CD4+CD25+ T Cells Regulate Airway
Eosinophilic Inflammation by Modulating the
Th2 Cell Phenotype
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J Immunol 2004; 172:3842-3849; ;
doi: 10.4049/jimmunol.172.6.3842
http://www.jimmunol.org/content/172/6/3842
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References
Zeina Jaffar, Thamayanthi Sivakuru and Kevan Roberts
The Journal of Immunology
CD4ⴙCD25ⴙ T Cells Regulate Airway Eosinophilic
Inflammation by Modulating the Th2 Cell Phenotype1
Zeina Jaffar,2* Thamayanthi Sivakuru,† and Kevan Roberts2*
I
n recent years, allergic asthma has considerably increased in
prevalence worldwide. The disease is characterized by airway hyperreactivity (AHR)3 and chronic mucosal inflammation mediated by CD4⫹ Th2 cells (1). Such events are associated
with pulmonary eosinophilia, mucus hypersecretion, and airway
remodeling (2). It has been suggested by several laboratories that
the chronic inflammation evident in asthma arises as a consequence of a defect in immune regulation. However, the specific
events that serve to down-regulate airway Th2-mediated inflammation are poorly understood. Certainly, the production of antiinflammatory cytokines TGF-␤ or IL-10 and specific prostanoids
at sites of inflammation serve to limit mucosal immune responses
(3, 4). More notably, regulatory T cells producing high levels of
IL-10 have recently been shown to modulate allergen-induced airway responses (5).
Regulatory T cells have been identified in mice and humans as
a distinct population of CD4⫹ T cells that constitutively express
the IL-2R ␣-chain (CD25) (6, 7). CD4⫹CD25⫹ T cells play an
essential role in the maintenance of peripheral self-tolerance (8, 9)
by preventing the activation and proliferation of autoreactive T
cells that have escaped thymic deletion (10). The seminal finding
*Center for Environmental Health Sciences, University of Montana, Missoula, MT
59812; and †Medical Specialties, Southampton General Hospital, Southampton,
United Kingdom
Received for publication October 24, 2003. Accepted for publication January
13, 2004.
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.
1
This work was kindly supported by grants from The Royal Society, Wellcome Trust
(U.K.) and the National Institutes of Health (EPSCOR, GC032-01-Z1886).
2
Address correspondence and reprint requests to Dr. Kevan Roberts or Dr. Zeina Jaffar,
Center for Environmental Health Sciences, 152A Skaggs Building, University of Montana, Missoula, MT 59812. E-mail addresses: [email protected] or zjaffar@
spahs.umt.edu
3
Abbreviations used in this paper: AHR, airway hyperreactivity; PLN, peripheral
lymph node; BAL, bronchoalveolar lavage; LMC, lung mononuclear cell; Penh, enhanced pause; EPO, eosinophil peroxidase.
Copyright © 2004 by The American Association of Immunologists, Inc.
that prompted speculation that these cells played a critical role in
preventing autoimmunity was the observation that depletion of
CD4⫹CD25⫹ T cells resulted in the development of organ-specific
autoimmune disorders which could be prevented by the adoptive
transfer of CD4⫹CD25⫹ T cells (6).
Following TCR engagement, CD4⫹CD25⫹ cells can suppress
the activation and proliferation of other CD4⫹ and CD8⫹ T cells
in an Ag-nonspecific manner (11–13). Murine CD4⫹CD25⫹ T
cells mediate the suppression of effector T cell function both in
vitro and in vivo via several mechanisms requiring either cell-cell
contact (14) or the production of immunosuppressive cytokines
such as IL-10 (15) and TGF-␤ (16). A role for glucocorticoidinduced TNFR in abrogating CD4⫹CD25⫹ T cell-mediated suppression has been proposed (17, 18). More recently, it was reported
that the forkhead transcription factor Foxp3 is specifically expressed in CD4⫹CD25⫹ T cells and is required for their development. An important observation was that Foxp3 mutant scurfy and
Foxp3-null mice developed a lethal autoimmune syndrome as a
consequence of a deficiency in CD4⫹CD25⫹ regulatory T cells.
Transfer of CD4⫹CD25⫹ T cells into neonatal Foxp3-deficient
mice prevented the development of disease (19, 20).
CD4⫹CD25⫹ T cells have been demonstrated to prevent the
onset of colitis, a Th1-mediated disease (21); however, their precise role in Th2-driven disease is unclear. In this study, we investigated whether Th2-mediated pulmonary inflammation is influenced by Ag-specific CD4⫹CD25⫹ T cells. In DO11.10 mice,
4 – 6% of CD4⫹ T cells constitutively expressed CD25 and coexpressed the transgenic TCR. Surprisingly, depletion of
CD4⫹CD25⫹ T cells before Th2 differentiation profoundly reduced the production of Th2 cytokines. However, the
CD4⫹CD25⫺-derived cells did not secrete IFN-␥, suggesting that
they were indeed Th2 polarized. Unexpectedly, transfer of
CD4⫹CD25⫺-derived Th2 cells into BALB/c mice resulted in a
heightened pulmonary eosinophilic inflammation following OVA
inhalation in these animals compared with recipients of total
CD4⫹ Th2 cells. This eosinophilia was associated with increased
0022-1767/04/$02.00
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We used a TCR-transgenic mouse to investigate whether Th2-mediated airway inflammation is influenced by Ag-specific
CD4ⴙCD25ⴙ regulatory T cells. CD4ⴙCD25ⴙ T cells from DO11.10 mice expressed the transgenic TCR and mediated regulatory
activity. Unexpectedly, depletion of CD4ⴙCD25ⴙ T cells before Th2 differentiation markedly reduced the expression of IL-4, IL-5,
and IL-13 mRNA and protein when compared with unfractionated (total) CD4ⴙ Th2 cells. The CD4ⴙCD25ⴚ-derived Th2 cells
also expressed decreased levels of IL-10 but were clearly Th2 polarized since they did not produce any IFN-␥. Paradoxically,
adoptive transfer of CD4ⴙCD25ⴚ-derived Th2 cells into BALB/c mice induced an elevated airway eosinophilic inflammation in
response to OVA inhalation compared with recipients of total CD4ⴙ Th2 cells. The pronounced eosinophilia was associated with
reduced levels of IL-10 and increased amounts of eotaxin in the bronchoalveolar lavage fluid. This Th2 phenotype characterized
by reduced Th2 cytokine expression appeared to remain stable in vivo, even after repeated exposure of the animals to OVA
aerosols. Our results demonstrate that the immunoregulatory properties of CD4ⴙCD25ⴙ T cells do extend to Th2 responses.
