cell cycle progression and differentiation
of immature thymocytes in vivo. J. Exp. Med. 191: 321–334.
15. Costello, P. S., S. C. Cleverley, R. Galandrini, S. W. Henning, and D. A. Cantrell.
2000. The GTPase Rho controls a p53-dependent survival checkpoint during
thymopoiesis. J. Exp. Med. 192: 77–85.
16. Bogue, M. A., C. Zhu, E. Aguilar-Cordova, L. A. Donehower, and D. B. Roth.
1996. p53 is required for both radiation-induced differentiation and rescue of
V(D)J rearrangement in scid mouse thymocytes. Genes Dev. 10: 553–565.
17. Guidos, C. J., C. J. Williams, I. Grandal, G. Knowles, M. T. Huang, and
J. S. Danska. 1996. V(D)J recombination activates a p53-dependent DNA dam-
age checkpoint in scid lymphocyte precursors. Genes Dev. 10: 2038–2054.
4824Wip1 REGULATION OF THYMOCYTE DEVELOPMENT
18. Haks, M. C., P. Krimpenfort, J. H. van den Brakel, and A. M. Kruisbeek. 1999. Download full-text
Pre-TCR signaling and inactivation of p53 induces crucial cell survival pathways
in pre-T cells. Immunity 11: 91–101.
19. Jiang, D., M. J. Lenardo, and J. C. Zuniga-Pflucker. 1996. p53 prevents matu-
ration to the CD4?CD8?stage of thymocyte differentiation in the absence of T
cell receptor rearrangement. J. Exp. Med. 183: 1923–1928.
20. Fiscella, M., H. Zhang, S. Fan, K. Sakaguchi, S. Shen, W. E. Mercer,
G. F. Vande Woude, P. M. O’Connor, and E. Appella. 1997. Wip1, a novel
human protein phosphatase that is induced in response to ionizing radiation in a
p53-dependent manner. Proc. Natl. Acad. Sci. USA 94: 6048–6053.
21. Choi, J., E. Appella, and L. A. Donehower. 2000. The structure and expression
of the murine wildtype p53-induced phosphatase 1 (Wip1) gene. Genomics 64:
22. Choi, J., B. Nannenga, O. N. Demidov, D. V. Bulavin, A. Cooney, C. Brayton,
Y. Zhang, I. N. Mbawuike, A. Bradley, E. Appella, and L. A. Donehower. 2002.
Mice deficient for the wild-type p53-induced phosphatase gene (Wip1) exhibit
defects in reproductive organs, immune function, and cell cycle control. Mol.
Cell. Biol. 22: 1094–1105.
23. Takekawa, M., M. Adachi, A. Nakahata, I. Nakayama, F. Itoh, H. Tsukuda,
Y. Taya, and K. Imai. 2000. p53-inducible wip1 phosphatase mediates a negative
feedback regulation of p38 MAPK-p53 signaling in response to UV radiation.
EMBO J. 19: 6517–6526.
24. Lu, X., D. Bocangel, B. Nannenga, H. Yamaguchi, E. Appella, and
L. A. Donehower. 2004. The p53-induced oncogenic phosphatase PPM1D inter-
acts with uracil DNA glycosylase and suppresses base excision repair. Mol. Cell.
25. Donehower, L. A., M. Harvey, B. L. Slagle, M. J. McArthur, C. A. Montgomery,
Jr., J. S. Butel, and A. Bradley. 1992. Mice deficient for p53 are developmentally
normal but susceptible to spontaneous tumours. Nature 356: 215–221.
26. Rincon, M., D. Conze, L. Weiss, N. L. Diehl, K. A. Fortner, D. Yang,
R. A. Flavell, H. Enslen, A. Whitmarsh, and R. J. Davis. 2000. Conference
highlight: do T cells care about the mitogen-activated protein kinase signalling
pathways? Immunol. Cell Biol. 78: 166–175.
27. Rincon, M. 2001. MAP-kinase signaling pathways in T cells. Curr. Opin. Im-
munol. 13: 339–345.
28. Bulavin, D. V., C. Phillips, B. Nannenga, O. Timofeev, L. A. Donehower,
C. W. Anderson, E. Appella, and A. J. Fornace, Jr. 2004. Inactivation of the Wip1
phosphatase inhibits mammary tumorigenesis through p38 MAPK-mediated ac-
tivation of the p16Ink4a-p19Arfpathway. Nat. Genet. 36: 343–350.
29. Murga, C., and D. F. Barber. 2002. Molecular mechanisms of pre-T cell receptor-
induced survival. J. Biol. Chem. 277: 39156–39162.
30. Bulavin, D. V., O. N. Demidov, S. Saito, P. Kauraniemi, C. Phillips,
S. A. Amundson, C. Ambrosino, G. Sauter, A. R. Nebreda, C. W. Anderson, et
al. 2002. Amplification of PPM1D in human tumors abrogates p53 tumor-sup-
pressor activity. Nat. Genet. 31: 210–215.
31. Laws, A. M., and B. A. Osborne. 2004. p53 regulates thymic Notch1 activation.
Eur. J. Immunol. 34: 726–734.
32. Ciofani, M., T. M. Schmitt, A. Ciofani, A. M. Michie, N. Cuburu, A. Aublin,
J. L. Maryanski, and J. C. Zuniga-Pflucker. 2004. Obligatory role for cooperative
signaling by pre-TCR and Notch during thymocyte differentiation. J. Immunol.
33. Huang, E. Y., A. M. Gallegos, S. M. Richards, S. M. Lehar, and M. J. Bevan.
2003. Surface expression of Notch1 on thymocytes: correlation with the double-
negative to double-positive transition. J. Immunol. 171: 2296–2304.
34. Sadot, E., B. Geiger, M. Oren, and A. Ben Ze’ev. 2001. Down-regulation of
?-catenin by activated p53. Mol. Cell. Biol. 21: 6768–6781.
35. Rother, K., C. Johne, K. Spiesbach, U. Haugwitz, K. Tschop, M. Wasner,
L. Klein-Hitpass, T. Moroy, J. Mossner, and K. Engeland. 2004. Identification of
Tcf-4 as a transcriptional target of p53 signalling. Oncogene 23: 3376–3384.
36. Levina, E., M. Oren, and A. Ben Ze’ev. 2004. Downregulation of ?-catenin by
p53 involves changes in the rate of ?-catenin phosphorylation and Axin dynam-
ics. Oncogene 23: 4444–4453.
37. Xiao, J. H., C. Ghosn, C. Hinchman, C. Forbes, J. Wang, N. Snider, A. Cordrey,
Y. Zhao, and R. A. Chandraratna. 2003. Adenomatous polyposis coli (APC)-
independent regulation of ?-catenin degradation via a retinoid X receptor-medi-
ated pathway. J. Biol. Chem. 278: 29954–29962.
38. Xu, Y., D. Banerjee, J. Huelsken, W. Birchmeier, and J. M. Sen. 2003. Deletion
of ?-catenin impairs T cell development. Nat. Immunol. 4: 1177–1182.
39. Bernards, R. 2004. Wip-ing out cancer. Nat. Genet. 36: 319–320.
40. Saito-Ohara, F., I. Imoto, J. Inoue, H. Hosoi, A. Nakagawara, T. Sugimoto, and
J. Inazawa. 2003. PPM1D is a potential target for 17q gain in neuroblastoma.
Cancer Res. 63: 1876–1883.
41. Landis, S. H., T. Murray, S. Bolden, and P. A. Wingo. 1999. Cancer statistics,
1999. CA Cancer J. Clin. 49: 8–31.
4825 The Journal of Immunology