Lymphopenic mice reconstituted with limited
repertoire T cells develop severe, multiorgan,
Th2-associated inflammatory disease
Joshua D. Milner*, Jerrold M. Ward†, Andrea Keane-Myers‡, and William E. Paul*§
*Laboratory of Immunology,†Infectious Disease Pathogenesis Section, Comparative Medicine Branch, and‡Laboratory of Allergic Diseases,
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
Contributed by William E. Paul, November 20, 2006 (sent for review November 3, 2006)
Lymphopenia and restricted T cell repertoires in humans are often
associated with severe eosinophilic disease and a T cell Th2 bias. To
examine the pathogenesis of this phenomenon, C57BL/6 Rag2?/?
mice received limited (3 ? 104) or large (2 ? 106) numbers of CD4
T cells. Three to 5 months after transfer, mice that had received 3 ?
104T cells, but not those that received 2 ? 106, developed
fulminant macrophage pneumonia with eosinophilia, Ym1 depo-
sition, and methacholine-induced airway hyperresponsiveness, as
well as eosinophilic gastritis; esophagitis and other organ damage
occurred in some cases. Donor cells were enriched for IL-4, IL-5, and
IL-13 producers. When 3 ? 104cells were transferred into CD3??/?
hosts, the mice developed strikingly elevated serum IgE. Prior
transfer of 3 ? 105CD25? CD4 T cells into Rag2?/? recipients
prevented disease upon subsequent transfer of CD25? CD4 T cells,
whereas 3 ? 104regulatory T cells (Tregs) did not, despite the fact
of transfer of CD25? CD4 T cells. Limited repertoire complexity of
Tregs may lead to a failure to control induction of immunopatho-
logic responses, and limitation in repertoire complexity of conven-
tional cells may be responsible for the Th2 phenotype.
eosinophils ? IgE ? IL-4 ? macrophages ? pneumonia
phenotype is eosinophilia, occasionally markedly elevated levels
of serum IgE, and lymphocytic infiltration of parenchymal
tissues. This phenotype is seen in Omenn’s syndrome, maternal
engraftment in SCID, and atypical complete DiGeorge syn-
drome (1). Omenn’s syndrome is perhaps the most well studied
of these diseases. It is a severe combined immunodeficiency
commonly caused by mutations in Rag 1 or Rag 2 that severely
reduce, but do not eliminate, the recombinase’s function (2). In
patients with Omenn’s syndrome the periphery is populated by
oligoclonal T cell populations heavily weighted toward expres-
sion of the Th2 phenotype, despite the fact that there is no
intrinsic defect in the peripheral T cells themselves. The limited
T cell repertoire seen in Omenn’s syndrome is also a feature of
the other immunodeficiencies associated with erythroderma,
hypereosinophilia and elevated serum IgE. In advanced HIV
infection, the eosinophilia that often develops is associated with,
and may be caused by, the limited T cell repertoire (3). Cuta-
neous T cell lymphoma (mycosis fungoides) has also been
reported to be associated with a limited peripheral T cell
receptor (TCR) repertoire, a Th2 phenotype, and peripheral
eosinophilia (4, 5).
Examples of lymphopenia associated with a Th2 phenotype,
hypereosinophilia, and/or elevated IgE have also been observed
in mice. This phenotype is seen in lat mutants (6, 7). It has
recently been reported that mice with limited numbers of CD4
T cells, such as MHC class II?/? mice and nu/nu mice, have
elevated serum IgE (8). There are no mouse models, however,
of lymphopenia in the context of normal thymic and peripheral
n primary human immunodeficiencies in which limited num-
bers of T cells are delivered to the periphery, a common
Upon transfer into lymphopenic hosts, T cells undergo a
process termed homeostatic or lymphopenia-induced prolifera-
tion. This proliferation is thought to be driven by cytokines as
well as TCR engagement. It is unclear whether the peptides
recognized by the proliferating T cells are derived from self-
proteins, from gut flora, or from foreign antigens (9).