Specifically, CD4ⴙCD25ⴙ T cells play a key role in modulating Th2-mediated pulmonary inflammation by suppressing the
development of a Th2 phenotype that is highly effective in vivo at promoting airway eosinophilia. Conceivably, this is partly a
consequence of regulatory T cells facilitating the production of IL-10. The Journal of Immunology, 2004, 172: 3842–3849.
The Journal of Immunology
levels of eotaxin but reduced amounts of IL-10 in the bronchoalveolar lavage (BAL). Our data demonstrate that the suppressive
properties of CD4⫹CD25⫹ T cells do extend to Th2 responses and
that these cells play a crucial role in modulating allergic lung
inflammation.
Materials and Methods
Animals
DO11.10 TCR-transgenic mice (originally developed by Dr. D. Y. Loh,
Howard Hughes Medical Institute, St. Louis, MO) were provided by Dr. E.
Shevach (National Institutes of Health, Bethesda, MD). These animals
were housed in microisolator cages under pathogen-free conditions at
Southampton University (Southhampton, U.K.). BALB/c mice were obtained from Harlan (Loughborough, U.K.). All experiments were performed according to the Home Office guidelines.
Preparation of CD4⫹CD25⫺ and CD4⫹CD25⫹ T cells
Measurement of cytokine production by Th2-polarized cells
To examine cytokine production, 8-day polarized cells (5 ⫻ 105/ml) were
stimulated with immobilized anti-CD3 (2 ␮g/ml) for 24 h, and the supernatants were harvested for measurement of IL-4, IL-5, IL-13, and IFN-␥ by
ELISA as described previously (4, 22). For IL-10 measurement by ELISA,
JES5-2A5 (BD PharMingen) was used as capture Ab and biotinylated
polyclonal anti-IL-10 Ab for detection (PeproTech, Rocky Hill, NJ).
Adoptive transfer of DO11.10 Th2 cells and OVA challenge of
recipient animals
DO11.10 Th2 cells derived from either CD4⫹CD25⫺ or total CD4⫹ T cells
were injected i.v. into BALB/c mice (107cells/mouse). Mice (four to six per
group) were then intranasally challenged by exposure to aerosolized solutions of OVA (0.5%, Grade V; Sigma-Aldrich, Poole, U.K.) for 20 min a
day over 7 consecutive days using a Wright’s nebulizer (Buxco Europe,
Petersfield, U.K.). Control mice were exposed to OVA aerosols but did not
receive DO11.10 Th2 cells. AHR was measured on day 7 in response to
methacholine inhalation by whole-body plethysmography (Buxco Europe,
Petersfield, U.K.). Animals were placed in chambers and exposed to nebulized PBS (baseline) followed by increasing concentrations of methacholine. Enhanced pause (Penh) was measured after each 3-min exposure.
Mice were killed on day 8, and BAL fluid was collected for analysis. Lung
tissue was dispersed by collagenase (Sigma-Aldrich) and the resultant lung
mononuclear cells (LMCs) were stimulated with OVA peptide or anti-CD3
for 24 h. Cytokine production by the lung cells was measured by ELISA (as
described above) and eosinophil peroxide (EPO) levels present in the
LMCs were determined by colorimetric analysis as previously described
(22). Macrophages were not depleted from LMC preparations since we
have previously shown in this model that interstitial macrophages suppress
IL-2 production and the associated T cell proliferation but do not affect Th2
cytokine expression (22).
Lung histology and immunohistochemical analysis
Preparation of effector DO11.10 Th2 cells
To drive T cell differentiation into Th2 effector phenotype, purified
CD4⫹CD25⫺ or unfractionated (total) CD4⫹ T cells (5 ⫻ 105/ml) were
incubated for 4 days in the presence of OVA323–339 peptide (1 ␮g/ml) and
murine IL-4 (2 ng/ml; R&D Systems, Abingdon, U.K.) plus anti-IFN-␥ Ab
(5 ␮g/ml, R4-6A2; American Type Culture Collection, Manassas, VA).
