Inducible costimulator expression regulates the magnitude of Th2-mediated airway inflammation by regulating the number of Th2 cells.
ABSTRACT Inducible Costimulator (ICOS) is an important regulator of Th2 lymphocyte function and a potential immunotherapeutic target for allergy and asthma. A SNP in the ICOS 5' promoter in humans is associated with increased atopy and serum IgE in a founder population and increased ICOS surface expression and Th2 cytokine production from peripheral blood mononuclear cells. However, it is unknown if increased ICOS expression contributes to disease progression or is a result of disease pathology.
We developed a mouse model in which ICOS surface expression levels are genetically predetermined to test our hypothesis that genetic regulation of ICOS expression controls the severity of Th2 responses in vivo. Using ICOS+/+ and ICOS+/- mice in a Th2 model of airway inflammation, we found that T cells from the ICOS+/- mice had reduced ICOS expression and decreased Th2-mediated inflammation in vivo. Although the activation status of the T cells did not differ, T cells isolated from the lungs and draining lymph nodes of ICOS+/- mice at the peak of inflammation produced less Th2 cytokines upon stimulation ex vivo. Using 4get mice, which express GFP upon IL-4 transcription, we determined that the decreased Th2 cytokines in ICOS+/- is due to reduced percentage of Th2 cells and not a defect in their ability to produce IL-4.
These data suggest that in both mice and humans, the level of ICOS surface expression regulates the magnitude of the in vivo Th2 response, perhaps by influencing Th2 differentiation.
- [show abstract] [hide abstract]
ABSTRACT: There has been a world-wide increase in the prevalence of atopic diseases. These atopic diseases, including asthma, allergic rhinoconjunctivitis and atopic eczema/dermatitis, are common in childhood and create a challenge of management for physicians and parents. MEDLINE was searched for articles related to atopy, allergy asthma, allergic rhinoconjunctivitis and atopic eczema/dermatitis. The conditions of asthma, allergic rhinoconjunctivitis and atopic eczema/dermatitis cause very significant burdens regarding the discomfort to the affected individual, management problems for the parent and physician and the economic cost to the family and the nation.Allergy 09/2004; 59 Suppl 78:7-11. · 5.88 Impact Factor
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ABSTRACT: The diseases of asthma, eczema and hay fever are typified by reactions to common allergens, which are mediated by immunoglobulin E. These allergic diseases are increasing in prevalence, and are now a major source of disability throughout the developed world. They are the result of complex interactions between largely unknown genetic and environmental mechanisms. The identification of the environmental factors offers the real possibility of prevention of disease, and unravelling the genetics of allergic illnesses is likely to change their classification and treatment. Early life seems particularly important, when the initiation of allergic disease may result from genetic and environmental modification of the immune interaction between mother and child.Nature 12/1999; 402(6760 Suppl):B5-11. · 38.60 Impact Factor
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ABSTRACT: Background: Several genomic regions have been identified that might contain genes contributing to the development of asthma and atopy. These include chromosome 2q33, where we have observed evidence for linkage for variation in total serum IgE levels in a Dutch asthma population. Two candidate genes, CTLA4 and CD28 , important homeostatic regulators of T-cell activation and subsequent IgE production, map within this candidate region. Objective: We sought to fine-map the chromosome 2q33 region and evaluate CTLA4 and CD28 as candidate genes for the regulation of total serum IgE levels and related phenotypes. Methods: The coding regions of CTLA4 and CD28 were resequenced in 96 individuals; 4 novel SNPs in CTLA4 and 10 in CD28 were identified. Polymorphisms in both genes were analyzed in 200 asthmatic probands and their spouses (n = 201). Results: Subsequent fine- mapping in this region has resulted in an increased log of the odds (lod) score (1.96 to 3.16) for total serum IgE levels. For CTLA4 , the +49 A/G single nucleotide polymorphism (SNP) in exon 1 and the 3 ′ untranslated region microsatellite were significantly associated with total serum IgE levels (P =.0005 and .006, respectively). For the combined +49 A/G and 3 ′untranslated region genotypes, individuals homozygous for the risk allele for both polymorphisms (AA and 86/86) had the highest total serum IgE values (87.1 IU/mL), whereas those individuals with the GG and XX/XX genotypes (anything but the 86-bp allele) had the lowest IgE values (29.3 IU/mL). Significant association was also observed for the CTLA4 −1147 C/T SNP with bronchial hyperresponsiveness (BHR) and asthma (P = .008 and .012, respectively), but not for allergy-related phenotypes. Promoter luciferase assays examining the −1147 polymorphism suggested that the T allele, which was associated with increased BHR susceptibility, was expressed at half the level of the C allele. Individuals with the risk genotypes for both BHR (−1147 CT or TT) and elevated IgE levels (+49 AA) were 4.5 times more likely to have asthma than individuals with both nonrisk genotypes (P = .0009). No significant associations were observed for SNPs in CD28. Conclusion: These data suggest that the costimulatory pathway, specifically CTLA4 , is important in the development of atopy and asthma. (J Allergy Clin Immunol 2002;110:743-51.)01/2002;
Inducible Costimulator Expression Regulates the
Magnitude of Th2-Mediated Airway Inflammation by
Regulating the Number of Th2 Cells
Bryan S. Clay1,2, Rebecca A. Shilling2, Hozefa S. Bandukwala1, Tamson V. Moore1, Judy L. Cannon2,
Andrew A. Welcher3, Joel V. Weinstock4, Anne I. Sperling1,2*
1Committee on Immunology, University of Chicago, Chicago, Illinois, United States of America, 2Section of Pulmonary and Critical Care Medicine, Department of
Medicine, University of Chicago, Chicago, Illinois, United States of America, 3Amgen Inc., Thousand Oaks, California, United States of America, 4Division of
Gastroenterology, Department of Internal Medicine, Tufts New England Medical Center, Boston, Massachusetts, United States of America
Background: Inducible Costimulator (ICOS) is an important regulator of Th2 lymphocyte function and a potential
immunotherapeutic target for allergy and asthma. A SNP in the ICOS 59 promoter in humans is associated with increased
atopy and serum IgE in a founder population and increased ICOS surface expression and Th2 cytokine production from
peripheral blood mononuclear cells. However, it is unknown if increased ICOS expression contributes to disease progression
or is a result of disease pathology.
