Adoptive Transfer of Induced-Treg Cells Effectively
Attenuates Murine Airway Allergic Inflammation
Wei Xu1,4., Qin Lan
Chun-Song Yan4, Jiu-Long Kuang4, David Warburton1, Dieudonne ´e Togbe5, Bernhard Ryffel5, Song-
Guo Zheng2*, Wei Shi1*
., Maogen Chen2, Hui Chen1, Ning Zhu2, Xiaohui Zhou2,3, Julie Wang2, Huimin Fan3,
1Developmental Biology and Regenerative Medicine Program, Department of Surgery, Children’s Hospital Los Angeles, Los Angeles, California, United States of America,
2Division of Rheumatology, Department of Medicine, The Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America,
3Institute of Immunology, Shanghai East Hospital of Tongji University, Shanghai, China, 4Department of Respiratory Medicine, The Second Affiliated Hospital of
Nanchang University, Nanchang, Jiangxi Province, China, 5Molecular Immunology, University and CNRS, Orleans, France
Both nature and induced regulatory T (Treg) lymphocytes are potent regulators of autoimmune and allergic disorders.
Defects in endogenous Treg cells have been reported in patients with allergic asthma, suggesting that disrupted Treg cell-
mediated immunological regulation may play an important role in airway allergic inflammation. In order to determine
whether adoptive transfer of induced Treg cells generated in vitro can be used as an effective therapeutic approach to
suppress airway allergic inflammation, exogenously induced Treg cells were infused into ovalbumin-sensitized mice prior to
or during intranasal ovalbumin challenge. The results showed that adoptive transfer of induced Treg cells prior to allergen
challenge markedly reduced airway hyperresponsiveness, eosinophil recruitment, mucus hyper-production, airway
remodeling, and IgE levels. This effect was associated with increase of Treg cells (CD4+FoxP3+) and decrease of dendritic
cells in the draining lymph nodes, and with reduction of Th1, Th2, and Th17 cell response as compared to the controls.
Moreover, adoptive transfer of induced Treg cells during allergen challenge also effectively attenuate airway inflammation
and improve airway function, which are comparable to those by natural Treg cell infusion. Therefore, adoptive transfer of in
vitro induced Treg cells may be a promising therapeutic approach to prevent and treat severe asthma.
Citation: Xu W, Lan Q, Chen M, Chen H, Zhu N, et al. (2012) Adoptive Transfer of Induced-Treg Cells Effectively Attenuates Murine Airway Allergic
Inflammation. PLoS ONE 7(7): e40314. doi:10.1371/journal.pone.0040314
Editor: Christian Taube, Leiden University Medical Center, The Netherlands
Received February 29, 2012; Accepted June 4, 2012; Published July 9, 2012
Copyright: ? 2012 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: National Institutes of Health (NIH) HL109932 and HL068597, and Webb Foundation grant (WS)NIH AR059103, the American College of Rheumatology
(Within Our Reach) and the Arthritis Foundation (SGZ). California Institute of Regenerative Medicine Training Grant (WX). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Co-authors Wei Shi and Song-Guo Zheng are PLoS ONE Editorial Board members. This does not alter the authors’ adherence to all the
PLoS ONE policies on sharing data and materials.
* E-mail: email@example.com SG); firstname.lastname@example.org (WS)
. These authors contributed equally to this work.
Allergic airway inflammation and airway hyperresponsiveness
(AHR) are characteristics of atopic asthma pathophysiology. More
than 7% of Americans suffer from asthma , and annual
expenditure for health and lost productivity due to asthma is
estimated at nearly $20 billion. The currently available therapeutic
approaches for asthma usually include quick symptomatic relief
measures directed to relaxation of airway smooth muscle
(bronchodilator) and long-term control with suppression of airway
inflammation . However, these existing standard asthma
therapies have several caveats and remain inadequate. For
example, inhaled anti-inflammatory corticosteroids only suppress
but do not cure asthmatic inflammation, and long-term use of
corticosteroids causes many pleiotropic side effects. Other more
recently developed therapies, including inhibitors of leukotriene
production and leukotriene receptor blockade, and anti-IgE
monoclonal antibody (Omalizumab), are used as alternative
treatments for persistent asthma. However, limited efficacy, high
cost, and lack of responsiveness in some asthma patients are the
major drawbacks. Thus, novel and more effective therapeutic
approaches for asthma are still needed.