Irradiated (3000 rad) splenic APCs (depleted of CD4⫹ and CD8⫹ cells
using complement) were also added to these cultures (5 ⫻ 105/ml). After
4 days of incubation, cells were restimulated as before for another 4 days,
but this time also in the presence of IL-2 (100 U/ml; Cetus, Emeryville,
CA). Following culture for a total of 8 days, polarized effector Th2 cells
prepared from either total CD4⫹ T cells or CD4⫹CD25⫺ T cells were
adoptively transferred into BALB/c mice as described below. To drive T
cell differentiation into a Th1 phenotype, PLN cells were incubated (5 ⫻
105/ml) in the presence of OVA323–339 peptide (1 ␮g/ml), and mouse IL-12
(1 ng/ml; R&D Systems) plus anti-IL-4 Ab (5 ␮g/ml, 11B11; American
Type Culture Collection). After 4 days of culture, cells were restimulated
as before for another 4 days but this time also in the presence of IL-2 (100
U/ml; Cetus). After 8 days, Th1 cells were analyzed for cytokine expression by real-time RT-PCR.
Expansion of CD4⫹CD25⫹ T cells
The limited numbers of CD4⫹CD25⫹ T cells in DO11.10 mice made a
complete analysis of the properties of these cells difficult. Consequently,
purified CD4⫹CD25⫹ T cells (5 ⫻ 105/ml) were expanded for 8 days as
described above in the presence of APCs, OVA peptide, anti-IFN-␥ Ab and
either IL-2 alone (added throughout the 8-day culture) or IL-2 plus IL-4.
The expansion of CD4⫹CD25⫹ T cells in the presence of exogenous IL-2
was limited (30-fold increase in cell numbers over 8 days) compared with
that observed in the presence of IL-2 plus IL-4 (80-fold increase over 8
days). The CD4⫹CD25⫹ T cells expanded in IL-2 plus IL-4 were used for
experiments to examine suppressor function. We were able to maintain
these regulatory T cells for up to 10 days in culture.
Suppressor function of CD4⫹CD25⫹ T cells
To monitor the effects of CD4⫹CD25⫹ cells on T cell proliferative responses, either freshly isolated (day 0) or expanded (day 8) CD4⫹CD25⫹
T cells were added at various concentrations to DO11.10 PLN cells (2 ⫻
105) and the proliferation in response to immobilized anti-CD3 (2 ␮g/ml)
or OVA peptide (1 ␮g/ml) was determined after 48 h by [3H]thymidine
incorporation.
Nonlavaged lungs were obtained and one part of the lung tissue was fixed
in 10% Formalin, embedded in paraffin, and then stained with H&E. Small
samples (3 mm2) from another part of the lung were fixed in acetone
(containing protease inhibitors), embedded in glycol methacrylate, and
then sections (2 ␮m) were stained with biotinylated anti-clonotypic Ab
KJ1-26 and avidin-peroxidase.
Level of inflammation in the airways
BAL was performed by cannulating the trachea of each animal and washing the airways with 3 ⫻ 0.5 ml of PBS to collect BAL fluid. BAL fluid of
four animals was pooled and EPO levels present in BAL cells were determined by colorimetric analysis as described before (22). Cell differential
percentages were determined by light microscopic evaluation of stained
cytospin preparations and expressed as absolute cell numbers. Levels of
cytokines IL-4, IL-5, IL-10, IL-13, IFN-␥, and the chemokine eotaxin in
the BAL were measured using sensitive commercially available ELISA
kits (all from BioSource International, Camarillo, CA, except IL-4 and
eotaxin which are from R&D Systems), according to the manufacturers’
instructions.
Cotransfer of CD4⫹CD25⫹ T cells
In certain experiments, 8-day expanded DO11.10 CD4⫹CD25⫹ T cells
(5 ⫻ 106 cells/mouse) were cotransferred into BALB/c recipients simultaneously with CD4⫹CD25⫺-derived Th2 cells (107cells/mouse). The level
of airway eosinophilia was assessed as described above.
Flow cytometry
Cells were stained and analyzed either on a FACSAria (BD Biosciences,
San Diego, CA) using FACSDiVa software for performing three-color
analysis to enumerate CD4⫹ T cells (using GK1.5-APC-Cy7), OVA-specific T cells (using KJ1-26-FITC) and CD25⫹ T cells (using 7D4-PE), or
on a FACSCalibur (BD Biosciences) using CellQuest software to enumerate CD4⫹ T cells (GK1.5-PE; BD PharMingen), clonotypic T cells (KJ126-FITC), and CD25⫹ T cells (anti-7D4-biotin and avidin-FITC).
Real-time RT-PCR
RNA was extracted from Th1- and Th2-polarized cells or purified
CD4⫹CD25⫹ T cells (following stimulation with immobilized anti-CD3)
using TRIzol reagent (Life Technologies, Renfrewshire, U.K.). Total RNA
(2 ␮g) was then reverse transcribed using Omniscript II (Qiagen, Crawley,
U.K.) at 37°C for 1 h using oligo(dT)15 as a primer and the cDNA was then
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To prepare CD4⫹CD25⫺ and CD4⫹CD25⫹ T cells, peripheral lymph node
(PLN) cells from DO11.10 mice were first depleted of B cells by panning
using anti-Ig Abs. Cells were then further depleted of both class II⫹ and
CD8⫹ cells by a second round of panning using both M5/114 and
YTS169.4 Abs (Serotec, Oxford, U.K.). The resultant CD4⫹ T cells were
either left unfractionated or separated into CD25⫹ and CD25⫺ T cells by
magnetic microbeads (Miltenyi Biotec, Bisley, Surrey, U.K.) using both
biotinylated anti-CD25 Abs 3C7 and 7D4, labeled with avidin-FITC (BD
PharMingen, San Diego, CA), and anti-FITC beads (Miltenyi Biotec).