We have reported that a reduced TCR repertoire with normal
numbers of memory phenotype CD4 cells can be achieved by
transferring small numbers of CD4? T cells into lymphopenic
recipients (10). The question arises as to whether this state of
reduced repertoire could have deleterious effects for the recip-
ient organism, much like the profound phenotype seen in the
immunodeficient conditions mentioned above. Here we show
that these mice develop a severe, multiorgan eosinophilic dis-
ease, strikingly elevated levels of serum IgE (when B cells are
present), and a memory population of Th2-phenotype CD4 T
cells. An important element in the development of this disease
appears to be the limited repertoire of regulatory T cells (Tregs).
Rag2?/? Mice Receiving a Small Number of CD4 T Cells Develop
Severe Multiorgan Inflammatory Disease. Transfer of CD4 T cells
from C57BL/6 donors into syngeneic Rag2?/? recipients leads
to rapid proliferation of a portion of the transferred cells. At 2
months after transfer, the number of CD44hiCD4 cells in the
lymph nodes of the recipients is ?1 ? 106, independent of the
number of cells transferred, over a range from 104to 107(10).
Although the number of CD44hiCD4 T cells present 6 weeks
after transfer was independent of the number of transferred
cells, the recipients of large and small numbers of cells showed
a striking difference in their subsequent development of an
eosinophilic inflammatory disease. C57BL/6 Rag2?/? mice
received either 3 ? 104or 2 ? 106CD4 lymph node T cells from
C57BL/6 donors. Three to 6 months after transfer, mice that had
received 3 ? 104, but not mice that had received 2 ? 106, CD4
T cells had severe macrophage pneumonia with eosinophilic and
lymphocytic infiltrates, mucus metaplasia of airway epithelium,
and eosinophilic crystal formation, both within pulmonary mac-
crystals were found to be Ym1-positive (Fig. 1 D and E). Ym1
is an eosinophilic and potentially eosinophilotactic crystal pro-
also displayed methacholine airway hypersensitivity (Fig. 3A).
Author contributions: J.D.M., A.K.-M., and W.E.P. designed research; J.D.M., J.M.W., and
A.K.-M. performed research; J.M.W. and A.K.-M. contributed new reagents/analytic tools;
J.D.M., J.M.W., A.K.-M., and W.E.P. analyzed data; and J.D.M. and W.E.P. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Abbreviations: Treg, regulatory T cell; TCR, T cell receptor; PE, phycoerythrin.
§To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
January 9, 2007 ?
vol. 104 ?
A marked eosinophilic gastritis with inflammation of the
glandular stomach and forestomach and with complete parietal
cell loss with some vacuolization was also present in mice that
received 3 ? 104, but not 2 ? 106, CD4 T cells (Figs. 1F and 2B).
Some recipients of 3 ? 104CD4 T cells also developed eosin-
ophilic esophagitis and variable small and large bowel lesions,
lesions commonly found in these mice were myeloid hyperplasia
and chronic inflammation, with occasional eosinophilic infiltra-
tion of the liver, and mesenteric lymph node granulomas with
fibrosis and giant cells [supporting information (SI) Fig. 6].
Sporadic lymphomas, sarcomatous change of mesenteric gran-
ulomata, conjunctivitis with eosinophils (SI Fig. 6), and spinal
neuritis were also noted.
Even at 1 month after transfer of 3 ? 104CD4 T cells, sparse
areas of perivascular lymphocytic infiltration in the lungs and
eosinophilic and lymphocytic inflammation in the stomach were
noted (SI Fig. 6). At 2 months after transfer of 3 ? 104CD4 T
cells, more pronounced lesions with macrophage infiltration in
the lungs and mucus metaplasia of bronchiolar endothelium
were found, in some instances encompassing entire lung lobes.
In the stomachs of these ‘‘2-month’’ mice there was more
pronounced eosinophilic and lymphocytic inflammation, partic-
ularly of the glandular stomach. The inflammatory response was
similar, if not more severe, in mice that had received 3 ? 104
CD25? ‘‘naı ¨ve’’ (CD44lo) CD4 T cells. More variably, transfer
of 2 ? 106CD25? naı ¨ve CD4 T cells also induced this inflam-
matory disease (Table 1 and Fig. 2). CD4 T cells derived from
AND TCR transgenic mice on a Rag2?/? background, whose T
cells are specific for a pigeon cytochrome c peptide, did not
induce disease when either 3 ? 104or 2 ? 106cells were
transferred, although these AND cells underwent vigorous pro-
liferation in the lymphopenic host. This finding indicates that not
all proliferating T cells with limited receptor diversity (here,
monoclonal) can induce disease (Table 1).