Recent studies have found that immune function dysregulation
is one of the key pathogenic mechanisms underlying asthma .
Reduction and/or defects in regulatory T (Treg) cells, which
function as negative regulators to suppress excessive immune
response and maintain immunological tolerance have been
detected in asthma patients . Therefore, replenishment of Treg
cells is thought to be a promising cell therapeutic approach.
However, the use of thymus-derived naturally occurring regula-
tory T (nTreg) cells has several caveats that may significantly
diminish their practical application for asthma treatment. These
include limited availability, susceptibility to inflammation-trig-
gered apoptosis, inability in suppressing pro-inflammatory Th17
cells, and self-conversion to Th17 and/or other T effector cells in
the milieu of inflammation. In contrast, Treg cells that are induced
by TGF-b and IL-2 in combination with low dose antigen
exposure have similar phenotypic and functional characteristics to
nTreg cells, without the caveats of nTreg cells mentioned above
. Herein, we report that adoptive transfer of the induced-Treg
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(iTreg) cells to OVA-sensitized mice either before or even after
allergen challenge effectively attenuates OVA-induced airway
allergic inflammation, AHR, and other asthma-like lung pathology
by modulating the systemic immune system.
Adoptive transfer of in vitro TGF-b-induced Treg (iTreg)
cells prior to OVA challenge effectively prevented allergic
inflammation in mouse respiratory airways and alveoli
iTreg cells were induced from splenic naı ¨ve CD4+CD252cells
in vitro with TGF-b, IL-2 and anti-CD3/28 antibodies, as
described previously . As show in Fig. 1, more than 70% of
the cells were induced to become iTreg cells. The phenotypes and
functions of these iTreg cells are similar to those of nTreg cells
In OVA-sensitized mice, repeated intra-nasal (i.n.) ovalbumin
(OVA) challenges at day 25–27 resulted in severe peri-vascular/
peri-bronchiolar and alveolar inflammation, indicated by excessive
inflammatory cell infiltration surrounding small airways and
vasculature, as well as alveolar septa (Fig. 2A, 2B). The serum
level of IgE was significantly increased, and infiltration of
eosinophil around airway was also verified by Discombe’s staining
(Fig. 2C, 2E). Consistent with the lung histological changes, the
total amount of proteins in bronchoalveolar lavage (BAL) fluid was
significantly increased (Fig. 2D). The number of cells in BAL also
increased more than 10-fold than the control groups (data not
shown). Moreover, epithelial cell hypertrophy with increased
mucin expression in small airways, thickened airway smooth
muscle cell (SMC) layer, and resultant smaller lumen with rippled
epithelial surface of small airways were also observed in OVA-
challenged mouse lungs (Fig. 3). Therefore, a typical OVA-allergic
airway inflammatory model was verified.
Figure 1. In vitro induction of regulatory T (iTreg) cells by TGF-b. Naive CD4+CD252cells were stimulated with anti-CD3/CD28 coated beads
with IL-2 in the presence (CD4TGF-b) and absence (CD4med) of TGF-b for 5–6 days. nTreg cells were splenic CD4+CD25+cells that were sorted and
expanded with anti-CD3/CD28 coated beads with IL-2 for 6–7 days. (A). FoxP3 expression was determined by flow cytometry with anti-Foxp3
antibody. cIgG, control IgG. (B). T cells labeling with CFSE were stimulated with anti-CD3 with or without CD4 condition cells (ratio of CD4 condition
to T responder=1:2) for three days and CFSE dilution was identified on the CD4+cell gate. (C). T cell proliferation was determined by3H-thymidine
incorporation assay. (D). The T cell proliferation was determined in the different ratios of CD4 conditioned cells and T responder cells. Data was
representative or mean 6 SEM of three independent experiments.
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Using this established OVA-allergic mouse model, the preven-
tive anti-inflammatory effect of adoptive transfer of iTreg cells was
first examined. Three days before OVA challenge (Day 22), a
single transfer of 56106iTreg cells was given to the mice via tail
vein injection. T cells cultured without TGF-b addition were used
as an additional specificity control. After three-day i.n. OVA
challenge, lung specimens were harvested for detailed analyses.
iTreg cells, but not control T cells, significantly attenuated OVA-
induced allergic inflammation including reduced infiltration of
inflammatory cells, particularly eosinophils, in airway and alveolar
septa, decreased levels of serum IgE and BAL proteins (Fig. 2), as
well as reduction in the number of cells in BAL by 2-fold (p,0.05).
