FOXP3, IL-10, and TGF-β genes expression in children with IgE-dependent food allergy.
ABSTRACT Regulatory T cells (Tregs) have an essential role in tolerance and immune regulation. However, few and controversial data have been published to date on the role and number of these cells in food allergic children. The forkhead/winged-helix transcription factor box protein 3 (FOXP3) is considered the most reliable marker for Tregs.
This study aims to investigate the FOXP3, interleukin (IL)-10, and transforming growth factor (TGF-β) genes expression in children with IgE-dependent food allergy.
The study group consisted of 54 children with IgE-dependent food allergy (FA) and a control group of 26 non-atopic healthy children. The diagnosis of FA was established using questionnaires, clinical criteria, skin prick tests, serum sIgE antibodies (UniCAP 100 Pharmacia Upjohn), and a double-blind placebo control food challenge. In order to assess gene expression, the isolation of nucleated cells was performed using Histopaque-1077 (Sigma-Aldrich, Germany). The concentration of RNA obtained was measured using a super-sensitive NanoDrop ND1000 spectrophotometer (Thermo Scientific, USA). A reverse transcription reaction was performed using a commercially available set of High Capacity cDNA Archive Kit (Applied Biosystems, USA). Analysis have been carried out in the genetic analyzer 7900HT Real-Time PCR (Applied Biosystems, USA).
The average level of the FOXP3 gene expression in the studied group was 2.19 ± 1.16 and in the control group 2.88 ± 1.66 (p = 0.03). The average level of IL10 mRNA expression in the study group was 13.6 ± 1.07 and was significantly lower than corresponding values in the control group 14.3 ± 1.1 (p = 0.01). There were no significant differences in the average level of the TGF-β mRNA expression in the study group (3.4 ± 0.4) and controls (3.5 ± 0.3; p > 0.05). The FOXP3 gene expression was the highest in children who acquired tolerance to food (3.54 ± 0.75), lower in heated allergen-tolerant children (2.43 ± 0.81), and the lowest in heated allergen-reactive children (1.18 ± 0.5; p = 0.001 control vs heated allergen reactive; p = 0.005 heated allergen tolerant vs heated allergen reactive; p = 0.001 outgrown vs heated allergen reactive). The significant tendency toward lower total IgE levels with a higher FOXP3 mRNA expression was detected (n = 54; Pearson r = -0.4393; p = 0.001).
Children with FA showed statistically significant lower level of the FOXP3 and IL10 gene expression than healthy children. Children acquiring tolerance to the food show significantly higher levels of the FOXP3 gene expression than children with active FA. The correlation between the level of FOXP3 and total IgE was detected.
- SourceAvailable from: Lucie Mondoulet[Show abstract] [Hide abstract]
ABSTRACT: Allergen-specific immunotherapy favours immune deviation from a Th2 to a Th1 response and increases the number of regulatory T cells (Tregs). Epicutaneous immunotherapy (EPIT) of sensitized mice decreases the clinical and the allergen-specific Th2 responses, and increases local and peripheral Foxp3(+) Tregs. To investigate the role of Tregs in EPIT, and characterize their phenotype and maintenance following EPIT. Tregs were investigated using in vivo depletion or adoptive transfer into BALB/c mice. Tregs were depleted using anti-CD25 antibody injection during EPIT and allergen-specific responses were compared to Sham, EPIT alone, and naïve mice. To demonstrate that Tregs can mediate protection by their own, and to study their maintenance following the end of EPIT, CD25(+) CD4(+) Tregs isolated from mice just after or eight weeks after EPIT were transferred into peanut-sensitized mice. Foxp3-IRES-mRFP mice were transferred with EPIT-induced Tregs to analyze the induction of host Tregs. The anti-CD25 antibody injection to EPIT mice abrogated the induction of Tregs in spleen and the expression of Foxp3 in oesophagus. This resulted in levels of peanut-induced eosinophilic infiltration in esophagus similar to Sham and significantly higher than EPIT. Whereas the transfer of Tregs from Sham-treated mice demonstrated no effect, the transfer of Tregs isolated just after EPIT prevented peanut-induced eosinophil infiltration and eotaxin expression, and induced Foxp3 in oesophagus. The transfer of Tregs isolated 8 weeks after EPIT suppressed allergen-specific responses as efficiently as did Tregs isolated just after EPIT, and increased spleen Foxp3(+) CD25(+) CD4(+) cells similarly. The use of reporter mice demonstrated an increase of host Tregs. These results confirm the Tregs-mediated mechanism of EPIT and demonstrate the persistence of efficient Tregs during a long period of time after treatment cessation. This suggests that EPIT induces long-term tolerance in peanut-sensitized mice. This article is protected by copyright. All rights reserved.Clinical & Experimental Allergy 03/2014; · 4.32 Impact Factor