Phenotype and Is Associated with Elevated Serum IgE in CD3??/?
from peripheral lymph nodes of mice that had received 3 ? 104
cells produced IL-4, IL-5, and IL-13 upon ex vivo stimulation
than those that had received 2 ? 106cells. The frequency of
IFN?-producing cells was similar in mice that had received small
or large numbers of cells (Fig. 3B).
When limited numbers of CD4 T cells were transferred into
CD3??/? recipients, which lack T cells but have B cells, striking
elevation in serum IgE levels was noted. Thirty thousand total
CD4 T cells or naı ¨ve CD25? CD4 T cells or 2 ? 106total CD4
T cells or naı ¨ve CD25? CD4 T cells were transferred into
CD3??/? recipients. By 1 week after transfer, mice that had
received the larger numbers of T cells displayed detectable
serum IgE. At 4 weeks after transfer, all mice had significant
amounts of serum IgE (1–10 ?g/ml). However, at 8 weeks and 12
weeks after transfer, mice that had received 3 ? 104CD4 T cells
displayed massive increases in serum IgE, with a mean concen-
tration of ?350 ?g/ml in mice that had received 3 ? 104naı ¨ve
CD25? CD4 T cells 12 weeks earlier (Fig. 3C). The induction of
IgE depended on IL-4 produced by the transferred cells, because
Il4?/? donor CD4? T cells were unable to elicit detectable IgE
Rag2?/? mice. (A) Gross pathology of lungs from normal Rag2?/? mice and
mice that had received 3 ? 104CD4 T cells. (B and C) H&E stain (B) and Luna
stain (C) for eosinophils (red arrow) and eosinophilic crystal-laden macro-
from mice that had received 3 ? 104CD4 T cells (D) and of lungs from normal
mice that had received 3 ? 104CD4 T cells showing eosinophilic and lympho-
cytic infiltrate with parietal cell loss.
Pathology at 4 months after transfer of 3 ? 104CD4 T cells into
glandular stomach and forestomach (B) from Rag2?/? mice 4 months after
transfer of 30,000 CD25? CD44loCD4 T cells, 30,000 CD4 T cells, 2 million
CD25? CD44loCD4 T cells, or 2 million CD4 T cells and from an age-matched
Rag2?/? control that had not received cells.
Representative H&E sections of whole lung (A) and junction of
Milner et al.PNAS ?
January 9, 2007 ?
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no. 2 ?
not shown). As noted above, recipients of 3 ? 104CD4 T cells
displayed airway hypersensitivity as did recipients of 3 ? 104and
2 ? 106naı ¨ve CD25? CD4 T cells. Mice that received 2 ? 106
CD4 T cells did not display airway hypersensitivity (Fig. 3A).
Failure of Lymphocytes from Affected Mice to Transfer Accelerated
Disease Onset to Naı ¨ve Recipients. To determine whether lymph
node T cells from mice that had severe macrophage pneumonia
would cause an accelerated onset of disease when transferred to
Rag2?/? recipients, 2 ? 105lymph node T cells from such mice
were transferred to Rag2?/? hosts. In two experiments involv-
ing 10 recipients, no disease was noted up to 1.5 months after the
transfer, implying that these cells did not cause an accelerated
onset of disease.
Disease Is Associated with Autoantibody Formation. To determine
whether CD3??/? recipients of small numbers of CD4 T cells
developed antiparietal cell antibodies, we incubated normal
total or CD25? naı ¨ve CD4 T cells had no detectable antiparietal
cell antibodies (0/4 and 0/3, respectively) whereas almost all of
those receiving either 3 ? 104total or CD25? naı ¨ve CD4 T cells
were strongly positive for such antibodies (3/4 and 4/4, respec-
tively) (Fig. 4).