Alterations of airway walls subsequent to allergic inflammation,
including epithelial hypertrophy and increased mucin production
(PAS-positive staining), as well as thickened smooth muscle cells in
small airways (Fig. 3A, 3B, 3C), were likewise significantly
attenuated. These results indicate that adoptive transfer of iTreg
cells prior to OVA allergic challenge can effectively prevent lung
inflammation and abnormal airway remodeling.
Adoptive transfer of iTreg cells prior to OVA challenge
effectively inhibited airway hyperresponsiveness (AHR) in
OVA-sensitized mice with repetitive i.n. administration of OVA
developed significant AHR to methacholine (MCh) challenge
compared to normal control mice (Fig. 3D). However, adoptive
transfer of iTreg cells prior to repetitive challenge of OVA
significantly inhibited AHR, although increased AHR was still
detected. In contrast, adoptive transfer of T control cells did not
significantly reduce AHR, although slight reduction in AHR was
detected in some mice. Thus, adoptive transfer of iTreg cells, but
not control T cells, prior to allergen exposure can also effectively
reduce AHR in addition to the reduction in airway inflammation
Adoptive transfer of iTreg cells prior to OVA challenge
modulated immune response to OVA-allergen
The infused exogenous iTreg cells, labeled with CFSE
(carboxyfluorescein succinimidyl ester) dye, were detected 8 days
after injection in mediastinal draining lymph nodes and lung
tissues with comparable frequencies between normal and OVA-
challenged mice (Fig. 4), suggesting that adoptively transferred
Figure 2. Attenuated allergic inflammation in lung tissues by adoptive transferring of iTreg cells prior to OVA challenge. (A) Lung
tissue sections from the mice with indicated treatments were stained with H&E. (B) Overall lung inflammation were graded with scores 0 to 15 (none
to severe inflammation, see Materials and Methods for details). (C) Eosinophil, detected by Discombe’s staining (red intracellular granules), was the
major type of cells that were infiltrated in small airways and adjacent vasculature. (D) Total proteins in BAL fluids from mice with different treatments
were quantified. (E) IgE level in serum from different groups of mice was quantified by an ELISA. *P,0.05, **P,0.01, n=5.
Induced-Treg Cells Attenuates Asthma
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iTreg cells are homing to these tissues independent of pulmonary
Numerous studies have found that asthmatic inflammation is
related to abnormal cellular immunity, including defective Treg
cells, inappropriate ratio of Th2 to Th1 cell population, and
dysfunction of Th17 and dendritic cells (DCs). Thus, we have
compared these immune cells and their related cytokine produc-
tion between iTreg cell-treated and untreated OVA-asthmatic
mice. In OVA-asthmatic mice that received exogenous iTreg cells
prior to OVA challenge, about 2-fold increase of Treg cells
(CD4+FoxP3+) was detected in the draining lymph nodes and the
spleen, while there were no significant changes in the number of
Treg cells in untreated or control T cell-treated OVA group
(Fig. 5). In mediastinal draining lymph nodes, increased Th1, Th2,
and Th17 cells caused by repetitive OVA challenge was
significantly attenuated by adoptive transfer of iTreg cells
(Fig. 6A, 6B), but not by control T cells. Moreover, in OVA-
challenged mouse lung tissues, increases expression of Th1 and
Th2 differentiation-related transcription factors T-bet1 and Gata-
3 was also significantly inhibited by infused iTreg cells, as
measured at the mRNA level (Fig. 6C). However, slight but not
statistically significant reduction of RORcT, a transcription factor
related to Th17 differentiation, was detected in iTreg-treated
OVA mouse lung compared to untreated OVA control. This
Figure 3. Abnormal airway wall remodeling and AHR were subsided with iTreg cell treatment. (A) Excessive mucin expression in small
airway epithelial cells was detected by PAS staining (red color). (B) The numbers of airways with PAS-positive epithelial cells per lung tissue section
were quantified in different experimental groups (n=5). (C) Clara cells in small airway epithelia were stained by CCSP immunofluorescence staining
(green) and the surrounding airway smooth muscle cells were immunostained by SMA (red). (D) Airway resistance was measured upon Mch (40mg/
ml) aerosol delivery. Although the airway resistance was still significantly higher in iTreg cell-treated group than that in normal control group,
significant reduction of airway resistance was achieved in iTreg cell-treated group compared to non-treated (OVA) or control T cell-treated group.