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ABSTRACT: Food allergy, other adverse immune responses to foods, inflammatory bowel disease, and eosinophilic esophagitis have become increasingly common in the last 30 years. It has been proposed in the “hygiene hypothesis” that dysregulated immune responses to environmental microbial stimuli may modify the balance between tolerance and sensitization in some patients. Of the pattern recognition receptors that respond to microbial signals, toll-like receptors (TLRs) represent the most investigated group. The relationship between allergy and TLR activation is currently at the frontier of immunology research. Although TLR2 is abundant in the mucosal environment, little is known about the complex relationship between bystander TLR2 activation by the commensal microflora and the processing of oral antigens. This review focuses on recent advances in our understanding of the relationship between TLR2 and oral tolerance, with an emphasis on regulatory T cells, eosinophils, B cells, IgA, intestinal regulation, and commensal microbes.Mediators of Inflammation 09/2014; · 2.42 Impact Factor
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ABSTRACT: Regulatory T cells play a critical role in the immune response to vaccination, but there is only a limited understanding of the response of regulatory T cells to aluminum adjuvants and the vaccines that contain them. Available studies in animal models show that although induced T regulatory cells may be induced concomitantly with effector T cells following aluminum-adjuvanted vaccination, they are unable to protect against sensitization, suggesting that under the Th2 immune-stimulating effects of aluminum adjuvants, Treg cells may be functionally compromised. Allergic diseases are characterized by immune dysregulation, with increases in IL-4 and IL-6, both of which exert negative effects on Treg function. For individuals with a genetic predisposition, the beneficial influence of adjuvants on immune responsiveness may be accompanied by immune dysregulation, leading to allergic diseases. This review examines aspects of the regulatory T cell response to aluminum-adjuvanted immunization and possible genetic susceptibility factors related to that response.Vaccine 07/2014; · 3.49 Impact Factor
FOXP3, IL-10, and TGF-β Genes Expression
in Children with IgE-Dependent Food Allergy
Aneta Krogulska & Maciej Borowiec &
Ewa Polakowska & Jarosław Dynowski &
Wojciech Młynarski & Krystyna Wasowska-Królikowska
Received: 27 September 2010 /Accepted: 3 November 2010 /Published online: 24 November 2010
# The Author(s) 2010. This article is published with open access at Springerlink.com
Background Regulatory T cells (Tregs) have an essential
role in tolerance and immune regulation. However, few
and controversial data have been published to date on
the role and number of these cells in food allergic
children. The forkhead/winged-helix transcription factor
box protein 3 (FOXP3) is considered the most reliable
marker for Tregs.
Objective This study aims to investigate the FOXP3,
interleukin (IL)-10, and transforming growth factor (TGF-
β) genes expression in children with IgE-dependent food
Material and Methods The study group consisted of 54
children with IgE-dependent food allergy (FA) and a
control group of 26 non-atopic healthy children. The
diagnosis of FA was established using questionnaires,
clinical criteria, skin prick tests, serum sIgE antibodies
(UniCAP 100 Pharmacia Upjohn), and a double-blind
placebo control food challenge. In order to assess gene
expression, the isolation of nucleated cells was performed
using Histopaque-1077 (Sigma-Aldrich, Germany). The
concentration of RNA obtained was measured using a
super-sensitive NanoDrop ND1000 spectrophotometer
(Thermo Scientific, USA). A reverse transcription reac-
tion was performed using a commercially available set of
High Capacity cDNA Archive Kit (Applied Biosystems,
USA). Analysis have been carried out in the genetic
analyzer 7900HT Real-Time PCR (Applied Biosystems,
Results The average level of the FOXP3 gene expression in
the studied group was 2.19±1.16 and in the control group
2.88±1.66 (p=0.03). The average level of IL10 mRNA
expression in the study group was 13.6±1.07 and was
significantly lower than corresponding values in the control
group 14.3±1.1 (p=0.01). There were no significant
differences in the average level of the TGF-β mRNA
expression in the study group (3.4±0.4) and controls (3.5±
0.3; p>0.05). The FOXP3 gene expression was the highest
in children who acquired tolerance to food (3.54±0.75),
lower in heated allergen-tolerant children (2.43±0.81), and
the lowest in heated allergen-reactive children (1.18±0.5;
p=0.001 control vs heated allergen reactive; p=0.005
heated allergen tolerant vs heated allergen reactive; p=
0.001 outgrown vs heated allergen reactive). The significant
tendency toward lower total IgE levels with a higher
FOXP3 mRNA expression was detected (n=54; Pearson
Conclusions Children with FA showed statistically signif-
icant lower level of the FOXP3 and IL10 gene expression
than healthy children. Children acquiring tolerance to the
food show significantly higher levels of the FOXP3 gene
expression than children with active FA. The correlation
between the level of FOXP3 and total IgE was detected.