CD25? Tregs Play a Role in Controlling Lymphopenia-Associated
Disease. When limited numbers of CD4 T cells are transferred
into lymphopenic recipients, the memory cells present 1 month
after transfer have very limited TCR repertoire diversity (10).
Because the relative expansion of CD25? and CD25? cells in
these mice is similar (10), it is likely that the TCR repertoires of
both the conventional and regulatory T cells in mice that
received small numbers of CD4 T cells are of limited complexity.
We asked whether pretransfer of CD25? cells could protect
mice against the induction of the eosinophilic inflammatory
disease induced by CD25? naı ¨ve CD4 T cells and whether the
number of initially transferred CD25? cells would determine
whether there was protection.
A total of 3 ? 104or 3 ? 105CD25? CD4 T cells were injected
into Rag2?/? mice. At 2 months, mice were killed and the total
number of CD25? T cells in the lymph nodes and spleen were
similar (Fig. 5A), consistent with a previous report (12). We then
introduced 2 ? 106CFSE-labeled CD25? naı ¨ve CD4 T cells into
other mice that had initially received similar transfers of CD25?
T cells. Although the number of CD25? cells at the time of the
secondary transfer was similar in mice that had initially received
3 ? 104or 3 ? 105CD25? cells, only in mice that had received
the larger number of CD25? cells was the rapid division of the
To test whether the pretransfer of Tregs would regulate
disease induction, 3 ? 104or 3 ? 105sorted, CD25? CD4 T cells
were injected into Rag2?/? mice. Two months later 2 ? 105
CD25? CD4 T cells were transferred into these mice, and the
animals were killed a further 2 months later. The number of
CD25? T cells was similar at the time of death, but there were
five times as many CD25? CD4 T cells in the mice that had
initially received 3 ? 104CD25? CD4 T cells as in those that
received an initial transfer of 3 ? 105CD25? CD4 T cells (Fig.
5C). Mice that had initially received 3 ? 104CD25? CD4 T cells
developed severe eosinophilic lung and stomach disease,
whereas the mice that had initially received 3 ? 105CD25? CD4
T cells developed far fewer lesions (Fig. 5 D–G).
We describe here a severe multiorgan eosinophilic disease that
develops in the context of lymphopenia with reduced T cell
repertoire. This disease appears to be, in part, due to the lack of
a diverse TCR repertoire among the Foxp3? Tregs. It can be
argued that limitation in TCR diversity in the Treg population
hypersensitivity. Mice were placed individually in a whole-body plethysmo-
graph (Buxco Electronics), and Penh values were calculated. There were three
a number of the mice in the groups receiving 30,000 T cells. (B) Peripheral
T cells 4 months earlier were stimulated with PMA and ionomycin for 6 h;
monensin was added for the last 2 h. Cells were stained for CD45.1 to identify
transferred cells. Cells were then fixed, permeabilized, and stained for intra-
cellular cytokines. Each group consisted of four to five mice; shown is a
representative experiment of three similar experiments. (C) Elevated serum
IgE. B10.A CD3??/? mice intravenously received the cells indicated and were
four mice in each group; this experiment was repeated once with similar
results. Standard error bars are shown.
Th2 phenotype predominates in diseased mice. (A) Methacholine
www.pnas.org?cgi?doi?10.1073?pnas.0610289104 Milner et al.
would make these cells less efficient in inhibiting the action of
conventional (effector) T cells that mediate the immunopathol-
ogy observed in this transfer system. The implication is not
necessarily that the conventional and regulatory T cells popu-
lations must have matching repertoires for normal control of
autoimmunity/immunopathology but rather that some level of
intersection of repertoires is needed, if only to ensure that
in the same place, at the same time, so that the regulatory cells
could have the opportunity to control the potential effectors.
Although the importance of regulatory T cells in the control
of the immunopathology observed when small numbers of CD4
T cells are transferred into lymphopenic recipients seems clear,
the reason for the Th2 diathesis in the resulting immunopathol-
ogy is not obvious. It is possible that the absence of effective
regulatory cells by itself is sufficient to induce a Th2-like
immunopathology, independent of the number of ‘‘effector’’
cells transferred. Foxp3?/? mice demonstrate severe Th2 dis-
ease with markedly elevated serum Th2 (and Th1) cytokines as
IgE levels are not as high in mice that received 2 ? 106sorted
naı ¨ve CD25? CD4 effector T cells than in mice that received 3 ?