Flow cytometry was used to determine the purity of CD4⫹ T cells (⬎98%)
and the fractionated CD4⫹CD25⫺ (⬎98%) or CD4⫹CD25⫹ T cells
(⬎82%).
3843
3844
PCR amplified and quantified using the TaqMan technique (Applied Biosystems, Warrington, U.K.). Real-time PCR was performed using the Perkin-Elmer AB1 Prism 7700 Sequence Detection System (Applied Biosystems). The expression of GAPDH (housekeeping gene), IL-4, IL-5, IL-10,
IL-13, and IFN-␥ was determined (4, 23). Equal amounts of cDNA were
used in triplicate and amplified with the TaqMan master mix according to
manufacturer’s instructions (Applied Biosystems). Thermal cycling conditions were 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of
two-step PCR consisting of 15 s at 95°C and 1 min at 60°C. Threshold
cycle was measured as the cycle number at which the reporter fluorescent
emission increased above a threshold level. The amount of mRNA was
expressed as fold difference relative to the amount obtained from unstimulated control cells. Amplification efficiencies were validated and normalized against GAPDH. For all samples, total RNA that was not reverse
transcribed was also analyzed to determine genomic DNA contamination,
which was negligible.
Statistical analysis
Data are summarized as means ⫾ SEM. Data obtained from adoptive transfer experiments were analyzed using the Mann-Whitney U test, and differences were considered statistically significant with p ⬍ 0.05.
CD4⫹CD25⫹ T cells from DO11.10 mice mediate regulatory
function
CD4⫹CD25⫹ regulatory T cells have been demonstrated to play a
critical role in preventing organ-specific autoimmune diseases (6).
We used DO11.10 mice to model allergic lung inflammation and
investigated whether a Th2 inflammatory response is influenced by
Ag-specific CD4⫹CD25⫹ T cells. We first assessed the characteristics of CD4⫹CD25⫹ T cells in nonimmunized DO11.10 mice.
Consistent with previous reports, FACS analysis revealed that
4 – 6% of CD4⫹ T cells from DO11.10 mice constitutively expressed CD25 (Fig. 1A), whereas in BALB/c mice 10 –15% of
CD4⫹ T cells express CD25 (11, 24, 25). It is likely that the
expression of the nonautoreactive transgenic TCR contributes to
the reduced number of CD4⫹CD25⫹ regulatory T cells present
in DO11.10 mice (26). The limited numbers of DO11.10
CD4⫹CD25⫹ cells made a complete analysis of the properties of
these cells difficult and necessitated the expansion of these cells in
vitro over 8 days. The majority of freshly isolated (day 0) and
expanded (day 8) CD4⫹CD25⫹ or CD4⫹CD25⫺ T cells were
OVA specific since they expressed the transgenic TCR i.e., KJ126⫹ (Fig. 1, A and B). Interestingly, both newly isolated and 8-day
expanded CD4⫹CD25⫹ T cells exhibited regulatory function by
suppressing proliferative responses of effector T cells to anti-CD3
or OVA323–339 peptide in a dose-dependent manner (Fig. 1C). The
inhibition elicited by OVA peptide was less potent than that observed with anti-CD3 stimulation. This may arise because antiCD3 is a more effective stimulus, being less dependent on APCs.
Second, the anti-CD3 used was immobilized on a plate and may
thus exert a cross-linking function between regulatory and nonregulatory CD4⫹ T cells. Such a bridging effect could potentially
augment suppressor function.
Depletion of CD4⫹CD25⫹ T cells results in the generation of a
Th2 cell phenotype characterized by reduced cytokine
expression
To evaluate whether regulatory T cells suppressed Th2 responses,
we initially determined the effect of depleting CD4⫹CD25⫹ T
cells on Th2 cell polarization in vitro. To this end, DO11.10
CD4⫹CD25⫺ or unfractionated (total) CD4⫹ T cells were differentiated for 8 days in the presence of IL-2 and IL-4 and the expression of cytokines in response to anti-CD3 stimulation was then
determined. The level of proliferation of CD4⫹CD25⫺-derived
and total CD4⫹ T cells were similar (80-fold increase in cell numbers over 8 days). Unexpectedly, 8-day polarized CD4⫹CD25⫺-
derived Th2 effector cells secreted significantly less IL-4, IL-5,
IL-10, and IL-13 than total CD4⫹ Th2 cells in response to immobilized anti-CD3 (2 ␮g/ml) stimulation for 24 h (Fig. 2A). Moreover, the difference in Th2 cytokine production was maintained
using different concentrations of immobilized anti-CD3, and the
addition of accessory cells had no effect on the level of cytokines
produced by these polarized cells (data not shown). Interestingly,
no IFN-␥ was secreted by either type of Th2-differentiated cells
(Fig. 2A). The lack of IFN-␥ production by CD4⫹CD25⫺-derived
Th2 cells implies that their reduced level of Th2 cytokine production did not arise as a consequence of incomplete Th2 polarization.
The expression of mRNA transcripts for IL-4, IL-5, IL-10, and
IL-13 were also markedly reduced in CD4⫹CD25⫺-derived Th2
cells compared with total CD4⫹ Th2 cells (Fig. 2B). This suggests
that the difference in the level of Th2 cytokine production is a
consequence of reduced transcription rather than consumption of
the cytokine. In comparison, Th1 cells and CD4⫹CD25⫹ regulatory T cells expressed small or negligible amounts of IL-4, IL-5,
and IL-13 mRNA (regulatory T cells did express IL-10 transcripts). Neither CD4⫹CD25⫺-derived, or total CD4⫹ Th2 cells
expressed any IFN-␥ mRNA (Fig. 2B). Thus, depletion of
CD4⫹CD25⫹ T cells before Th2 differentiation resulted in markedly reduced cytokine expression by the polarized cells.