Keywords FOXP3.Treg cells.IL-10.TG-β.children.
Source of support: This study is supported by the grant no. 502-11-
750 from the Medical University of Lodz.
A. Krogulska (*):J. Dynowski:K. Wasowska-Królikowska
Department of Pediatric Allergology,
Gastroenterology and Nutrition, Medical University of Lodz,
91-738, Lodz, Poland
M. Borowiec:E. Polakowska:W. Młynarski
Laboratory of Immunopathology and Genetics,
Department of Pediatrics, Oncology, Hematology and Diabetes,
Medical University of Lodz,
J Clin Immunol (2011) 31:205–215
Cow milk allergy
Regulatory T cells
Forkhead/winged-helix transcription factor
box protein 3
Transforming growth factor
Double-blind placebo control food challenge
European Academy of Allergy and Clinical
Peripheral blood mononuclear cell
Polymerase chain reaction
A failure in the establishment and maintenance of oral
tolerance in infancy may result in food allergy (FA). The
recent reports have supported the hypothesized role for
regulatory T cells (Tregs) in the development of normal
tolerance or the spontaneous resolution of milk allergy [1–
3]. Indirect evidence exists to suggest that the development
of FA may be controlled by CD4+CD25+ Treg cells .
Generally, several Tregs have been found to be important
for oral tolerance: Th3 cells, a population of CD4+ cells
that secrete transforming growth factor (TGF-β); T regula-
tory type 1 (Tr1) cells, cells that secrete IL-10; CD4+CD25
+ regulatory T cells, cells that express the transcription
factor forkhead/winged-helix transcription factor box pro-
tein 3 (FOXP3); CD8+ suppressor Tcells; and gamma-delta
T cells. The best characterized Tregs are CD4+ T cells that
constitutively express high levels of surface CD25 (the IL-2
receptor chain: CD25hi T cells). Unlike CD4+ Th cells,
these CD25hi cells do not proliferate or produce cytokines
when stimulated with antigen but actively suppress prolif-
eration and cytokine production from effector T cells
involved in mediating inflammation [5, 6]. Current evi-
dence suggests that CD4+CD25+ Treg cells employ several
mechanisms to suppress immune responses: via direct cell
contact, indirectly by reducing the antigen-presenting
capacity of antigen-presenting cells (APCs)  or by
suppressive cytokines [8, 9]. Candidate molecules respon-
sible for the immunosuppressive function of Tregs include
inhibitory cytokines IL-10 and TGF-β. TGF-β has been
demonstrated to play a key role in the regulation of the
immune response, mainly by inhibiting proliferation of
lymphocytes T, lymphocytes B, NK cells, and expression
of major histocompatibility complex (MHC) class II
molecules on APCs. In turn, IL-10 inhibits pro-
inflammatory cytokine production by effector lympho-
cytes, monocytes, and macrophages and also reduces the
ability of APCs to express MHC class II molecules.
Several studies indicate the role of TGF-β and IL-10 in
the pathogenesis of allergic diseases [1, 3, 10–13];
however, recent data are conflicting .
The CD4+CD25+ Tregs owe their suppressive pheno-
type in a large part to the expression of high levels of
FOXP3 [7, 15, 16]. The FOXP3 is considered the most
reliable marker for Tregs performing a major role in the
formation and functioning of lymphocytes T CD4+CD25+
[16–19]. The level of FOXP3 expression has been shown to
correlate with suppressive activity [16, 19]. The expression
of FOXP3 primarily concerns nTreg, but it is also found to
a lesser extent and temporarily in effector T cells. Tr1 cells
show no FOXP3 expression. Furthermore, FOXP3 over-
expression increases the number of Tregs .
