Cellular and Molecular Characterization of the scurfy Mouse
Lisa B. Clark,* Mark W. Appleby,* Mary E. Brunkow,* J. Erby Wilkinson,†
Steven F. Ziegler,1* and Fred Ramsdell2*
Mice hemizygous (Xsf/Y) for the X-linked mutation scurfy (sf) develop a severe and rapidly fatal lymphoproliferative disease
mediated by CD4?CD8?T lymphocytes. We have undertaken phenotypic and functional studies to more accurately identify the
immunologic pathway(s) affected by this important mutation. Flow cytometric analyses of lymphoid cell populations reveal that
scurfy syndrome is characterized by changes in several phenotypic parameters, including an increase in Mac-1?cells and a
decrease in B220?cells, changes that may result from the production of extremely high levels of the cytokine granulocyte-
macrophage CSF by scurfy T cells. Scurfy T cells also exhibit strong up-regulation of cell surface Ags indicative of in vivo
activation, including CD69, CD25, CD80, and CD86. Both scurfy and normal T cells are responsive to two distinct signals provided
by the TCR and by ligation of CD28; scurfy cells, however, are hyperresponsive to TCR ligation and exhibit a decreased re-
quirement for costimulation through CD28 relative to normal controls. This hypersensitivity may result, in part, from increased
costimulation through B7-1 and B7-2, whose expression is up-regulated on scurfy T cells. Although the specific defect leading to
this hyperactivation has not been identified, we also demonstrate that scurfy T cells are less sensitive than normal controls to
inhibitors of tyrosine kinases such as genistein and herbimycin A, and the immunosuppressant cyclosporin A. One interpretation
of our data would suggest that the scurfy mutation results in a defect, which interferes with the normal down-regulation of T cell
activation. The Journal of Immunology, 1999, 162: 2546–2554.
animal models for human immunologic disorders. These include
xid, the murine equivalent of X-linked agammaglobulinemia (1, 2),
beige (the equivalent of Chediak-Higashi syndrome) (3), lpr and
gld (defects in fas and fas ligand), X-linked severe combined im-
munodeficiency (4), and the hemopoietic cell phosphatase mutant
motheaten (SHP-1) (5). We have chosen to study an as yet un-
cloned X-linked mouse mutant, scurfy (sf). Mice hemizygous for
the scurfy mutation exhibit a severe lymphoproliferative disorder
(6–9). It is our hypothesis that the cloning of the gene responsible
for the scurfy syndrome will result in the discovery of a critical
component in the regulation of the immune system.
Males hemizygous (Xsf/Y) for the scurfy mutation develop a
progressive lymphocytic infiltration of the lymph nodes, spleen,
liver, and skin, resulting in gross morphologic symptoms that in-
clude splenomegaly, hepatomegaly, greatly enlarged lymph nodes,
runting, exfoliative dermatitis, and thickened malformed ears (7,
8). Other clinical symptoms include elevated leukocyte counts,
hypergammaglobulinemia, and severe anemia (6); the death of af-
fected males usually occurs by 3 wk of age. The sf locus has been
mapped to the extreme proximal region of the X chromosome,
approximately 0.7 cM from the locus for sparse-fur (spf) (6, 10),
itself a point mutation within the ornithine transcarbamylase gene
nherited mutations affecting the murine immune system have
proven to be a rich source of novel genes critical to the reg-
ulation of the immune system and have furnished important
(Otc) (11). The sf locus is also tightly linked to the murine Gata1,
Tcfe3, and Wasp loci (10, 12). Similarities between scurfy and
human Wiskott-Aldrich syndrome have been noted (6), and the
mouse Wasp gene has been proposed as a candidate for scurfy (6,
12). Closer biologic examination reveals significant differences be-
tween Wiskott-Aldrich syndrome and scurfy, however, and the
two loci have been demonstrated to be nonallelic (Jeffery &
Brunkow, unpublished data). Thus, the identity of the scurfy gene
remains to be determined.
Disease in scurfy mice has been shown to be primarily mediated
by CD4?CD8?T lymphocytes (8, 9, 13), suggesting that the sf
gene plays an important role in regulating T cell function. This T
cell defect is primarily manifested as a generalized overproduction
of cytokines. Dysregulated expression of a variety of cytokine
genes, including IL-2, IL-4, IL-5, IL-6, IL-10, IFN-?, and TNF-?,
has been demonstrated in scurfy mice at the levels of mRNA and
protein expression (13, 14). Excessive production of these cyto-
kines correlates well with the range of pathologic changes ob-
served in scurfy mice and may be the proximal cause of scurfy
immunopathology, yet the cellular and molecular mechanism(s)
leading to cytokine dysregulation and ultimately to pathology in
scurfy mice remain unknown.