104sorted cells could possibly be accounted for not by an
but rather by the transfer of more contaminating Tregs with the
larger number of sorted cells. There is a population of Foxp3?,
CD25? CD4 T cells (?10% of Foxp3? cells) (14). Thus,
substantially more regulatory cells (?1 ? 104, assuming that
Foxp3? cells are ?5% of CD44dullCD4 T cells) (B. Min,
personal communication) would be transferred with the larger
number of sorted cells than with the smaller number, where the
number of Foxp3? cells may be anticipated to have been ?150.
The latter may be too small a frequency to inhibit a Th2 response
even after homeostatic expansion whereas the former may have
been partially active. Furthermore, lymphopenia-driven expan-
sion has been associated with conversion of CD25? Foxp3? to
the Foxp3? phenotype (15), suggesting that the diversity of
TCRs in the regulatory population may be increased by the
presence of a complex TCR repertoire among conventional cells
undergoing rapid proliferation. In a mouse immunization model,
elevated serum IgE and allergy have been noted when only
monoclonal T and B cell populations specific for the antigen are
present, but the phenotype is markedly inhibited when regula-
tory T cells are present (16). Although the absence or reduced
repertoire of natural or peripherally converted regulatory T cells
alone may explain the Th2 disease, it is still possible that an
effector population of limited TCR diversity may intrinsically
differentiate to the Th2 phenotype much more efficiently than
a similar population of greater TCR diversity.
There are a number of possible explanations to account for
Th2 pathology as due to an intrinsic property of effector cells
transferred at lower frequencies. It may well be that the larger
number of divisions that would be required in mice receiving
fewer CD4 T cells may in some way predispose the cells to Th2
differentiation, perhaps in part because of a greater propensity
of differentiating Th1 cells to undergo apoptosis (17). Alterna-
tively, TCR affinity for antigen may determine that a Th2-like
differentiation is dominant when there is a limited TCR diver-
sity. Thus, on the average any newly formed pMHC complex is
less likely to find a complementary high-affinity TCR when
diversity is low, and thus most interactions in mice that receive
small numbers of CD4 T cells are likely to be of low affinity. It
has been shown in several systems that priming with peptides of
low affinity for the receptor of the responding T cell (i.e., altered
peptide ligands) (18) or with low concentrations of peptides (19,
20) favors Th2 differentiation. If such low-affinity responses are
normally ‘‘outcompeted’’ when high-affinity interactions also
occur, then one could anticipate that, in a population of limited
diversity, on the average, Th2 differentiation of activated CD4 T
cells would be favored. A related possibility is raised by a recent
report that Tregs exert preferential control of CD5loweffectors
(21). Such cells presumably have lower affinity for self; thus, in
the absence of Tregs their ‘‘self’’ response would be relatively
favored, and, if they tended to develop into Th2 cells, then a
preferential self-specific Th2 response in the absence of Tregs
would be expected, which might be even more marked with a
limited repertoire diversity.
Many of the lymphopenic C57BL/6 mice receiving CD25?
Treg-depleted cell populations did not develop colitis, which
would have been expected with such transfers (22). Differences
in gut microflora could potentially explain this. Interestingly, in
a different National Institutes of Health animal facility, transfer
of 5 ? 105CD45RBhiCD4 cells into C57BL/6 Rag2?/? recip-
ients resulted in colitis only 20% of the time, but in C57BL/10
Rag2?/? recipients such transfer resulted in colitis 80% of the
time. Furthermore, an eosinophilic, Ym1? pneumonia was seen
Table 1. Summary of disease induced by transfer of CD4 or CD25? CD44loCD4 T cells into Rag2?/? mice
30,000 CD25? CD44locells
30,000 CD4 cells
2 million CD25? CD44locells
2 million CD4 cells
AND 30,000 CD4 cells
AND 2 million CD4 cells
Shown are results from three or more separate experiments for each group, with pathology determined at least 4 months after transfer.