Figure 4. Comparison of infused CFSE-labeled iTreg cells in the
absence and presence of OVA-induced allergic inflammation.
No significant difference was detected in mediastic draining lymph
nodes and lung tissues for infused iTreg cells between normal control
mice and mice that had OVA-induced allergic inflammation.
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suggests that adoptive transfer of iTreg cells may suppress Th1 and
Th2 cell differentiation in OVA mouse lungs.
Consistent with the above changes, serum Th2 cytokines
including IL-5 and IL-13 were significantly increased upon
OVA challenge (Fig. 7A). Adoptive transfer of iTreg cells, but
not T control cells, was able to partially block these increases in
mice. Recently studies reported that a crucial role of Treg cells in
suppressing Th2-driven mucosal inflammation, and the inhibitory
properties of the Treg cells were mediated by enhancing
expression of IRF-4 and IL-10 [7–9]. Interestingly, a 7-fold
increase of IRF-4 mRNA and a 14-fold increase of IL-10 mRNA
expression were also detected in total lymphocytes isolated from
iTreg cell-treated OVA lung compared to those from untreated
OVA mice (Fig. 7B).
Since migration of respiratory tract DCs to the draining
mediastinal lymph node was reported to be an important
mechanism to activate antigen-specific T cells during airway
allergic inflammation , the effect of adoptively transferred
iTreg cells on alteration of DCs in the OVA-challenged mice were
investigated. Compared to untreated OVA-challenged mice,
expression of both CD80+and CD86+of CD11c+cells in
mediastinal lymph nodes were significantly attenuated by infused
iTreg cells (Fig. 8A). Moreover, the level of DC-related cytokine
IL-23 expression in draining lymph nodes was also significantly
reduced by injected iTreg cells compared to untreated OVA mice
(Fig. 8B). This suggests that suppression of DC-initiated allergic
response may be one of the mechanisms underlying iTreg-
mediated attenuation of airway allergic inflammation. Therefore,
adoptive transfer of iTreg cells may modulate both local and
systemic immunity, and thus prevent excessive cellular response
against OVA-induced allergic reaction and inflammation.
Adoptive transfer of iTreg cells during OVA challenge
also effectively attenuated airway inflammation and
reduced airway hyperresponsiveness
In addition to the effects of iTreg cells in preventing allergen-
induced airway inflammation and ARH, we also evaluated the
therapeutic potential of adoptive transfer of iTreg cells by giving
iTreg cells after first allergic challenge. Interestingly, the lung
inflammation, particularly in peri-vascular and peri-bronchiolar
area, was significantly attenuated in the mice received iTreg cells
injection compared to those of the OVA-challenged mice received
no cells or control T cells (Fig 9A). Moreover, adoptive transfer of
expanded nTreg cells also had reduced lung inflammation,
comparable to that in iTreg-treated lung. The inflammatory
histopathology was further quantified using the same scale
described above (Fig 9B). Both iTreg and nTreg cell treatments
had comparable anti-inflammatory effects in this acute allergic
inflammatory model. Consistent with the changes in lung
inflammation, AHR to Mch was also significantly attenuated in
the mice that received either iTreg or nTreg injection after first
time OVA challenge (Fig 9C). In addition, both iTreg and nTreg
cell treatment significantly increased Foxp3+ Treg cells and
decreased Th1 and Th2 cell frequency in spleens (Fig. 9D),
draining lymph nodes and lungs (data not shown). Conversely,
treatment with CD4 control cells did not cause significant changes
in Treg and Th1 cell frequency, while it increased Th2 cell
numbers in the OVA-asthma mouse model (Fig. 9D). As IL-17A
and IL-17F cell frequency was very low in the OVA-asthma mice
(data not shown), we were not able to detect alteration of Th17 cell