Consequences of deficiency or dysfunction of Treg cells
CD4+CD25+ in the development of allergic diseases are
the subject of many studies [21–25]; however, studies
assessing the importance of Tregs in children with allergies,
especially with FA, are rare [26–29]. Previous studies have
tried to find the answers to the main questions: whether
there is any relationship between Tregs during prenatal life
and the risk of FA, as well as FA occurrence and FA
outgrowing, and whether the effects of Tregs depend on
their number or function [26–28, 30–33].
Based on these considerations, the aim of the present
study was to determine the (1) gene FOXP3 expression
(representing Tregs) in children with IgE-dependent cow’s
milk or egg’s allergy in comparison to healthy non-atopic
children, (2) IL-10 and TGF-β mRNA expression in the
same groups, (3) relationship between these immunologic
indicators and the course of food allergy, and (4) probable
correlations between them. This is the first study which
evaluates the FOXP3, IL-10, and TGF-β gene expression in
children with IgE-dependent FA in Polish children.
Materials and Methods
Subjects with IgE-dependent FA were recruited from the
Department of Pediatric Allergology, Gastroenterology and
Nutrition of the Medical University of Lodz and the
Outpatient Allergology Clinics. The study was approved
by the institutional ethics committee, and informed consent
was obtained before enrollment.
Between January 2004 and December 2008, we enrolled
679 children with a suspicion of cow milk or egg allergy. A
lack of data or cooperation resulted in exclusion of 61
cases. Skin prick tests and/or specific IgE revealed
206J Clin Immunol (2011) 31:205–215
sensitization to food allergens in 193 children. In 143
children, elimination diet caused improvement. Double-
blind placebo control food challenge (DBPCFCs) were
planned for 135 children. The DBPCFC was not carried out
in five of children because of a well-documented history of
reaction and reported anaphylactic reactions. Seven chil-
dren were lost, so 143 food challenges were carried out in
131 children. The food allergy was confirmed in 59
children. All together, food allergy was diagnosed in 64
children. A lack of data or cooperation resulted in exclusion
of ten cases. Therefore, a group of 54 children with IgE-
dependent cow milk or egg allergy was subjected to further
investigations. In the study group, ten children had
concomitant atopic dermatitis (AD); six AD and allergic
rhinitis (AR); nine AD and asthma; eight AD, asthma, and
AR; one asthma; 11 AR; and nine digestive tract complaints
—seven due to immediate type of food allergy, i.e., nausea,
vomiting, diarrhea, abdominal pain, and two due to
eosinophilic esophagitis. The control group comprised 26
non-atopic children without FA. Characteristics of the study
and control groups were shown in Table I.
The diagnosis of FA was established using questionnaires,
clinical criteria, skin prick tests to specific food allergens,
serum sIgE antibodies directed against the food allergens,
and the DBPCFC. Exclusion of FA was confirmed by
questionnaires, skin prick tests to specific food allergens,
and serum sIgE antibodies directed against the food
allergens and food challenge. The allergens responsible
for positive results in the food challenge were milk in 27
children and egg in 24 children. In three children, one
allergic to milk and two to eggs, the DBPCFC was not
carried out because of a well-documented history of
reaction and reported anaphylactic reactions. Blood samples
for evaluation of FOXP3, IL-10, and TGF-β genes
expression were collected from patients who had been in
a stable condition for ≥6 weeks, out of season in the case of
concomitant seasonal allergy, and at least 2 weeks after
entering elimination diet.
Skin Prick Test
Standardization of the method was in line with the
position papers of the European Academy of Allergy and
Clinical Immunology (EAACI) . Standard allergen
extracts were provided by Allergopharma (Reinbek,
Germany). Positive control was 10 mg/ml histamine
(Allergopharma). As a negative control, 50% glycerosa-
line solution was used. Reactions to each allergen were
measured 15 min after the pricks. The food allergens
included milk and egg; cereals: wheat flour and rye flour;
fish, soy, cocoa, sesame, peanut, and hazelnut; fruits:
apple, peach, and orange; and vegetables: celery, carrot,
potato, and tomato. Skin prick test positivity was defined
as a wheal of at least 3 mm being larger than the negative
control. Data were excluded if the saline control was
≥3 mm, the histamine control was <3 mm, or if the
difference of histamine minus saline was <3 mm.