Fluorescence microscopy to detect antiparietal cell antibodies in serum from
CD3??/? recipient mice 12 weeks after transfer of 30,000 (Upper Left) or 2
million (Upper Right) sorted CD44loCD25? CD4 T cells. Mouse serum was
anti-mouse IgG antibody was used for detection. Results from the staining
experiment are summarized in Lower.
Antiparietal cell antibodies in mice receiving 30,000 CD4 T cells.
Milner et al.PNAS ?
January 9, 2007 ?
vol. 104 ?
no. 2 ?
20% of time in C57BL/10 recipients but not in C57BL/6,
Rag2?/? recipients (B. Kelsall, personal communication).
Although we were able to detect specific antiparietal cell
autoantibodies in CD3??/? recipient mice with eosinophilic
immunopathology, we were not able to rapidly induce the
disease by transferring cells from the draining nodes of affected
mice to healthy Rag2?/? mice. This finding suggests that the
immunopathologic response may be due to ‘‘bystander’’ effector
functions as opposed to cognate TCR-based recognition of
‘‘autoantigens.’’ This is not surprising given the uniform induc-
tion of disease despite the very low numbers, and hence the
limited TCR diversity, of cells transferred. Bystander, noncog-
nate antigen-driven hyperIgE production in nu/nu or MHC class
II?/? mice has recently been reported and has been attributed
of IL-13 or IL-4 into airways can induce airway hypersensitivity
and mucus metaplasia (23), implying that local cytokine pro-
duction in a ‘‘sensitive’’ environment in and of itself can induce
While not mimicking every phenotype of disease associated
with primary human immune deficiencies associated with mark-
edly reduced T cell repertoires, we have created a model for
lymphopenia and Th2 disease. This model may also be useful in
studying other Th2-associated immunopathologic states such as
allergy and ‘‘extrinsic’’ asthma. Antigen encounters during pe-
riods of limited TCR repertoire may predispose CD4 T cells
toward Th2 differentiation, due to the lack of TCR specificity-
matching between regulatory and effector T cells, an effector T
cell intrinsic predisposition toward Th2 phenotype when TCR
repertoires are reduced, or both. Future study of such antigen-
specific encounters in the context of lymphopenia are needed to
elucidate the mechanism by which the Th2 phenotype emerges.
Materials and Methods
Mice. B10.A, Ly5.1 B10.A, C57BL/10, C57BL/10 AND TCR Tg,
C57BL/10 Rag2?/?, B10.A Rag 2?/?, B10.A CD3??/?, Ly5.1
C57BL/6, C57BL/6 Rag2?/?, and C57BL/6 IL-4?/? mice were
obtained from the National Institute of Allergy and Infectious
Diseases contract facility at Taconic Farms (Germantown, NY).
C57BL/6 mice were obtained from The Jackson Laboratory (Bar
Harbor, ME). Mice were maintained under pathogen-free con-
ditions at the National Institute of Allergy and Infectious
Diseases animal facility.
Adoptive Transfer. CD4, CD25? CD4, CD25? CD4, or CD25?/
CD44dullCD4 lymph node cells were obtained by sorting on a
FACSVantage SE or FACSAria (Becton Dickinson, Franklin
Lakes, NJ). Purity was ?99%. In some cases, cells were labeled
with CFSE (Molecular Probes, Carlsbad, CA) at a final concen-
tration of 1.25 ?M. Cells suspended in PBS were transferred via
tail vein injection into recipient mice.
Flow Cytometry. Anti-CD25-allophycocyanin (APC) (PC61),
CD4-FITC (3T4), CD44-phycoerythrin (PE), CD44 PE-cy5.5,
CD45.1-PE, CD45.2-FITC, CD45.1 PE-cy5, IL-4-PE, IL-5-PE,
and IFN?-APC were purchased from BD Pharmingen (San
Diego, CA). Anti-FoxP3 was purchased from eBiosciences (San
Diego, CA). Anti-IL-13 (clone 38213) was purchased from R &
D Systems (Minneapolis, MN) and conjugated to APC at the
CD25? CD4 T cell transfer, 1 million CFSE-labeled CD45.1 CD25? CD4 T cells were transferred, and lymph nodes were harvested 1 week later. Representative
CFSE profiles of transferred CD45.1 T cells are shown. (C) Ten weeks after primary CD45.2? Treg transfer, mice received a second transfer of 1 ? 105CD25?