frequency after cell therapy. This suggests that iTreg cells
generated in vitro have therapeutic effects comparable to nTreg
cells in controlling asthma progression. Thus, the use of both Treg
cell subsets could provide an effective approach to diminish the
allergic inflammation already occurred in the lung, and to improve
Asthma is an inflammatory disorder of the conducting airways
with abnormal airway hyper-responsiveness and remodeling,
resulting in airflow restriction during breathing [3,11]. Studies
have found that immune function dysregulation is one of the key
pathogenic mechanisms underlying airway allergic inflammation,
in particular imbalance between Th2 cell and Th1 cell responses
[12,13]. More recently, another pivotal subset of CD4+T cells
(Treg cells), which have been related to control immune tolerance
and subsequent autoimmune diseases, were also found to be
important in suppressing allergic responses [14,15]. For example,
CD4+CD25+Treg cells from grass pollen-allergic individuals were
less able to suppress proliferative responses and IL-5 production by
CD4+CD25+Treg cell frequency in peripheral blood was detected
in patients with persistent or exacerbation of asthma when
compared to control groups . In addition, Treg cells in
patients with asthma were also decreased in bronchoalveolar
Figure 5. Increased total CD4+FoxP3+Treg cells in the spleen and draining lymph nodes of the mice that received exogeneous iTreg
cells. In contrast, the frequencies of CD4+FoxP3+Treg cells did not show significant changes between normal mice and OVA mice, as well as control T
cell-treated mice. The experiments were repeated with consistent results.
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Figure 6. Adoptive transfer of iTreg cells significantly diminished Th1/Th2/Th17 cell frequencies in draining lymph nodes in
asthmatic mice. Intracellular expression of IFN-c, IL-5, and IL-17A in CD3+T cells were determined by FACS. (A) A representative of 9 mice in each
group. Cells were gated on CD3+cells. (B) Results were mean 6 SEM of values of 9 mice in each group. *P,0.05, **P,0.01, ***P,0.001. (C) Significant
reductions of T cell differentiation markers, including T-bet1 and Gata-3, but not RORcT, were detected in iTreg-treated OVA mice compared to iTreg-
untreated OVA control.
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lavage (BAL) fluid [18,19]. In mouse asthma models, naturally
occurring Treg (nTreg) cells are present in the lung tissue of
sensitized mice and increase upon allergen inhalation. Inhibition
of nTreg cells augments respiratory allergen-induced AHR and
IgE production, as well as Th2 cytokine levels in BAL fluid .
Therefore, cell therapy by replenishing functional Treg cells may
be a new promising strategy for asthma prevention and treatment.
It also have advantages by correcting the immune cell proportions
and functions, which restore appropriate cytokine productions,
compared to just targeting a single or a few cytokines produced by
these dysregulated immune cells.
Figure 7. Adoptive transfer of iTreg cells altered cytokine production. (A) iTreg cells inhibited OVA-induced increase of Th2 cytokines. IL-5
and IL-13 in mouse serum were quantified by specific ELISA. Significant reduction of IL-5 and IL-13 in the group receiving iTreg treatment was
detected compared to OVA challenge only control group. **P,0.01. (B) Expression of IRF-4 and IL-10 at the mRNA level of lung lymphocytes was
significantly increased in iTreg-treated OVA mice compared to untreated OVA mice. *P,0.05.
Figure 8. Alteration of dentritic cells and related cytokine IL-23. Significant reduction of both CD11c+CD86+and CD11c+CD80+subsets of
DCs in mediastinal lymph nodes were detected in iTreg-treated OVA mice compared to untreated OVA mice. (B) IL-23 expression at the mRNA level in
draining lymph nodes was also significantly reduced by iTreg treatment compared to untreated OVA mice (Fig. 8B).