Adoptive transfer of CD4⫹CD25⫺-derived Th2 cells causes a
heightened pulmonary eosinophilic inflammation to inhaled OVA
We next studied allergic pulmonary inflammation by adoptively
transferring DO11.10 Th2 cells, generated from unfractionated
CD4⫹ T cells, into BALB/c mice (107 cells/animal) which were
then exposed to OVA aerosols for 7 consecutive days. To investigate how regulatory T cells influence airway inflammation,
CD4⫹CD25⫹ T cells were removed before Th2 polarization, and
the Th2 cells prepared from CD4⫹CD25⫺ T cells were also transferred into BALB/c mice. Following exposure to OVA aerosols, a
pronounced peribronchial and perivascular eosinophilic inflammation and an increase in KJ1-26⫹ T cells was observed in the lung
parenchyma of recipients of Th2 cells generated from either
CD4⫹CD25⫺ or total CD4⫹ T cells compared with animals that did
not receive any cells (Fig. 3A). We next examined the influx of
inflammatory cells into the BAL. Paradoxically, recipients of
CD4⫹CD25⫺-derived Th2 cells displayed a striking increase in the
number of eosinophils compared with recipients of total Th2 cells
(Fig. 3B). Consistently, there was a significant rise in the level of EPO
activity in the BAL of animals injected with CD4⫹CD25⫺-derived
Th2 cells (Fig. 3C) and in LMCs of these animals (14.8 ⫾ 2.6 ng/ml
EPO for CD4⫹CD25⫺ Th2 recipients vs 7.4 ⫾ 1.7 ng/ml for total
CD4⫹ Th2 recipients). However, the AHR was not significantly
elevated in recipients of CD4⫹CD25⫺-derived Th2 cells compared
with recipients of total CD4⫹ T cells (Fig. 3D).
Interestingly, when monitoring pulmonary inflammation at earlier time points, a lag in the onset of pulmonary eosinophilia mediated by Th2 cells derived from CD4⫹CD25⫺ T cells was observed. However, invariably the eosinophilic inflammation
observed in mice that have received CD4⫹CD25⫺-derived Th2
cells was markedly more intense following 7 days of aerosol exposure (Table I). The delayed kinetics in the onset of eosinophilia
by CD4⫹CD25⫺ Th2 cells is likely to reflect decreased IL-5 production by these cells and the consequent slower maturation of
eosinophil precursors. The cotransfer of expanded CD4⫹CD25⫹
regulatory T cells along with CD4⫹CD25⫺-derived cells did not
reduce the airway inflammatory response (data not shown). Thus,
although removal of CD4⫹CD25⫹ T cells before Th2 polarization
resulted in a Th2 phenotype with reduced cytokine expression,
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Results
CD4⫹CD25⫹ T CELLS REGULATE AIRWAY Th2 RESPONSES
The Journal of Immunology
3845
these cells were effective at inducing a heightened airway eosinophilic inflammation following OVA inhalation for 7 days.
Depletion of CD4⫹CD25⫹ T cells alters cytokine and
chemokine production in vivo
FIGURE 1. CD4⫹CD25⫹ T cells are present in DO11.10 mice and mediate suppressor function. A, FACS analysis of the expression of CD25 by
CD4⫹ T cells in PLN of nonimmunized DO11.10 mice (day 0). Using
three-color analysis, CD4⫹ T cells were gated and the proportion of OVAspecific CD25⫹ T cells was assessed by staining with FITC-conjugated
KJ1-26, an Ab specific for DO11.10-transgenic TCR. The limited numbers
of CD4⫹CD25⫹ T cells in DO11.10 mice (4.9%), necessitated their expansion for 8 days in the presence of OVA323–339 peptide and IL-2 plus
IL-4. B, The expression of transgenic TCR (KJ1-26) by expanded
CD4⫹CD25⫹ and CD4⫹CD25⫺ T cells (day 8). C, CD4⫹CD25⫹ regulatory T cells suppressed T cell proliferative responses. Either freshly
Since DO11.10 Th2 cells prepared from CD4⫹CD25⫺ T cells secreted lower amounts of IL-4, IL-5, and IL-13 in vitro, yet caused
an elevated airway eosinophilia, we examined levels of Th2 cytokines expressed in vivo in the BAL and lung tissue. The levels of
IL-13 were significantly lower in the BAL of recipients of
CD4⫹CD25⫺-derived Th2 cells compared with recipients of total
CD4⫹ Th2 cells, whereas the amounts of IL-4 and IL-5 were only
marginally reduced (Fig. 4A). Moreover, BAL levels of the antiinflammatory cytokine IL-10 were also decreased in recipients of
CD4⫹CD25⫺-derived Th2 cells (Fig. 4B). No IFN-␥ was present
in the BAL of these two experimental groups (data not shown).
Interestingly, although there was a reduction in cytokine production in mice that have received Th2 cells prepared from
isolated or expanded DO11.10 CD4⫹CD25⫹ T cells were added at various
concentrations to DO11.10 PLN cells (2 ⫻ 105) and the proliferation in
response to OVA323–339 peptide (1 ␮g/ml) or immobilized anti-CD3 (2
␮g/ml) was determined after 48 h by [3H]thymidine incorporation. Data
denote means ⫾ SEM (n ⫽ 3). Flow cytometric and proliferation results
are representative of three independent experiments.