The following functional and phenotypic studies of scurfy T
cells were undertaken to further our understanding of the disease
mechanism(s) underlying scurfy syndrome. Using flow-cytometric
analysis, we demonstrate that the onset of scurfy disease is char-
acterized by an increased relative abundance of Mac-1?cells and
a corresponding drop in the relative abundance of B220?cells in
lymphoid tissues. We also show that scurfy T cells produce ex-
tremely high levels of the cytokine GM-CSF,3a differentiation
factor for granulocytic and monocytic cells that is produced by
activated T cells and is known to suppress B lymphopoiesis (15).
*Chiroscience R&D, Inc., Seattle, WA 98021; and†Oak Ridge National Laboratory,
Oak Ridge, TN 37830
Received for publication July 13, 1998. Accepted for publication November 4, 1998.
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.
1Current address: Virginia Mason Research Center, Seattle, WA.
2Address correspondence and reprint requests to Dr. Fred Ramsdell, Chiroscience
R&D, Inc., 1631 220th Street S.E., Bothell, WA 98021. E-mail address:
3Abbreviations used in this paper: GM-CSF, granulocyte-macrophage colony-stim-
ulating factor; cRPMI, complete RPMI; CsA, cyclosporin A; NLC, normal littermate
control; PTK, protein tyrosine kinase.
Copyright © 1999 by The American Association of Immunologists0022-1767/99/$02.00
Cell surface staining of CD4?T cells from scurfy mice and normal
controls shows that scurfy T cells express elevated levels of acti-
vation-related Ags, including CD69, CD25 (IL-2R), and B7-1/B7-2.
Although CD4?T lymphocytes from scurfy mice exhibit an
activated surface phenotype, they are not constitutively activated,
but rather are hyperresponsive to TCR stimulation. In this study,
we demonstrate that scurfy CD4?cells exhibit the same require-
ment as normal T cells for two activation signals, yet differ mark-
edly from normal cells in the magnitude of responsiveness to these
signals. The heightened expression of B7 on scurfy cells may con-
tribute to the hypersensitivity of scurfy T cells to activation in
vitro. Our data are consistent with a model in which scurfy syn-
drome results from a biochemical defect that interferes with the
normal down-regulation of T cell activation responses.
Materials and Methods
The original scurfy mutation arose spontaneously in the partially inbred
MR stock at Oak Ridge National Laboratory (Oak Ridge, TN) in 1949. A
strain doubly mutant for sf and the closely-linked sparse-fur (spf) mutation
was generated (7) and has been maintained by mating sf spf/?? females
to (C3Hf/Rl ? 101/Rl)F1or (101/Rl ? C3Hf/Rl)F1males. The spf muta-
tion appears to have no effect on the scurfy phenotype. Doubly mutant
carrier females were obtained from Oak Ridge National Laboratory, and
the stock continued to be maintained by crossing to (101/Rl ? C3Hf/Rl)F1
males. Animals were housed in a conventional environment with a standard
pelleted diet and reverse osmosis water fed ad libitum. All animals used in
these functional studies were obtained from doubly mutant progeny-tested
females. Carriers of spf were identified directly by PCR amplification of a
171-bp fragment from the Otc gene (11). Upon digestion of the PCR prod-
ucts with MseI, nonmutant DNA produced fragments of 43 and 128 bp,
while mutant DNA produced fragments of 43, 93, and 35 bp. The primers
GAGAAGCATCA. Mice aged 10–15 days were used for all experiments,
except where noted.
T cell activation cultures
Lymph node, spleen, and thymic tissues removed from scurfy mice and
normal littermate control (NLC) animals were macerated in culture media
between the ground glass ends of sterile microscope slides, filtered through
a sterile 70-?m nylon mesh, collected by centrifugation, and cultured at
37°C in complete RPMI (cRPMI) (10% FBS, 0.05 mM 2-ME, 15 mM
HEPES, 100 U/ml penicillin, and 100 ?g/ml each streptomycin and glu-
tamine) in 96-well round-bottom tissue culture plates. Culture wells were
prepared for T cell activation cultures by preincubation with purified anti-
TCR? Ab (clone H57-597) in sterile PBS for 2 h at 37°C. Each well was
rinsed twice with sterile PBS to remove nonimmobilized Ab before the
initiation of T cell cultures. Purified anti-mouse CD28 (clone 37.51) and
hamster IgG isotype control (anti-mouse keyhole limpet hemacyanin)
(PharMingen, San Diego, CA) were coimmobilized on culture wells when
used in functional assays.