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Treg cells can be either derived from the thymus (nTreg cells),
or induced in the periphery in a TGF-b-dependent fashion (iTreg
cells) [4,21]. Both nTreg and iTreg cells share similar phenotypic
characteristics and immune suppressive functions. nTreg cells are
rare cell population so that it is difficult to obtain sufficient
numbers of nTreg directly for the therapeutic need. Although
repetitive expansion in vitro is able to generate enough nTreg cells,
recent study has indicated that the phenotypes and functional
characteristics of the nTreg cells after the repetitive expansion
have been altered . Moreover, intrinsic CD4+CD25+nTreg
cells are often defective in suppressing allergic immune responses
in asthma patients, which limits the clinical use of these nTreg cells
as autograft therapeutic agents. Our study has shown that iTreg
cells are effective in both preventing and treating airway allergic
responses, and that in vitro induced-iTreg has a comparable anti-
inflammatory activity to that of nTreg cells. iTreg cells may
migrate to inflammatory sites in airways, and likely suppress Th1
Figure 9. Adoptive transfer of Treg cells after first OVA challenge still effectively suppressed airway inflammation and AHR, as well
as Th1/Th2 cell frequencies. (A) Histopathological changes of lungs from the mice that received no cell, iTreg, nTreg, or control T cells after first
OVA challenge (day 25). Lung specimens were taken on day 28 after another two daily OVA challenges on day 26 and 27. (B) Accumulated
inflammatory scores of peri-vascular, peri-bronchiolar and alveolar regions of different groups. (C) Airway resistance was measured to evaluate lung
functional changes among different groups. (D) Altered frequencies of Treg, Th1, and Th2 subsets in the spleens of the mice receiving different
treatments. *P,0.05, **P,0.01.
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and Th2 immune response directly or indirectly through inhibiting
Induction of iTreg cells in vitro not only avoids systemic
application of cytokines and growth factors, but also generates
an approach similar to an autograft, by which sufficient iTreg cells
can be easily induced from the CD4+cells in individual asthmatic
patient in vitro, and then adoptively transferred back to the same
patient to induce immune tolerance to allergens and to suppress
abnormal inflammatory responses in the airway without significant
side effects. Moreover, iTreg cells also have several superior
functional features compared to nTreg cells, including anti-
apoptosis and resistance to Th17 conversion [21,23]. Conversely,
nTreg cells are more plastic and unstable under pro-inflammatory
conditions [23,24]. Therefore, adoptive transfer of iTreg cells may
be a better approach as a novel cell therapy in airway allergic
Although many studies have demonstrated that adoptive
transfer of iTregs can control lupus, colitis, gastritis and diabetes
in animal models [25–29], it is unknown whether infusion of iTreg
cells can attenuate airway allergic inflammation and improve
respiratory function after asthma onset, We have now shown that
adoptive transfer of iTreg cells before allergen challenge effectively
prevented airway allergic inflammation and improved airway
function in an OVA model. In addition to local relief of
inflammation, abnormal systemic immune functions were also
corrected to some extent. Of importance, the infusion of iTreg
cells during allergen challenge still had significant effects on
reducing lung inflammation and ARH, although to a less degree
compared to iTreg cell administration prior to allergen challenge.
The different efficacies between these two regimens can be caused
with these possibilities. (1) Exogenous iTreg cells are more effective
to attenuate the initiation of the inflammatory process, but less
effective for the inflammation already occurred; (2) It needs certain
time for infused iTreg cells to be effective, as all lung specimens
were taken on day 28, regardless of when iTreg cells were given on
day 22 before allergen challenge or on day 25 after first allergen
challenge. Nevertheless, both iTreg and nTreg cells displayed the
similar therapeutic effects on asthma progression. Mechanistically,
Treg subset infusion regulates the local and systemic immune
balance by increasing Treg and decreasing Th1/Th2 cell
frequencies in the ongoing asthma.
In summary, adoptive transfer of in vitro induced iTreg cells is an
effective way to both prevention and treatment of airway allergic
disease, such as asthma, in a mouse OVA-asthma model, which
will be a promising therapeutic approach for airway allergic
OVA-sensitized mouse asthma model and exogenous cell
6 to 8-week-old female C57BL/6 mice weighing 20–25g were
used for the experiments. Mice were sensitized by intraperitoneal
(i.p.) injections of 25 mg OVA mixed with aluminum hydroxide
(Pierce) at day 1, and followed by another booster i.p. injection at
day 14. These sensitized mice then were challenged with 20 mg of
OVA through an intranasal (i.n.) route for three consecutive days
(days 25, 26, and 27). 56106of Treg cells or control T cells were
intravenously injected into mice before allergen challenge (day 22),
or after first allergen challenge (Day 25). Lung functional test and
specimen were all performed 24 hours after last allergen challenge
(day 28). Experiments were approved by IACUC at Children’s
Hospital Los Angeles.