Methodology/Principal Findings: We developed a mouse model in which ICOS surface expression levels are genetically
predetermined to test our hypothesis that genetic regulation of ICOS expression controls the severity of Th2 responses in
vivo. Using ICOS+/+and ICOS+/2mice in a Th2 model of airway inflammation, we found that T cells from the ICOS+/2mice
had reduced ICOS expression and decreased Th2-mediated inflammation in vivo. Although the activation status of the T
cells did not differ, T cells isolated from the lungs and draining lymph nodes of ICOS+/2mice at the peak of inflammation
produced less Th2 cytokines upon stimulation ex vivo. Using 4get mice, which express GFP upon IL-4 transcription, we
determined that the decreased Th2 cytokines in ICOS+/2is due to reduced percentage of Th2 cells and not a defect in their
ability to produce IL-4.
Conclusion: These data suggest that in both mice and humans, the level of ICOS surface expression regulates the
magnitude of the in vivo Th2 response, perhaps by influencing Th2 differentiation.
Citation: Clay BS, Shilling RA, Bandukwala HS, Moore TV, Cannon JL, et al. (2009) Inducible Costimulator Expression Regulates the Magnitude of Th2-Mediated
Airway Inflammation by Regulating the Number of Th2 Cells. PLoS ONE 4(11): e7525. doi:10.1371/journal.pone.0007525
Editor: Derya Unutmaz, New York University School of Medicine, United States of America
Received April 30, 2009; Accepted August 27, 2009; Published November 4, 2009
Copyright: ? 2009 Clay 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: This work was supported by R01 AI50180 and P01 A156352. Support for BSC and TVM was provided by 5T32AI07090 and a supplement to P01-
AI56352. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
The steady increase in the number of asthmatic and allergic
individuals that react to common allergens in industrialized countries
allergen specific IgE, total serum IgE and augmented levels of blood
eosinophils that are induced by the Th2 subset of T cells. There is a
genetic link to the development of atopy and asthma as the
concordance rate is significantly higher for monozygotic twins than
dizygotic twins . Multiple regions of the genome have been linked
with increased atopy in humans including a region on chromosome
2q33. Specifically, this region has been associated with increased
eosinophilia, serum IgE and atopy [3–5]. Among the candidate genes
located in this genomic region that may be responsible for this
phenotype are members of the CD28 family of T cell costimulatory
molecules including Inducible Costimulator, ICOS.
ICOS is expressed on the surface of T cells and has been implicated
in several immune-mediated diseases [6–9]. Although ICOS is
expressed at low levels on naı ¨ve T cells, its surface expression greatly
increases upon activation [10,11] and studies using ICOS-deficient
animals have shown that ICOS is critical for Th2 differentiation,
germinal center formation and Th2-mediated antibody class switching
[12–14]. In addition to these ICOS-knockout studies, we and others
have shown that blocking ICOS inhibits Th2-mediated lung
inflammation, and ICOS blockade in vitro resulted in a corresponding
decrease in IL-4 and IL-5 production by Th2 cells [15–17].
Murine Th2 cells have increased ICOS cell-surface expression
compared to Th1 cells  and ICOS surface expression levels
have been associated with distinct T cell cytokines. For example, T
cells expressing ICOS at low levels produced IFN-c, T cells that
expressed ICOS at mid levels produced IL-4 and IL-5, while T
cells expressing ICOS at high levels were shown to produce IL-10
. ICOS signaling has also been shown to influence Th2
differentiation by enhancing IL-4 production and IL-4 receptor
signaling [10,19]. Thus, ICOS has a critical role for Th2
differentiation and cytokine production.
PLoS ONE | www.plosone.org1November 2009 | Volume 4 | Issue 11 | e7525
We have recently shown that a single nucleotide polymorphism
(SNP) in the 59 human ICOS promoter region associates with
increased ICOS surface expression and Th2 cytokine production.
Importantly, the same SNP was associated with increased atopy in
the Hutterite population  suggesting a potential link between
ICOS surface expression and Th2 responses. Furthermore, in
addition to our findings, both rheumatoid arthritis and lupus patients
have higher ICOS cell-surface expression than healthy individuals
[21,22]. However, these models cannot determine if increased ICOS
surface expression is contributing to disease progression or
alternatively, if it is a result of augmented T cell activation.
To definitively demonstrate whether differences in ICOS cell-
surface expression alone can have global effects on a Th2 immune
response, we have used a mouse model in which the experimental
group had its ICOS surface expression fixed at a lower level than
wild-type T cells. We find that ICOS+/2T cells have decreased
ICOS surface expression compared to ICOS+/+T cells although
activation status was equal, and therefore we used ICOS+/+and
ICOS+/2mice to test our hypothesis that genetically decreasing
ICOS surface expression levels could directly result in diminished
Th2 response. Herein we demonstrate that ICOS+/2mice have
decreased Th2 immune responses in vivo resulting in decreased
airway eosinophilia and defective T cell cytokine production. These
results suggest that a genetic predisposition resulting in reduced
ICOS expression on the surface of T cells can directly result in
diminished Th2 responses.