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FIGURE 2. Depletion of CD4⫹CD25⫹ T cells before Th2 differentiation results in the generation of a Th2 cell phenotype characterized by
reduced cytokine expression. CD4⫹CD25⫺ T cells were prepared by depletion of CD4⫹CD25⫹ T cells using magnetic beads. CD4⫹CD25⫺ or
unfractionated (total) CD4⫹ T cells were differentiated into Th2 phenotype
by culture for 8 days in the presence of OVA323–339 peptide, splenic APCs,
and IL-2 plus IL-4. A, The Th2-polarized cells (5 ⫻ 105/ml) were stimulated for 24 h with immobilized anti-CD3 (2 ␮g/ml) and production of
cytokine protein was measured by ELISA. B, Cytokine mRNA expression
by total CD4⫹ or CD4⫹CD25⫺-derived Th2-polarized cells as compared
with Th1 or freshly isolated CD4⫹CD25⫹ T cells following stimulation
with immobilized anti-CD3. Cells were analyzed by quantitative real-time
RT-PCR. mRNA levels were expressed relative to nonstimulated control
cells. Data are means ⫾ SEM. (n ⫽ 3). AU, Arbitrary units.
3846
CD4⫹CD25⫹ T CELLS REGULATE AIRWAY Th2 RESPONSES
CD4⫹CD25⫺ T cells, the amount of the chemokine eotaxin
present in the BAL was elevated in these animals (Fig. 4C).
To determine the cytokine production by DO11.10 T cells recruited to the lung, the lung tissue was enzymatically digested and
the production of cytokines by LMCs was assessed after restimulation with OVA323–339 peptide or immobilized anti-CD3 for 24 h.
Following OVA inhalation, there was a reduced production of both
IL-4 and IL-5 by stimulated LMCs from animals injected with
CD4⫹CD25⫺-derived Th2 cells compared with recipients of total
CD4⫹ Th2 cells (Fig. 5). However, the frequency of KJ1-26⫹ T
cells in the lungs of these mice, as assessed by FACS, was comparable (2.5% in recipients of total CD4⫹ cells, vs 2.6% in recipients of CD4⫹CD25⫺ cells). Negligible IFN-␥ was secreted by
LMCs from these animals in response to OVA peptide stimulation.
These results suggest that the OVA-specific CD4⫹CD25⫺-derived
Th2 cells retained their Th2 phenotype in vivo and their reduced
cytokine production. In contrast, anti-CD3 stimulation did elicit an
increase in IFN-␥ production by LMCs from recipients of
CD4⫹CD25⫺-derived Th2 cells, presumably reflecting increased
secretion of the Th1 cytokine by the host T cells as a consequence
of reduced IL-4 secretion by DO11.10 T cells present (i.e.,
cross-regulation).
Discussion
Allergic asthma is characterized by airway hyperresponsiveness
and mucosal inflammation mediated by CD4⫹ Th2 cells. That
these events arise as a consequence of a defect in immune regulation is implied from the observation that lung mucosal immune
responses are normally tightly regulated (22). However, the mechanisms that transpire to resolve, or possibly prevent, airway mucosal Th2-mediated inflammation remain poorly defined. In this
context, both TGF-␤ and IL-10 have been shown to regulate mucosal inflammation (3). In addition, PGI2 generated during allergic
pulmonary inflammation serves to selectively limit the progression
of Th2, but not Th1, responses (4).
Recent work has shown that following Ag inhalation,
CD4⫹CD25⫹ regulatory T cells play a key immunomodulatory
role (5). Such suppressor T cells have been shown to prevent autoimmunity (6, 27) and the development of colitis induced by Helicobacter hepaticus (3, 28). CD4⫹CD25⫹ T cells regulate both
CD4⫹ and CD8⫹ T cell responses, partly by inhibiting IL-2 production, the subsequent clonal expansion of T cells, and the development of memory (11, 12). In addition, it has been proposed
that both IL-10 and TGF-␤ can mediate suppression in vivo. However, regulation can take place in the absence of either of these
cytokines (29). In this instance, cell-cell contact forms a prerequisite for mediating immune regulation (11, 14) which can be reversed by Abs to glucocorticoid-induced TNFR (17).
It has been reported that Th1 responses are more prone to regulation by CD4⫹CD25⫹ T cells than Th2 responses (30). Nevertheless, CD4⫹CD25⫹ T cells can suppress Th2 maturation (31),
possibly by inhibiting IL-4 production (32) and the development of
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FIGURE 3. CD4⫹CD25⫺-derived
Th2 cells elicit increased pulmonary eosinophilic inflammation. CD4⫹CD25⫺derived Th2 cells and total CD4⫹ Th2
cells were injected (107 cells/animal) into
BALB/c mice that were then exposed to
OVA aerosols for 7 consecutive days.
Control mice did not receive Th2 cells
(None). A, Lung tissue were stained with
H&E (magnification, ⫻25) or prepared
for immunohistochemistry and stained
with biotinylated anti-clonotypic KJ1-26
Ab. Recipients of total CD4⫹ Th2 or
CD4⫹CD25⫺-derived Th2 cells displayed peribronchiolar and perivascular
eosinophilic inflammation and an infiltration of KJ1-26⫹ T cells. B, BAL fluid
was collected and cell differential counts
were determined by light microscopic
evaluation of cytospin preparations. Results are expressed as absolute number of
lymphocytes (Lym), eosinophils (Eos),
and neutrophils (Neu). C, The level of
EPO activity in the BAL was determined
by colorimetric analysis. D, Changes of
Penh measurements in response to inhaled methacholine. Exaggerated increases in Penh following exposure to
OVA aerosols indicate airway hyperreactivity. Data represent means ⫾ SEM
(n ⫽ 3) and are representative of four
separate experiments. ⴱ, p ⬍ 0.05 compared with recipients of total Th2 cells.