Serum Specific IgE Antibodies
Specific IgE for milk, egg, wheat flour and rye flour, fish,
shrimp, soy, cocoa, sesame, peanut, and hazelnut; fruit:
apple, peach, and orange; and vegetables: celery, carrot,
potato, and tomato were measured using the UniCAP 100
Pharmacia Upjohn (Pharmacia Diagnostics AB, Uppsala,
The oral challenge tests were performed using the DBPCFC
method. The trials were carried out in hospitalized patients
according to EAACI recommendations, after a minimum 2-
week eliminating diet . Systemic corticosteroids were
contraindicated, and systemic antihistamines were with-
drawn according to their half-life. The use of topical
corticosteroids for the airways was no reason to discontinue
testing; topical corticosteroids for skin complaints were
tapered to the minimum dose and kept constant throughout
the challenge procedure. The β2-adrenergic agonists and
theophylline were avoided for 48 h prior to the challenge.
Table I Patients’ characteristics
Study groupControl group
Age (years), mean ± SD
Male sex, n (%)
Family history of atopy, n (%)
Total IgE (IU/ml)
Mean ± SD
Mean ± SD
Cow milk allergy subjects, n (%)
Egg allergy subjects, n (%)
J Clin Immunol (2011) 31:205–215 207
In the provocative procedures, the native foods were used:
milk and egg. The patients were observed for at least 4 h
after the completion of the final challenge. The food
challenge results were scored as negative, mild to moderate,
or severe using a clinical reference table adapted from
Sampson and Benhamou et al. [36, 37]. The oral challenge
tests were performed in two time points: at the onset and
the end of the study. At the end of the study, all children
underwent a standardized food challenge on two sequenced
days: the first day with extensively heated products and the
next day with the unheated products. The patients were
characterized by a heated allergen product such as heated
allergen reactive (n=22) or heated allergen tolerant (n=32)
and by an unheated allergen product such as unheated
allergen reactive (heated allergen tolerant; n=19) or as
allergen tolerant (i.e., outgrown; n=13).
Isolation of nucleated cells from peripheral blood
collected for the patient Vacutiner vacuum system was
performed with the addition of an anti-coagulant EDTA.
The isolation was carried out in a strictly conforming to
study density gradient according to the standard protocol
provided with the isolation system. The isolation was
carried out using Histopaque®-1077 (Sigma-Aldrich,
Germany) with a gradient of density 1.077. RNA was
isolated from these nucleated cells in peripheral blood
of patients using the modified method of Chomczynski.
TRI reagent (Invitrogen, Germany) was used for proper
isolation, and the process was conducted according to
the standard protocol. The concentration of RNA
obtained was measured using a super-sensitive Nano-
Drop ND1000 spectrophotometer (Thermo Scientific,
Reverse transcription reaction was performed using a
commercially available set of High Capacity cDNA
Archive Kit (Applied Biosystems, USA). cDNA was
prepared from 1 μg of mRNA, with random hexamer
primers, according to the manufacturer’s instructions—
10 min 25°C, 2 h 37°C, and 4°C thereafter on a PCR
thermocycler Gene (Applied Biosystems, USA). The
resulting cDNA was diluted to a final concentration of
5 ng/μl and constituted a matrix in further experiments.
The analysis of FOXP3 gene expression was constructed
using a human commercial available assay Hs01085830_m1
(Applied Biosystems, USA), IL-10 assay Hs99999035_m1,
Hs00610319_m1 TGF-β assay, and human beta-actin
(Applied Biosystems, USA), which were controlled by
an internal reaction while allowing a reliable determi-
nation of absolute values and the expression of FOXP3
genes, IL-10, and TGF-β. The analysis was carried out
in the genetic analyzer 7900HT Real-Time PCR
(Applied Biosystems, USA). Comparative analyses of each
of these genes in individual patients were performed using
specialized computer programs SDS2.3 and RQ 2.1 (Applied
calculatedbyusingthe2?ΔΔCT(comparative threshold cycle,
or CT) method, as detailed by the manufacturer (Technical
Bulletin 2; Applied Biosystems).
For calculation of absolute values, data were logarithmically
transformed to assure normality of distribution. The mean
values of measurable features (arithmetic means), variable
ranges (min-max), and their internal differentiation with
standard deviations were calculated. In order to determine
the significance of differences between mean values of the
parameters in different groups of children, the analysis of
the variance test of F-Snedecor was used with post hoc
analysis performed with honestly significant difference
Tukey’s test. The Pearson’s r rank correlation coefficients
were used to evaluate relationships between continuous
variables and the Student’s test to verify the significance of
the correlation coefficient. The value of p≤0.05 was
considered statistically significant. The program STATIS-
TICA 8.0 (StatSoft, Tulsa, OK, USA) was used for
Among the study group, there were 28 cow’s milk
allergy children and 26 egg’s allergy children. Clinical
signs observed after food challenges in the study group
are shown in Table II. The average level of FOXP3 gene
expression in the studied group was 2.18±1.16 and in the
control group 2.88±1.66 (p=0.03). In turn, the average
level of IL-10 mRNA expression in the study group was
13.6±1.07 and was significantly lower than the
corresponding values in the control group 14.3±1.1 (p=
0.01). There were no significant differences in the
average level of the TGF-β mRNA expression in the
study group (3.4±0.4) and controls (3.5±0.3; p>0.05;
In 19 children with a mild anaphylactic reaction after
oral food challenge, the average level of FOXP3 gene
expression was 2.26±1.16; in 25 children with a moderate
208J Clin Immunol (2011) 31:205–215
response, it was 2.4±1.2; and in ten children with a severe
reaction, it was 1.5±0.8 (p=0.05; Table III).