Materials and Methods
ICOS2/2mice were generated as previously described  and
backcrossed 8 generations on the C57BL/6 background. ICOS+/2
micewereintercrossed to create ICOS+/+, ICOS+/2and ICOS2/2
littermates. B7RP-12/2mice were a gift in kind by Andrew
Welcher at Amgen. C.129-Il4tm1Lky/J (4get) mice were purchased
from The Jackson Laboratory (Bar Harbor, ME) and bred to
BALB/c ICOS+/2mice in our facility. The resulting ICOS+/+.4get
and ICOS+/2.4get littermates were used for the experiments in this
manuscript. Animals were housed in a specific pathogen-free facility
maintained by the University of Chicago Animal Resources Center
(Chicago, IL). The studies detailed herein conform to the principles
set forth by the Animal Welfare Act and the National Institutes of
Health guidelines for the care and use of animals in biomedical
In vitro stimulation of enriched T cells and flow
Mice were sacrificed and their lymph nodes were collected and
a single cell suspension was made. The cells were incubated with
an anti-HSA antibody (J11D, American Type Culture Collection
[ATCC], Manassas, VA) for 1 hr at 4uC followed by incubation
with sterile-filtered rabbit complement (Pel-Freez, Rogers, AR) for
30 min. at 37uC. Dead cells were removed via centrifugation
through Ficoll-Histopaque 1059 (Sigma, St. Louis, MO). The live
cells were then stimulated in the presence of plate-bound anti-CD3
and anti-CD28 antibodies for 48 hr in 24 well plates in media
alone, IL-4 or anti-IL-4 (11B11). Cells were suspended in 100 mL
FACS Buffer (PBS containing 0.1% sodium azide and 1% BSA,
and incubated with the anti Fc receptor antibody, 2.4G2 (ATCC),
for five minutes at room temperature. Cells were stained with anti-
CD3, CD4, ICOS, CD25, CD40L, CD28, CD44 or CD62L
antibodies. Flow cytometric analysis was performed on a BD
LSRII or FacsCanto (BD Pharmingen, San Diego, CA) and the
data analyzed with FlowJo software (Tree Star, Ashland, OR).
In vivo model of airway inflammation
Mice were sensitized and challenged with S. mansoni eggs as
previously described . Briefly, mice were injected with S.
mansoni eggs i.p. on day 0, challenged with soluble egg antigen
(SEA) intratracheally (i.t.) on day 7, and the mice were sacrificed
on day 11. Post sacrifice, blood was isolated via cardiac puncture.
Bronchoalveolar lavage (BAL) was performed by delivering
0.8 mL PBS in the airway via a cannulated trachea. The lavage
was repeated three times for a final volume of 2.5–3.0 mL and
cells were counted using a hemacytometer. The percentage of
CCR3+eosinophils, CD4+T cells and CD8+T cells in the BAL
were determined using flow cytometry.
In experiments using 4get mice, the mice were injected with S.
mansoni eggs i.p. on day 0, challenged with soluble egg antigen
(SEA) intratracheally (i.t.) on day 7 and day 14, and the mice were
sacrificed on day 18. The percentage of GFP+cells were
determined by flow cytometry.
Isolation of lung cells
Lungs were digested by agitating the tissue for 1 hr in 20 mL
digestion buffer (collagenase P at 1.0 mg/mL (Boehringer Manheim,
Manheim, Germany), DNaseI at 50 U/mL (Boehringer Manheim),
nytex filter, and RBCs were depleted with ammonium chloride-
potassium lysing buffer. Dead cells were removed via centrifugation
through Ficoll-Histopaque 1059 (Sigma).
Cells from the lungs and draining lymph nodes were analyzed via
ELISPOT or ELISA as described in the results. For ELISPOT, cells
were placed on plates coated with either anti-IL-4 or anti-IL-5
antibodies and stimulated overnight in the presence or absence of
anti-CD3 antibody. After 24 hours the number of antigen specific
spots was analyzed according to the manufacture’s instructions (BD
Pharmingen). For ELISA, the draining mediastinal lymph nodes
were removed, and the cells were stimulated with SEA (5 mg/mL) in
96 well plates at 46105cells/mL for 48 hours. Cytokine levels were
analyzed via ELISAaccording to the manufacturer’s instructions (IL-
5, IFN-c (BD Pharmingen) and IL-13 (R&D Systems Minneapolis,
MN)). In experiments using 4Get mice, cells isolated from both the
lungs and mediastinal lymph nodes were stained with anti-CD4
antibodies. The CD4+GFP2and CD4+GFP+cells were sorted using
a FACSAria (BD Pharmingen, San Diego, CA). The number of each
cell type was equalized and stimulated in the presence of anti-CD3
and anti-CD28 antibodies for 48 hours and IL-4 production was
analyzed via ELISPOT according to the manufacturer’s instructions.
Blood was isolated from mice and sera was isolated by
centrifugation in serum separator tube. IgE levels were analyzed via
ELISA according to manufacturer’s instructions (BD Pharmingen).
All statistical analysis was performed using Graphpad Prism
software and significance was determined using unpaired student’s t
test. In all figures and tables, *=p,0.05, **=p,0.01, ***=p,0.001.
ICOS+/2T cells have less cell surface ICOS expression
than wild-type T cells
To determine if T cells from ICOS heterozygous animals have
decreased ICOS expression on their cell surface, T cells were
ICOS Regulates Th2 Responses
PLoS ONE | www.plosone.org2November 2009 | Volume 4 | Issue 11 | e7525
enriched from the lymph nodes from ICOS+/+and ICOS+/2
animals and stimulated with plate-bound anti-CD3 and anti-
CD28 antibodies. After a 48 hour stimulation, ICOS+/2T cells
have decreased ICOS cell-surface expression compared to their
wild-type littermates (Fig. 1A). Previous reports have shown that
IL-4 may augment ICOS surface expression , however,
stimulating the cells in the presence of exogenous IL-4 did not
significantly alter ICOS surface expression on either ICOS+/+or
ICOS+/2T cells (Fig. 1A). Thus, the difference in ICOS surface
expression in ICOS+/2T cells was not influenced by IL-4 levels.