The Journal of Immunology
3847
Table I. Time course for the development of airway inflammation
BAL Cell Number (⫻103)
OVA Aerosol
Exposure (days)
3
5
7
Th2 cells
Injected
None
Total CD4⫹
CD4⫹CD25⫺
None
Total CD4⫹
CD4⫹CD25⫺
None
Total CD4⫹
CD4⫹CD25⫺
Lymphocytes
22.2 ⫾ 0.2
54.1 ⫾ 5.5
41.3 ⫾ 4.5
60.5 ⫾ 4.6
391.4 ⫾ 71.1
214.4 ⫾ 52.6
62.6 ⫾ 25.0
700.5 ⫾ 85.7
520.7 ⫾ 95.0
Eosinophils
Neutrophils
11.0 ⫾ 0.7 9.7 ⫾ 0.5
29.9 ⫾ 7.1 9.8 ⫾ 4.8
21.4 ⫾ 0.4 10.9 ⫾ 4.2
48.9 ⫾ 4.5 50.3 ⫾ 3.2
711.9 ⫾ 64.6 38.1 ⫾ 5.4
375.9 ⫾ 81.5 33.8 ⫾ 3.7
57.2 ⫾ 6.7 50.1 ⫾ 8.1
980.5 ⫾ 88.4 82.4 ⫾ 15.7
1782.0 ⫾ 92.8 91.1 ⫾ 6.2
FIGURE 4. Adoptive transfer of CD4⫹CD25⫺derived Th2 cells results in reduced cytokine levels
in the BAL but increased levels of the eosinophil
chemoattractant eotaxin. CD4⫹CD25⫺-derived Th2
cells or total CD4⫹ Th2 cells were injected (107
cells/animal) into BALB/c mice that were then exposed to OVA aerosols for 7 days. Control mice did
not receive Th2 cells (None). BAL fluid was analyzed by ELISA for levels of Th2 cytokines IL-4,
IL-5, and IL-13 (A), the anti-inflammatory cytokine
IL-10 (B), and the chemokine eotaxin (C). Data denote means ⫾ SEM (n ⫽ 3) and are representative
of four separate experiments. ⴱ, p ⬍ 0.05 compared
with recipients of total Th2 cells
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
pulmonary eosinophilic inflammation (33). The mechanism by
which regulatory T cells mediate these effects remains unresolved.
This is, in part, due to the difficulty in obtaining Ag-specific
CD4⫹CD25⫹ T cells.
In our study, we used the DO11.10 TCR-transgenic mouse to
examine the role of Ag-specific regulatory T cells in a model of
allergic pulmonary inflammation. In DO11.10 mice, 4 – 6% of
CD4⫹ T cells constitutively express CD25, which was consistent
with previous findings (25, 34). Our analysis revealed that the
majority of CD4⫹CD25⫹ T cells in these animals express the
transgenic TCR as defined by staining with the KJ1-26 Ab. The
low frequency of CD4⫹CD25⫹ T cells in DO11.10 mice made it
necessary to expand these cells over 8 days in culture before they
could be characterized in detail. Although these cells are anergic,
they could be rapidly expanded by stimulation with OVA323–339
peptide in the presence of exogenous IL-2 and IL-4. Our results
showed that freshly isolated and 8-day expanded CD4⫹CD25⫹ T
cells exhibited regulatory function by suppressing proliferative responses of DO11.10 T cells to OVA323–339 peptide or anti-CD3
stimulation. Consequently, the regulatory activity of these cells was
not notably affected by expansion in the presence of Ag or IL-4.