CD45.1? CD4 T cells. Cells were harvested 10 weeks later. CD45.1? and CD45.2? CD4 T cells were enumerated, and pathology of mice initially receiving 3 ? 104
Tregs (D and F) or 3 ? 105Tregs (E and G) was examined. Cell yields and representative pathology are shown. There were two to three mice in each group. This
experiment was repeated once with similar results. Standard error bars are shown.
Transfer of large but not small numbers of Tregs controls disease. CD45.2 Rag2?/? mice received either 3 ? 104or 3 ? 105sorted CD25? CD45.1 CD4
www.pnas.org?cgi?doi?10.1073?pnas.0610289104Milner et al.
National Institute of Allergy and Infectious Diseases core cus-
tom antibody facility. All flow cytometry was performed on a
Becton Dickinson FACSCalibur and analyzed by using FloJo
Pathology/Immunohistochemistry. Immediately after killing mice
in a CO2 chamber, organs were removed, fixed in neutral
buffered formalin, and embedded in paraffin. Sections were
stained with hematoxylin and eosin. Selected tissues were
eosinophils). Immunohistochemistry was performed on some
Laboratories, Burlingame, CA). Antiparietal cell antibodies
were detected by immunofluorescence on cryostat sections of
normal BALB/c stomach as described (25). Briefly, sections
were blocked with 2% FBS in 5% dry milk in PBS and incubated
with a 1/50 dilution of serum for 1 h at room temperature. The
presence of autoantibodies was visualized by adding FITC-goat
F(ab?)2 anti-mouse Ig (BioSource, Camarillo, CA). Slides were
examined under a fluorescence microscope and given a score of
0–4 depending on the extent of parietal cell staining by an
observer who did not have knowledge of the treatment the mice
Methacholine Hypersensitivity. Airway hyperresponsiveness of
control Rag2?/? mice and recipients of cell transfers was
measured by challenging the mice with increasing doses of
nebulized methylcholine (0, 6, and 12 mg/ml) in PBS and
measuring ‘‘enhanced pause’’ by using whole body plethysmog-
raphy (Buxco Electronics, Wilmington, NC) following the man-
ufacturer’s instructions. Doses above 12 mg/ml resulted in
significant morbidity in compromised mice consistent with
IgE ELISA. Immulon 4 96-well microtiter plates were coated with
100 ?l of anti-mouse IgE (Pharmingen) per well at 2 ?g/ml in
PBS overnight at 4°C. Plates were washed with wash buffer (PBS
plus 0.05% Tween 20) and blocked for 1 h at room temperature
with diluent/blocking buffer (PBS plus 0.5% BSA plus 0.05%
Tween 20). Samples [1/50 or 1/500 dilutions of mouse serum or
mouse IgE standard (Pharmingen)] were added and incubated
for 2 h at room temperature. Plates were washed, and biotin-anti
mouse IgE (Pharmingen) was added in diluent/blocking buffer
for 2 h at room temperature. Plates were washed again, and
streptavidin-HRP (Pharmingen) (1/4,000 dilution in diluents/
blocking buffer) was added for 1 h. Plates were washed again,
TMB solution (Sigma, St. Louis, MO) was added for 2–3 min,
and Stop Solution (2N H2SO4) was then added. Plates were read
at 450 nm, and the optical densities of the samples were then
plotted on the linear part of the standard curve and multiplied
by the dilution factor to calculate IgE concentration in serum
The excellent histotechnology assistance of Larry Faucette and Cindy
Erexson is greatly appreciated. We thank Drs. Brian Kelsall, Warren
Strober, and Hidehiro Yamane for critical readings of the manuscript.
J.D.M. is a fellow of the Pediatric Scientist Development Program (K12
HD00850). This research was supported in part by the Intramural
Research Program of the National Institutes of Health, National Insti-
tute of Allergy and Infectious Diseases and, in part, by a National
Institute of Allergy and Infectious Diseases contract to SoBran.