Another reason for the decreased ICOS expression on the
surface of ICOS+/2could be a result of insufficient activation of
the ICOS+/2T cells. However, ICOS+/2T cells did not have
decreased surface expression of the activation markers CD25 and
CD40L. T cells from ICOS+/+and ICOS+/2mice also express
equal levels of CD28, suggesting that disruption of the ICOS gene
did not negatively influence the expression of genes near the ICOS
locus (Fig. 1B). These data indicate that ICOS+/2T cells have
decreased capacity to express ICOS compared to ICOS+/+T cells,
and this diminished surface expression is not due to a difference in
T cell activation or IL-4 production.
ICOS expression regulates the magnitude of the Th2
As ICOS+/2T cells have decreased ICOS expression on their
surface upon activation and ICOS has an important role in Th2
responses, we hypothesized that ICOS+/2mice would have
decreased Th2-mediated airway inflammation in our model. To
test ourhypothesisweanalyzed theTh2invivoresponseofICOS+/+,
ICOS+/2, and ICOS2/2mice using an established Th2-mediated
airwayinflammationmodel.Inourmodelwe sensitizethe mice
i.p. with inactivated S. mansonii eggs and challenge the mice with
soluble egg antigen (SEA) intratracheally (i.t) resulting in a robust
influx of eosinophils into the lungs. We find that ICOS+/2mice had
a significant decrease in total cells, eosinophils, CD4+T cells and
CD8+T cells in the airways compared to ICOS+/+mice, but
ICOS+/2mice still had significantly more total cells and eosinophils
as compared to ICOS2/2mice(Fig. 2A). While ICOS+/2mice had
a significant reduction of CD4+and CD8+T cells compared to
ICOS+/+mice, the CD4+and CD8+T cell response was not
significantly different than the ICOS2/2mice. In concordance with
our in vitro data, CD4+T cells in the bronchoalveolar lavage (BAL)
fluid of sensitized and challenged ICOS+/2mice had a 50%
reduction in surface ICOS expression compared to CD4+T cells
from ICOS+/+mice (Fig. 2B). Interestingly, we observed a slight but
not significant decrease in PAS-positive cells in the airways of
ICOS+/2mice compared to ICOS+/+mice suggesting that
Figure 1. ICOS+ +/ /2 2T cells have decreased ICOS expression but
equal activation status. ICOS+/+(black lines) and ICOS+/2(grey line) T
cells were isolated and stimulated with anti-CD3 and anti-CD28
antibodies in media alone or in the presence of IL-4 or anti-IL-4
antibodies. After 48 hours the cells were removed and expression of the
indicated surface markers were analyzed via flow cytometry. (A) ICOS
expression on CD4+T cells stimulated under the indicated conditions.
(B) Expression of the indicated cell-surface markers after stimulation for
48 hours in media alone cultures.
Figure 2. ICOS cell-surface expression regulates the magnitude
of a Th2 response in vivo. ICOS+/+(black bars), ICOS+/2(grey bars) and
ICOS2/2(white bars) mice were activated as indicated in Materials and
Methods. (A) Total cells in BAL fluid were counted and percent CCR3+
eosinophils, CD4+T cells and CD8+T cells were analyzed via flow
cytometry and used to calculate total cell numbers. (B) ICOS expression
on BAL CD4+T cells from ICOS+/+(black line), ICOS+/2(grey line), ICOS2/2
(dotted line) is shown. (C) Serum IgE levels were analyzed via ELISA.
ICOS Regulates Th2 Responses
PLoS ONE | www.plosone.org3 November 2009 | Volume 4 | Issue 11 | e7525
decreased ICOS expression induces a defect in the cellular immune
response (data not shown).
ICOS2/2mice have been shown to have a severe defect in
germinal center formation and serum IgE levels . To
determine whether decreased ICOS surface expression also affects
humoral immune responses, serum IgE levels were analyzed in
ICOS+/2mice after sensitization and challenge. We find that
ICOS+/2mice have significantly less serum IgE than ICOS+/+
mice but still produce significantly more IgE than ICOS2/2mice
(Fig. 2C). The difference between ICOS+/+and ICOS+/2mice
was not strain specific as BALB/c ICOS+/2mice also had
decreased in vivo airway inflammation as compared to BALB/c
ICOS+/+mice (data not shown). Together, these data confirm that
the level of ICOS costimulation on T cells correlates directly to the
severity of Th2 mediated airway inflammation and the levels of
serum IgE produced during a Th2 immune response.
Reduced Th2 response in ICOS+/2mice
The finding that ICOS+/2mice have decreased lung eosino-
philia led us to hypothesize that T cells from these mice have
defects in Th2 differentiation and/or effector function. To test this
hypothesis, cells from the lung tissue and mediastinal lymph nodes
were isolated, and the number of cytokine producing cells was
analyzed by ELISPOT after 24 hour stimulation in the presence
or absence of anti-CD3 antibodies. ICOS+/2mice had signifi-
cantly less IL-4 and IL-5 producing cells in both the lungs and
draining lymph nodes than ICOS+/+mice (Fig. 3A, B). To
determine the cytokine production from antigen-specific T cells,
mediastinal lymph nodes cells were stimulated with SEA for
48 hours and their cytokine production was analyzed via ELISA.