T cells were cultured for proliferation assays at a cell density of 5 ? 104
cells/well (sort purified) or 105cells/well (whole tissue suspensions) in 200
?l of cRPMI and incubated at 37°C for 72 h. Individual wells were pulsed
with 1 ?Ci/well of [3H]thymidine (Amersham Life Science, Arlington
Heights, IL) for the last 8–12 h of culture. Proliferation data reported are
based upon mean value of triplicate wells and represent a minimum of
Supernatants (100 ?l) were collected from activation cultures at 48 h post-
stimulation, and their cytokine levels were determined by the following
IL-2 levels were determined using a Cytoscreen murine IL-2 immunoassay
kit from BioSource International (Camarillo, CA), according to the man-
ufacturer’s directions. The minimum detection limit for this assay is 8
The combined production of IL-2 and IL-4 was measured using a cellular
proliferation assay utilizing the IL-2-dependent cell line HT-2 (clone A5E;
American Type Culture Collection, Manassas, VA), which also proliferates
in response to IL-4. A total of 104HT-2 cells/well was cultured in 96-well
round-bottom plates together with 100 ?l of conditioned supernatants re-
moved from 3-day scurfy or NLC T cell cultures. HT-2 assays were incu-
bated at 37°C for 72 h in 200 ?l final volume cRPMI and pulsed with
[3H]thymidine for the last 8–12 h of culture. The minimum detection limit
for this assay is 0.2 ng/ml (IL-2).
GM-CSF levels were determined using a Quantikine murine GM-CSF im-
munoassay kit from R&D Systems (Minneapolis, MN), according to the
manufacturer’s instructions. The minimum detection limit for this assay is
Thymus, lymph node, and splenic tissues collected as described above
were resuspended for fluorescence staining in staining buffer (1% BSA,
0.1% sodium azide in PBS) at a cell density of 20 ? 106/ml. Cell aliquots
were treated with 2% normal mouse serum (Sigma, St. Louis, MO) to block
nonspecific binding, then stained by incubation on ice for 30 min with
combinations of the following fluorochrome-conjugated anti-mouse mAbs:
B220/Ly-5 (clone RA3-6B2, rat IgG2a), CD3 (clone 500-A2, hamster
IgG), CD8? (clone CT-CD8b, rat IgG2a), CD4 (clone CT-CD4, rat IgG2a),
CD11b/Mac-1 (clone M1/70.15, rat IgG2a), CD25 (clone PC61 5.3, rat
IgG1), IgG2a control (Caltag Laboratories, Burlingame, CA); CD28 (clone
37.51, hamster IgG), CD45RB (clone 23G2, rat IgG2a,?), CD69 (clone
H1.2F3, hamster IgG), CD80/B7-1 (clone 16-10A1, hamster IgG), CD86/
B7-2 (clone GL1, rat IgG2a,?), and CTLA-4 (clone UC10, 4F10, hamster
The fluorescence intensity of approximately 105cells was examined
using a MoFlo flow cytometer (Cytomation, Fort Collins, CO) and ana-
lyzed with Cyclops (Cytomation) software. Cell doublets and monocytic
cells were eliminated from the analysis on the basis of forward and side
light scatter gates, and dead cells were excluded by propidium iodide (10
?g/ml) staining. Data typically are shown for pooled cells from two to four
mice per experiment.
Fluorescence-activated cell sorting
CD4?T lymphocytes were sort purified from lymph nodes for functional
assays, as follows: lymph node cells were fluorescence stained by incuba-
tion on ice at a density of 20 ? 106/ml in sterile staining buffer (no sodium
azide) for 20 min with FITC-conjugated anti-mouse B220 and anti-mouse
CD8? (Caltag Laboratories) at a final concentration of 5 ?g/ml. The FITC-
stained target population consisting of B cells and CD8?T lymphocytes
(approximately 30–40% of the total cell population) was negatively sorted
from the desired CD4?T lymphocyte population by cell sorting using
MoFlo and Cyclops (Cytomation) software. Typical sort purities, as deter-
mined by postsort analysis, were 97–99% of the target population and
approximately 90–95% CD4?.
Onset of scurfy is accompanied by alterations in relative
abundance of Mac1?and B220?cells in lymphoid tissues
The gross features of the scurfy mutation have been described
previously (6–8). Neonatal scurfy pups are indistinguishable from
NLC until several days of age, but rapidly exhibit runting and
failure to thrive. Scurfy mice subsequently become increasingly
moribund until their deaths at 2–3 wk of age. To define better the
changes in the lymphoid system that accompany this rapid disease
progression, we performed flow cytometric analysis of immuno-
cytes from normal and scurfy animals. Although disease progres-
sion in the scurfy mouse has been attributed to CD4?T cells, the
disease was most noticeably manifest by alterations in the relative
abundance of B cells and macrophages between scurfy mice and
normal littermates (Fig. 1).
Both spleen cells and lymph node cells from scurfy animals are
characterized by a progressive increase in the proportion of
Mac-1?and a concomitant decrease in the proportion of B220?
2547 The Journal of Immunology
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2554 CHARACTERIZATION OF THE scurfy MOUSE MUTANT