Generation of in vitro TGF-b-induced regulatory T (iTreg)
Splenic CD4+CD252D62L+CD44lownaive T cells were isolated
by autoMACS (Miltenyi Biotech) from the littermates to the mice
used for generating the OVA-asthma model. iTreg cells were then
prepared as previously described . Briefly, CD4+CD252cells
were treated with anti-CD3/CD28 coated beads and IL-2 in the
presence of TGF-b for 5 days, the TGF-b-induced Treg cells
(CD4+CD25+FoxP3+) cells were then sorted by flow cytometry.
Splenic CD4+CD25+cells were sorted and expanded
with anti-CD3 and CD28 coated beads as described previously
. Foxp3 expression of nTregs was more than 75% and
sustained the suppressive activity after expansion (Fig. 1).
Determination of airway hyperresponsiveness (AHR) in
The mice were anesthetized with i.p. injection of sodium
pentobarbital (90mg/kg). A tracheostomy was performed. The
mice were then connected to a computer controlled small animal
ventilator (FlexiVent, SCIREQ) and ventilated at 150 breath/min
with a tidal volume of 10ml/Kg and a positive end-expiratory
pressure of 3 cmH2O. Methacholine (MCh, 40 mg/ml in PBS)
was then delivered to the subject by nebulized aerosol. The
frequency-independent airway resistance in mouse lung was
measured by FlexiVent/SCIREQ software, and the MCh
challenge experiments are repeated at least three times.
Lung histopathology and immunohistochemistry
Discombe’s and Periodic-Acid-Schiff staining were used detect
eosinophil and glycoprotein, respectively . Lung inflammation
was evaluated using a semi-quantitative method . Inflamma-
tion foci were scored 0–5 (0=no foci, 1=#5, 2=6–15, 3=16–
25, 4=26–35, 5=$35) separately for peri-vascular and peri-
bronchiolar, as well as alveolar regions. Thus, the total inflam-
mation score is the sum of these three measurements in a range of
0–15. At least four mice in each group were selected for this
analysis, as presented by mean 6 SEM.
Serum and intracellular cytokine analysis
The levels of cytokines (IL-4, IL5 and IL-13) and IgE in sera
were measured using ELISA kits (Invitrogen). Lymphocytes in
axillary draining lymph nodes and spleen were collected and
stained for markers. In the case of intracellular IL-4, IL-17A and
IFN-c, cells werestimulatedwith 0.25 mg/ml PMA and 0.25 mg/ml
ionomycin (Calbiochem), and followed by incubation with
brefeldin A (5 mg/ml) for additional 4 hours. The phenotypes
and intracellular cytokine expression were analyzed using a LSRII
Real-Time PCR analysis and primers
Total tissue RNAs were isolated form snap-frozen lung tissue or
lymph nodes using a RNeasy kit (Qiagen, Valencia, CA). Synthesis
of cDNA and quantitative reverse transcriptase analysis were
performed using iScript cDNA synthesis kit, and real-time
quantitative PCR was performed using SYBR Green I and
iCycle-iQ system (Bio-Rad) as previously published . The
PCR primers were: T-bet (59-TCAACCAGCACCAGACAGAG-
39, 59-AAACATCCTGTAATTGGCTTGTG-39), Gata3 (59-
CAATTT-39), IRF-4 (59-CAATGTCCTGTGACGTTTGG-39,
Induced-Treg Cells Attenuates Asthma
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59-GTTCCTGTCACCTGGCAAC-39), IL-10 (59-ACTGCACC- Download full-text
CCTTGTGGGTCACAACCAT-39), respectively. GAPDH was
used to normalize equal addition of template cDNAs.
The quantitative data are presented by mean 6 SEM.
Statistical analyses were performed by ANOVA. Differences were
considered statistically significant when a P value is less than 0.05.
Conceived and designed the experiments: HF CY JK DW DT BR SZ WS.
Performed the experiments: WX QL MC HC NZ XZ JW. Analyzed the
data: WX QL HC NZ XZ JW SZ WS. Contributed reagents/materials/
analysis tools: DT BR SZ WS. Wrote the paper: WX DW BR SZ WS.
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Induced-Treg Cells Attenuates Asthma
PLoS ONE | www.plosone.org10 July 2012 | Volume 7 | Issue 7 | e40314