ICOS+/2T cells had decreased Th2 cytokine production after
SEA restimulation but equal IFN-c production compared to
ICOS+/+T cells (Fig. 3C). Although we observed decreased IL-5
and IL-13 production by ICOS+/2T cells, there was not a
200.0647.1 ng/mL; ICOS+/2=121.0633.5 ng/mL). These da-
ta suggest a decrease in ICOS expression on ICOS+/2T cells
resulted in a proportional reduction in Th2 differentiation and
The decreased immune response and lung eosinophilia
observed in the ICOS+/2mice may also be caused by decreased
activation of ICOS+/2T cells. To ensure that the differences
observed in Figure 3 were not due to a decreased presence of
activated T cells in the ICOS+/2mice, lymphocytes were isolated
at the peak of the immune response and analyzed via flow
cytometry. ICOS+/2and ICOS+/+mice had similar percentage of
CD4+T cells in both the lungs and mediastinal lymph nodes
(Table 1). Furthermore, among the CD4+T cells in the lungs and
lymph nodes, ICOS+/+and ICOS+/2T cells had an equal
percentage of CD44 high cells and CD25+cells. Interestingly,
among the CD4+CD44 high cells ICOS+/2T cells have
decreased ICOS surface expression compared to ICOS+/+T cells
(Table 1). Together, these data indicate that varying the level of
surface ICOS expression does not affect global T cell activation in
ICOS downregulates regulates B7RP-1 expression on
It is known that ICOS2/2mice have defective Th2 responses,
and it has been published that IL-4 can induce B7 Related Protein-1
(B7RP-1) surface downregulation on B cells and peritoneal
macrophages [25,26]. Thus, we hypothesized that antigen present-
ing cells of ICOS2/2mice would have augmented B7RP-1
expression compared to B6 mice. To test this hypothesis we
performed our S. mansonii-based airway inflammation model in B6,
ICOS+/2and ICOS2/2mice and analyzed the dendritic cell
populations of both the mediastinal lymph nodes and spleen at the
peak of inflammation. ICOS2/2mice had a dramatic increase in
B7RP-1 expression on dendritic cells compared to B6 and ICOS+/2
mice (Fig. 4A). ICOS+/2mice had intermediate B7RP-1 expression
levels between that of ICOS+/+and ICOS2/2mice suggesting that
B7RP-1 expression inversely correlates with ICOS expression.
Furthermore, B cells and CD11chiDC from uninflamed mice also
had increased B7RP-1expression on bothcelltypesinthe ICOS2/2
micecompared to wild-type mice(Fig. 4B).Hence,inboth naı ¨veand
inflamed mice ICOS2/2APCs had greatly increased B7RP-1
expression compared to ICOS+/+APCs. However, the B7RP-1
expression on ICOS+/2APCs was only slightly elevated compared
Since the absence of ICOS on T cells results in increased B7RP-1
expression, we tested whether T cells are necessary to reduce B7RP-
Figure 3. Defective Th2 cytokine production in ICOS+ +/ /2 2mice.
(A) Four days after challenge the lungs were harvested and isolated
cells were stimulated with anti-CD3 for 24 hours. Number of cells
producing IL-4 and IL-5 were quantified via ELISPOT. (B) Mediastinal
lymph nodes were stimulated with anti-CD3 for 24 hours, and the
number of cells producing IL-4 and IL-5 were quantified via ELISPOT or
(C) The cells were stimulated with SEA for 48 hours and cytokine
supernatant levels were analyzed via ELISA.
ICOS Regulates Th2 Responses
PLoS ONE | www.plosone.org 4November 2009 | Volume 4 | Issue 11 | e7525
1 expression by analyzing the B7RP-1 expression in Rag2/2mice.
Rag2/2mice lack T cells and therefore lack ICOS+cells. As shown
in Fig. 4C, dendritic cells from naı ¨ve Rag2/2mice have increased
B7RP-1 expression compared to B6 mice further supporting that
ICOS expression results in decreased B7RP-1 surface expression.
To determinewhether itwas the absence of T cells or the absenceof
ICOS expression on T cells that lead to increased B7RP-1
expression, naı ¨ve B6 or ICOS2/2T cells were transferred into a
naı ¨ve Rag2/2mouse and four weeks later, we analyzed the B7RP-1
expression on their dendritic cells. Rag2/2mice which received
ICOS-positive B6 T cells had decreasedB7RP-1 expression on their
splenic dendritic cells as compared to Rag2/2mice which did not
receive any cells (Fig. 4D). Interestingly, Rag2/2mice that received
ICOS2/2T cells had augmented B7RP-1 expression compared to
Rag2/2mice which did not receive any T cells. Thus the transfer of
ICOS2/2T cells results in an environment that induces B7RP-1
upregulation. Overall, these data indicate that ICOS surface
expression on T cells can regulate B7RP-1 expression on dendritic
cells in an active manner.
Reduced B7RP-1 expression results in diminished Th2
Since ICOS has a single known ligand, B7RP-1, we
hypothesized that reduced B7RP-1 expression would result in
reduced ICOS costimulation; leading to reduced Th2 responses.
We tested this hypothesis by comparing the lung inflammatory
response of B7RP-1+/+, B7RP-1+/2and B7RP-12/2mice. To
first determine whether B7RP-1+/2mice actually have decreased
surface expression on their cells, we analyzed B7RP-1 expression
on splenic B cells and dendritic cells. Our results indicate that both
CD19+B cells and CD11c+cells from B7RP-1+/2spleens have
decreased surface B7RP-1 expression (Fig. 5A) compared to
Since we observe reduced B7RP-1 expression on B cells and
monocytes in B7RP-1+/2
mice, we performed our airway
inflammation model in B7RP-1+/+, B7RP-1+/2and B7RP-12/2
mice. Similar to ICOS+/2mice, B7RP-1+/2mice had decreased
total cells and eosinophils in the BAL than wild-type mice and
significantly increased number of total cells and eosinophils than
B7RP-12/2mice (Fig. 5B). B7RP-1+/2mice also had significantly
less CD4+T cells in the BAL compared to wild-type mice. However,
B7RP-1+/2mice had significantly more CD4+T cells than B7RP-
12/2mice; different than the ICOS+/2mice which had similar
numbers of CD4+T cells compared to ICOS2/2T cells. In
concordance to what we observed in ICOS+/2mice, B7RP-1+/2
Table 1. Activation and ICOS expression on T cells in ICOS+/+
ICOS+ +/ /+ +
ICOS+ +/ /2 2
Total Cells (6106)
The cell percentages are shown as mean 6
(a) percent of CD4+cells within CD3+population.