To investigate whether CD4ⴙCD25ⴙ T cells regulated Th2-mediated pulmonary inflammation, it was important to evaluate how
these cells influenced either the initial Th2 polarization stage or the
subsequent development of allergic inflammation in vivo. The role
of CD4ⴙCD25ⴙ T cells in modulating these processes was examined separately by monitoring the effect of depleting these cells on
both Th2 differentiation in vitro and the inflammation elicited in
vivo. CD4ⴙCD25ⴙ T cells had to be removed before polarization
of DO11.10 T cells for 8 days, since activated T cells express
CD25. CD4ⴙCD25ⴙ T cells, both freshly isolated or expanded in
the presence of IL-2 plus IL-4, expressed only low levels of IL-4,
IL-5, and IL-13 mRNA, implying that these cells do not differentiate into Th2 cells. Unexpectedly, depletion of regulatory T cells
markedly reduced the cytokine production by Th2 cells generated
in vitro. Most notably, the expression of mRNA and protein for the
cytokines IL-4, IL-5, IL-10, and IL-13 by Th2 cells derived from
CD4ⴙCD25⫺ T cells was markedly lower than that generated from
total CD4ⴙ T cells. This effect was observed irrespective of the
level of anti-CD3 stimulation used to induce cytokine secretion
and the addition of accessory cells had no effect. Such differences
did not seem to reflect different levels of proliferation or the
amount of IL-2 present given that exogenous IL-2 was added and
the level of proliferation was similar. Conceivably, CD4ⴙCD25ⴙ
T cells may influence either the maturation of Th2 cells directly or
act by inhibiting Th1 development (i.e., cross-regulation). Using
the DO11.10 model of airway inflammation, it has been suggested
that regulatory T cells favor Th2 polarization by inhibiting Th1
development (35). However, in our experiments, the Th2 cells prepared from CD4⫹CD25⫺ T cells did not produce any detectable
IFN-␥, even after adoptive transfer into mice that were then repeatedly exposed to OVA aerosols. This implies that the reduced
3848
cytokine expression by CD4⫹CD25⫺-derived Th2 cells was not
simply a consequence of incomplete Th2 polarization or a shift
toward Th1 maturation
We next examined whether the inflammatory response mediated
by CD4⫹CD25⫺-derived Th2 cells in vivo was similarly compromised. Surprisingly, when transferred into BALB/c hosts, the Th2
cells prepared from CD4⫹CD25⫺ T cells were capable of eliciting
a pronounced increase in the number of eosinophils present in the
BAL following OVA inhalation for 7 days compared with that
observed following injection of total CD4ⴙ Th2 cells. However,
AHR was not significantly different between the two groups. This
observation is consistent with the recent findings of Hadeiba and
Locksley (36) that CD4⫹CD25⫹ regulatory T cells suppress type
2 immune responses but not AHR. It is possible that CD4⫹CD25⫹
T cells are effective at regulating effector Th2 responses but are
unable to curtail certain events in vivo that involve other cell types
that influence AHR. The increased inflammation did not appear to
be a consequence of more effective expansion or recruitment of
DO11.10 T cells in recipients of CD4⫹CD25⫺-derived Th2 cells,
since the number of KJ1-26ⴙ T cells in the airways was not notably different from that observed following transfer of total CD4ⴙ
Th2 cells. Stimulation of LMCs with OVA323–339 peptide revealed
that the lung cells from recipients of CD4⫹CD25⫺ Th2 cells con-
sistently produced lower amounts of IL-4 and IL-5 when compared
with recipients of total CD4ⴙ Th2 cells. However, the frequency of
CD4⫹KJ1-26⫹ T cells in the lungs of these two groups of mice
was comparable. Thus, the Th2 phenotype characterized by reduced cytokine production appeared to remain stable in vivo even
after repeated exposure of the animals to OVA aerosols. Anti-CD3
stimulation, however, did elicit an increase in IFN-␥ production by
LMCs from recipients of CD4⫹CD25⫺-derived cells, presumably
reflecting increased secretion of this cytokine by the host T cells as
a consequence of reduced IL-4 secretion by DO11.10 T cells
present (i.e., cross-regulation). Consistent with the reduced cytokine expression by the CD4⫹CD25⫺ Th2 cells, there was a
marked decrease in IL-13 levels in the BAL of recipients of these
cells. The amounts of IL-4 or IL-5 were only marginally lower,
possibly reflecting the different cellular provenance of the cytokines or production by host T cells.
The observation that CD4⫹ helper T cells producing low levels
of cytokine are particularly effective in vivo has been previously
reported (37). These authors demonstrated that Th1 cells could be
categorized with respect to the level of IFN-␥ they produce, with
cells deficient in secreting IFN-␥ surviving for long periods in
vivo. Such IFN-␥⫺ cells retained their Th1 phenotype in vivo and
were effective Th1 memory cells (37). Conceivably a similar classification may exist for CD4⫹ Th2 cells, with such memory-type
responses being suppressed by CD4⫹CD25⫹ regulatory T cells.
Alternative explanations for the reduced cytokine expression could
be that removal of CD4⫹CD25⫹ T cells resulted in an increase in
the proportion of anergic T cells, or that the regulatory T cells
themselves may have a positive influence on cytokine production
by Th2 cells. The effectiveness of CD4⫹CD25⫺-derived Th2 cells
in vivo thus suggests that these cells possess additional proinflammatory characteristics that compensate for an apparent deficiency
in their Th2 cytokine production. In this context, an increased level
of eotaxin, a potent chemoattractant of eosinophils in allergic inflammation (38), was observed in the BAL of mice that have received CD4⫹CD25⫺-derived Th2 cells. Moreover, this was associated with a decrease in IL-10 production in the BAL of these
mice. Whether reduced IL-10 levels were directly responsible for
elevated eotaxin release in the airways is unclear. Certainly, IL-10
has been suggested to inhibit the production of a range of chemokines (39). The cotransfer of cultured CD4⫹CD25⫹ T cells was
found to be unable to reverse the severe eosinophilic inflammation
elicited by CD4⫹CD25⫺-derived Th2 cells. Consequently, it
would seem unlikely that heightened inflammation elicited by the
CD4⫹CD25⫺-derived Th2 cells arises because this response is not
modulated by Ag-specific CD4⫹CD25⫹ T cells in vivo.
In summary, CD4⫹CD25⫹ T cells play a key role in regulating
airway eosinophilic inflammation. Our results demonstrate that the
immunomodulatory properties of CD4⫹CD25⫹ T cells do extend
to Th2 responses, most notably by limiting the development of a
proinflammatory CD4⫹ Th2 phenotype characterized by reduced
cytokine production. An understanding of the regulation of Th2
responses in vivo could provide better insight into the design of
novel approaches to modulate the chronic airway inflammatory
reaction evident in bronchial asthma.
Acknowledgments
We thank Drs. Ethan Shevach and Peter Kilshaw for valuable discussions
and gratefully acknowledge the Biomedical Imaging and Histology Research Units (Southampton General Hospital) for their technical assistance.
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