1. Markert ML, Alexieff MJ, Li J, Sarzotti M, Ozaki DA, Devlin BH, Sempowski
GD, Rhein ME, Szabolcs P, Hale LP, et al. (2000) J Allergy Clin Immunol
2. Villa A, Santagata S, Bozzi F, Giliani S, Frattini A, Imberti L, Gatta LB, Ochs
HD, Schwarz K, Notarangelo LD, et al. (1998) Cell 93:885–896.
3. Tietz A, Sponagel L, Erb P, Bucher H, Battegay M, Zimmerli W (1997) Eur
J Clin Microbiol Infect Dis 16:675–677.
TS (2005) Clin Cancer Res 11:5748–5755.
5. Yawalkar N, Ferenczi K, Jones DA, Yamanaka K, Suh KY, Sadat S, Kupper
TS (2003) Blood 102:4059–4066.
XJ, Sainty D, He HT, Malissen B, Malissen M (2002) Science 296:2036–2040.
7. Sommers CL, Park CS, Lee J, Feng C, Fuller CL, Grinberg A, Hildebrand JA,
Lacana E, Menon RK, Shores EW, et al. (2002) Science 296:2040–2043.
8. McCoy KD, Harris NL, Diener P, Hatak S, Odermatt B, Hangartner L, Senn
BM, Marsland BJ, Geuking MB, Hengartner H, et al. (2006) Immunity
9. Surh CD, Boyman O, Purton JF, Sprent J (2006) Immunol Rev 211:154–163.
10. Min B, Foucras G, Meier-Schellersheim M, Paul WE (2004) Proc Natl Acad Sci
11. Raes G, De Baetselier P, Noe ¨l W, Beschin A, Brombacher F, Hassanzadeh G
(2002) J Leukocyte Biol 71:597–602.
12. Almeida AR, Legrand N, Papiernik M, Freitas AA (2002) J Immunol
13. Lin W, Truong N, Grossman WJ, Haribhai D, Williams CB, Wang J, Martin
MG, Chatila TA (2005) J Allergy Clin Immunol 116:1106–1115.
14. Fontenot JD, Rasmussen JP, Williams LM, Dooley JL, Farr AG, Rudensky AY
(2005) Immunity 22:329–341.
15. Curotto de Lafaille MA, Lino AC, Kutchukhidze N, Lafaille JJ (2004)
J Immunol 173:7259–7268.
16. Curotto de Lafaille MA, Muriglan S, Sunshine MJ, Lei Y, Kutchukhidze N,
Furtado GC, Wensky AK, Olivares-Villagomez D, Lafaille JJ (2001) J Exp Med
17. Wu CY, Kirman JR, Rotte MJ, Davey DF, Perfetto SP, Rhee EG, Freidag BL,
Hill BJ, Douek DC, Seder RA (2002) Nat Immunol 3:852–858.
18. Pfeiffer C, Stein J, Southwood S, Ketelaar H, Sette A, Bottomly K (1995) J Exp
19. Yamane H, Zhu J, Paul WE (2005) J Exp Med 202:793–804.
20. Constant S, Pfeiffer C, Woodard A, Pasqualini T, Bottomly K (1995) J Exp Med
21. Shen S, Ding Y, Tadokoro CE, Olivares-Villagomez D, Camps-Ramirez M,
Curotto de Lafaille MA, Lafaille JJ (2005) J Clin Invest 115:3517–3526.
22. Powrie F, Leach MW, Mauze S, Caddle LB, Coffman RL (1993) Int Immunol
23. Wills-Karp M, Luyimbazi J, Xu X, Schofield B, Neben TY, Karp CL,
Donaldson DD (1998) Science 282:2258–2261.
24. Ward JM, Yoon M, Anver MR, Haines DC, Kudo G, Gonzalez FJ, Kimura S
(2001) Am J Pathol 158:323–332.
25. Suri-Payer E, Cantor H (2001) J Autoimmun 16:115–123.
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vol. 104 ?
no. 2 ?