(b) percent CD44highare within CD3+/CD4+population.
(c) ICOS MFI is ICOS expression on CD3+/CD4+/CD44highcells.
2SD as determined via flow cytometry.
Figure 4. B7RP-1 downregulation on APCs is regulated by ICOS
expression. (A) Cells were isolated from the mediastinal lymph node
and spleen of ICOS+/+(Black line), ICOS+/2(Grey line), and ICOS2/2
(dashed line) mice at the peak of inflammation. The B7RP-1 expression
on CD11chicells is shown. (B) Cells were isolated from the spleens of
naı ¨ve mice (ICOS+/+n=3; ICOS2/2n=4). The B7RP-1 MFI on the
indicated cell types is shown. (C) Left- Cells were isolated from the
spleens of naı ¨ve C57BL/6 (n=3) and Rag2/2(n=3). The B7RP-1 MFI on
CD11chicells is shown. Right. T cells were isolated from naı ¨ve ICOS+/+
and ICOS2/2mice and transferred to Rag2/2mice. Eight wks later the
mice were sacrificed and B7RP-1 expression was analyzed via flow
ICOS Regulates Th2 Responses
PLoS ONE | www.plosone.org 5November 2009 | Volume 4 | Issue 11 | e7525
and B7RP-12/2mice (Fig. 5B). Previous reports and our data
suggest B7RP-1 expression is inversely proportional to ICOS
expression [27,28]. Thus, we tested whether B7RP-1+/2mice have
altered ICOS expression. CD4+T cells were isolated from the
mediastinal lymph nodes of B7RP-1+/+and B7RP-1+/2mice at the
peak of inflammation and thelevel ofICOS expression was analyzed
via flow cytometry. The B7RP-1+/+and B7RP-1+/2T cells had
similar ICOS surface expression levels; indicating that unlike ICOS
expression, reduced B7RP-1 expression does not alter ICOS
expression on T cells (Fig. 5B). These data indicate that similar to
decreased ICOSexpressiononTcells,decreased B7RP-1expression
on APCs can also result in diminished Th2-mediated responses in
Decreased Th2 immune response in ICOS+/2mice is a
result of reduced number of Th2 cells
Our previous experiments determined that ICOS+/2mice have
a decreased Th2 response. However, from these experiments we
cannot determine whether the difference is due to a reduced
number of Th2 cells in the ICOS+/2mice, or if ICOS+/2Th2
cells secrete reduced Th2 cytokines during the effector response.
We used the IL-4 Green Enhanced Transcription (4get) mice to
determine the difference between these options. We performed an
airway inflammation protocol similar to that used in Figure 2 in
mice that were ICOS+/+.4get and ICOS+/2.4get littermates. If
reduced ICOS cell-surface expression results in reduced number
of Th2 cells, a reduced percentage of CD4+GFP+cells should be
observed. On the other hand, if decreased ICOS expression results
in diminished Th2 cytokine production, the ICOS+/2GFP+T
cells will produce less IL-4.
The results in Figure 6A indicate that the ICOS+/2.4get mice
have a significant reduction in the number of eosinophils and
CD4+GFP+cells in the BAL fluid compared to ICOS+/+.4get
mice. These results indicate that ICOS+/2mice have fewer cells
that have transcribed IL-4 in the airways compared to ICOS+/+
mice. Importantly, there are similar numbers of CD4+GFP2cells
in the BAL, indicating that ICOS+/2mice have a specific defect in
the number of eosinophils and Th2 cells in the airways.
In previous results, we demonstrated ICOS+/2mice had
decreased Th2 cytokine production in cells isolated from the
mediastinal lymph nodes and stimulated ex vivo with the SEA
antigen that was used previously to challenge the mice (Fig. 3).
Thus, we also analyzed the presence of CD4+GFP+cells in
ICOS+/+.4get mice and ICOS+/2.4get mice at the peak of the
inflammatory response. Similar to the results observed in the
airways, ICOS+/2mice had a significantly reduced percentage of
CD4+GFP+T cells (Fig. 6B). However, these mice did not have
reduced numbers of CD4+CD44highCD62Llow
(20.75%61.1 vs. 21.22%62.4). Indicating the difference in Th2
cells in ICOS+/2mice is not due to decreased T cell activation.
There was no significant difference in the number of GFP+cells in
the spleens of ICOS+/+and ICOS+/2mice (Fig. 6B) indicating
that the difference observed was localized to the site of the immune
An alternate explanation for the decreased Th2 cytokine
production upon ex vivo restimulation is that ICOS+/2T cells
have decreased number of IL-4 transcripts on a per cell basis. To
determine if ICOS+/2Th2 cells also have decreased IL-4
transcription on a per cell basis, we compared the GFP MFI of
the CD4+GFP+cells between the two groups. ICOS+/+and
ICOS+/2T cells in both the BAL and mediastinal lymph nodes
have similar GFP MFI (data not shown).
Figure 5. Decreased Th2 response in B7RP-1+ +/ /2 2mice. Spleno-
cytes from Wild-type and B7RP-1+/2mice were removed and single-cell
suspensions were made. The cells were stained with B7RP-1 and either
CD19 or CD11c. Black=WT Grey=B7RP-1+/2. (B) B6. B7RP-1+/+(black
bars), B6. B7RP-1+/2(grey bars), and B6. B7RP-12/2(white bars) mice
were sensitized and challenged as indicated in Materials and Methods.
Total cells in BAL fluid were counted and percent CCR3+eosinophils,
CD4+T cells and CD8+T cells were analyzed via flow cytometry and
used to calculate total cell numbers and normalized to the average of
the wild-type value. ICOS expression on BAL CD4+T cells isolated from
the mediastinal lymph nodes of mice is shown. Serum IgE levels were
analyzed via ELISA.
ICOS Regulates Th2 Responses
PLoS ONE | www.plosone.org 6November 2009 | Volume 4 | Issue 11 | e7525
To confirm the notion that equal ICOS+/+GFP+and ICOS+/2
GFP+cells have similar capacity to produce IL-4, we sorted
CD4+GFP+and CD4+GFP2cells from the lungs and mediastinal
lymph nodes of ICOS+/+and ICOS+/24get mice at the peak of
inflammation, equalized their numbers and restimulated them
with anti-CD3 and anti-CD28 to analyze their IL-4 production. In
contrast to Figure 3 where we stimulated all whole lung and
mediastinal lymph nodes, when we normalized the number of
cells, ICOS+/2T cells did not have reduced IL-4 production
(Figure 6C). Therefore, our data suggests that the decreased lung
inflammatory response observed in the ICOS+/2mice is due to
reduced number of Th2 cells and not in the ability of those Th2
cells to produce cytokines.
Multiple studies have shown that ICOS is important for Th2
immune responses and cytokine production [12–14]. The majority
of these studies compare ICOS+/+and ICOS2/2mice, but to date
only a small number of ICOS-deficient patients have been
identified . Our novel study comparing the immune responses
between ICOS+/+and ICOS+/2mice bears broader physiological
relevance to human diseases in which variances in ICOS
expression levels have been found. Tafuri et al. demonstrated
that ICOS+/2mice have decreased serum antibodies , but did
not explore the influence of T cell ICOS surface expression on
tissue inflammation. Our data adds to the study by Tafuri et al.
and demonstrates that the decreased ICOS cell-surface expression
leads to decreased lung eosinophilia, numbers of Th2 cells, and
Th2 cytokine production. To our knowledge, this is the first study
demonstrating that reducing the expression of a single costimu-
latory molecule alone (versus total deletion) can have a significant
effect on a tissue-specific Th2 immune response without
influencing T cell activation. Yagi et al. showed that ICOS
expression varies between numerous murine strains and there is a
positive correlation between ICOS expression and IL-4 produc-
tion across strains . However, there are numerous genetic
differences between the strains that may influence both ICOS
expression and Th2 differentiation. In this report we have
minimized the background genetic differences outside of ICOS
surface expression by using littermate ICOS+/+and ICOS+/2
mice; thus any defects observed are due to differences in this
specific costimulatory pathway and not background genetic
To explore how the level of ICOS-B7RP-1 interactions can
regulate Th2 responses, we used a model in which ICOS cell-
surface expression levels are pre-determined and compared the
responses of ICOS+/+mice and ICOS+/2mice in an established
airway inflammation model. We demonstrate that ICOS+/2mice
had less surface ICOS expression on CD4+T cells than ICOS+/+
T cells. The reduction in ICOS surface expression did not alter
global T cell activation but had a substantial influence on Th2
immune responses in vivo. The ICOS+/2mice had decreased
cellular infiltrates in the airways, decreased airway eosinophilia,
decreased serum IgE, and defective Th2 cytokine production upon
in vitro restimulation of T cells from the lungs and draining lymph
nodes. These results show the reduced ICOS surface expression
observed in the ICOS+/2mice leads to defective Th2 immune
Figure 6. Reduced number of Th2 cells in the airways of ICOS+ +/ /2 2mice. (A) ICOS+/+.4get (black bars), ICOS+/2.4get (grey bars) mice were
activated as indicated in Materials and Methods. Total cells in BAL fluid were counted and percent CCR3+eosinophils, total CD4+T cells, CD4+GFP+
and CD4+GFP2T cells were analyzed via flow cytometry and used to calculate total cell numbers. (B) The percent of CD4+GFP+cells from the
indicated organs are shown. The results are from separate experiments that have been combined (ICOS+/+n=7, ICOS+/2n=9). (C) CD4+GFP2and
CD4+GFP+cells were sorted from the mediastinal lymph nodes and lungs of ICOS+/+.4get and ICOS+/2.4get mice. The cells were equalized and
stimulated with anti-CD3 and anti-CD28 antibodies for 48 hours and the IL-4 production was analyzed via ELISPOT.
ICOS Regulates Th2 Responses
PLoS ONE | www.plosone.org7November 2009 | Volume 4 | Issue 11 | e7525
responses. Since both IL-5 and IL-13 are classical Th2 cytokines
and potent activators of eosinophil differentiation and recruitment,
the decrease in these cytokines likely contributes to the decrease in
airway eosinophilia observed in the ICOS+/2mice. By using this
model we are able to demonstrate that reduction in ICOS
expression on the surface of T cells resulted in reduced Th2
responses in vivo.
ICOS expression also regulates B7RP-1 expression in addition
to Th2 cytokine responses. We observed that B6 dendritic cells
and B cells have decreased B7RP-1 surface expression compared
to ICOS2/2DCs in both untreated and inflamed mice; hence,
providing new insight into the ability of T cells to regulate
dendritic cells. Interestingly, the B7RP-1 expression on ICOS+/2
APCs is slightly augmented but similar to ICOS+/+APCs and is
greatly reduced compared to ICOS2/2APCs. Thus we believe
the decreased Th2 response observed in ICOS+/2mice is not due
to differences in B7RP-1 expression. It is often discussed how naı ¨ve
T cells need to interact with dendritic cells in the periphery to
survive via interactions between the TCR and MHC. However,
my data suggests that the T cell/dendritic cell interaction can also
influence the dendritic cells and the presence or absence of ICOS
can change the status of the resident DCs. Therefore, these data
provide unique insights into the communication between T cells
and dendritic cells.
We utilized the 4get mice that express GFP upon IL-4
transcription to determine at what stage during the immune
response that decreased cell surface ICOS expression by ICOS+/2
T cells lead to decreased Th2 responses. The ICOS+/2mice had
fewer CD4+GFP+cells in both the airways and mediastinal lymph
nodes, but when the number of GFP+CD4+T cells isolated from
ICOS+/+and ICOS+/2mice were equalized, the cells produced
similar amounts of IL-4. These data indicate that if an ICOS+/2T
cell becomes a Th2 cell it can transcribe IL-4 as well as an ICOS+/+
Th2 cell. However, there are fewer Th2 cells in the ICOS+/2mice
compared to ICOS+/+mice indicating that ICOS cell-surface
expression levels regulate Th2 differentiation in vivo. Previous
reports have demonstrated that 4get T cells with increased GFP
expression also have increased IL-4 protein expression as indicated
by intracellular staining . Therefore, it is reasonable to presume
that the ICOS+/+.4get and ICOS+/2.4get T cells that are GFP+
have similar IL-4 producing capabilities. From our data, we
conclude that ICOS+/2mice have a decreased effector response
due to decreased number of Th2 cells but not a defect in the
cytokine production of effector T cells.
The human genome project demonstrated that the majority of
the differences among humans were due to SNPs in the genome,
and less than 1% of the SNPs are predicted to result in a mutation
in protein function . It is likely that SNPs in the regulatory
regions of genes will influence protein expression and this
difference in protein expression will have a dramatic impact on
the individual. In our model there is a 50% reduction in ICOS
surface expression on the T cells of ICOS+/2mice. This difference
results in a 50% reduction in the number of Th2 cells in the BAL
and a significant reduction in the percentage of Th2 cells in the
mediastinal lymph nodes. This difference in Th2 cells may have
greater consequences. For example, previous reports have
indicated that IL-4 can upregulate eotaxin expression [32,33],
which will recruit eosinophils into the lung. Thus, a reduction in
the number of Th2 cells in the lungs leads to a decreased number
of eosinophils that arrive in the lung. These eosinophils can
augment the existing Th2 response by secreting their own IL-4.
Similar to other reports, we observed that the eosinophils in the
BAL in the 4get mice are greater than 95% GFP+(data not shown,
). Remarkably, this large difference in eosinophils and Th2
cytokine production all results from a small difference in ICOS
Interestingly, the defect in the number of Th2 cells in ICOS+/2
was only observed at the site of the immune response as there were
equal numbers of CD4+GFP+cells in the spleens of ICOS+/+and
ICOS+/2mice. We are currently investigating whether, in
addition to their defect in Th2 differentiation, ICOS+/2mice
also have a defect in Th2 migration that may also contribute to the
reduced number of CD4+ GFP+ cells observed in the airways of
ICOS+/2mice. It has previously been demonstrated that IL-4 can
contribute to the induction of the Th2 chemokine, CCL17, in both
human and murine lung epithelial cells [35,36]. Therefore, the
defect in Th2 differentiation in ICOS+/2mice may also result in
decreased chemotaxis of existing Th2 cells to the site of
We recently published that in humans an ICOS promoter region
single nucleotide polymorphism is associated with allergy and
increased serum IgE in a founder population . The SNP also
correlates with increased ICOS surface expression and increased
Th2 cytokine production from peripheral blood mononuclear
cells. The data in this report provides a possible functional
explanation for the findings in Shilling et al. and suggests that a
genetic predisposition to express higher ICOS levels is a marker
for an augmented Th2 inflammatory response, and this augment-
ed response may lead to Th2-mediated diseases such as atopy. A
recent report identified a novel ICOS repressor named Roquin
. The sanroque mice, which lack Roquin resulting in augmented
ICOS cell-surface expression, acquire a lupus like phenotype
characterized by increased dsDNA antibodies, increased serum
IgG and IgE and splenomegaly. Thus, in both mice and humans,
the level of ICOS surface expression regulates the magnitude of
the in vivo Th2 response, perhaps by influencing Th2 differenti-
ation. Furthermore, genetic predisposition to express higher levels
of costimulatory molecules may result in differences in likelihood
of developing Th2-type diseases.
Since there is a correlation between ICOS surface expression
and in vivo responses, ICOS blockade may be a possible
therapeutic target to inhibit diseases initiated by immune
hyperresponsiveness. A critical component of rheumatoid arthritis,
lupus and myasthemia gravis is the production of anti-self
antibodies. Because ICOS costimulation is associated with
enhanced B cell isotype switching, blockade of the ICOS/B7RP-
1 protein may effectively inhibit the production of harmful anti-self
antibodies and reduce disease severity. These data indicate that
partially blocking ICOS-B7RP-1 interactions may be an effective
therapy for this and other chronic diseases. The partial blockade of
this pathway during a hyperactive Th2 immune response may
reduce Th2 cytokine production without completely debilitating
the immune response, allowing the immune system to maintain a
normal immune response against pathogens.
The authors would like to acknowledge Chen Dong and Richard Flavell
for providing the ICOS2/2mice. We also kindly thank Purvi Mody and
Kelly Blaine for editing the manuscript.
Conceived and designed the experiments: BSC RAS HSB TVM JLC AIS.
Performed the experiments: BSC RAS HSB TVM JLC. Analyzed the data:
BSC RAS HSB AIS. Contributed reagents/materials/analysis tools: AAW
JW AIS. Wrote the paper: BSC AAW JW AIS. Contributed significant
editing of the manuscript: AAW. Helped edit the paper: JW.
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