In order to assess FOXP3 gene expression, depend-
ing on the clinical course of allergy and the develop-
ment of tolerance, three groups of children were
selected: (1) children who did not tolerate the food
allergen in any form, i.e., heated allergen reactive (n=
22); (2) children with heated allergen tolerance, i.e.,
children with a milder course of food allergy (n=19); and
(3) children who acquired tolerance to food (n=13). A
statistically significant difference in the FOXP3 gene
expression among heated allergen-reactive children (n=
22; 1.18±0.5) and the other food allergic children (n=32;
3.08±0.87) was observed (p=0.001). The FOXP3 gene
expression was the highest in children who acquired
tolerance to food (3.54±0.75), lower in heated tolerant
children (2.4±0.8), and the lowest in heated reactive
children (1.18±0.5) (p=0.001 control vs heated allergen
reactive; p=0.005 heated allergen tolerant vs heated
allergen reactive; p=0.001 outgrown vs heated allergen
reactive; Fig. 2).
There was no statistically significant correlation between
FOXP3, IL-10, or TGF-β expression and age (r=−0.0605;
r=0.2478; r=−0.1651; p>0.05), although there was a
significant negative correlation between the average level
of expression of IL-10 and age in children up to 3 years of
life, which was not found in older children (r=−0.3876; p=
0.04). The significant tendency toward lower total IgE
(tIgE) levels with higher FOXP3 mRNA expression was
detected (r=−0.4393; p=0.001; Fig. 3a). There was no
correlation between IL-10 or TGF-β expression and tIgE
levels (r=0.0185; r=0.0193; p>0.05). Likewise, there was
no statistically significant correlation between FOXP3, IL-
10, or TGF-β expression and sIgE level (r=−0.0285; r=
0.0449; r=0.0873; p>0.05).
A statistically significant correlation between FOXP3
expression and tIgE levels depending on reaction grade
during food challenge was also demonstrated (moderate
reaction r=−0.4178; p=0.03; severe reaction r=−0.6602;
p=0.03; Fig. 3b).
On the other hand, insignificant differences between the
average level of IL-10 and TGF-β expression, depending
on the type of response during food challenge (Table III),
were beyond stating a negative relationship between the
average level of the expression of TGF-β and the age in
children with mild reactions after the oral challenge test
with food (r=−0.4623; p=0.04).
There were no significant differences between the
average level of expression of IL-10 and TGF-β
depending on the course of FA in children (Fig. 2).
However, the correlation between the average level of
IL10 expression and age in children with mild food
allergy (heated allergen tolerant) was detected (r=0.4994;
There was no statistically significant correlation
between FOXP3 expression and IL-10 mRNA expression
(r=−0.0315; p>0.05). The same lack of correlation
between FOXP3 mRNA expression and TGF-β expres-
sion was detected (r=−0.1527; p>0.05), as well as
between TGF-β expression and IL-10 expression (r=
Ten children in the study group used inhaled cortico-
steroids (ICS). Of these, six children had a severe
reaction in the oral challenge. The average level of
FOXP3 in ten children taking ICS was 1.7, while in
other children was 2.8.
Fig. 1 FOXP3, IL10, and TGF-β expression in control and study group
Table II The course of food challenges in the study group
One organ system involved (n=37)
≥2 organ system involved (n=17) Total,
Skin, n Gastrointestinal, n Respiratory, nSkin +
Skin + gastrointestinal +
Skin + respiratory, n
Total, n (%)
J Clin Immunol (2011) 31:205–215 209
Our study shows that the average levels of FOXP3 and IL-
10 gene expression were significantly lower in children
with IgE-dependent FA than in the control group. There
were no differences of TGF-β expression between groups.
These results may suggest diminished number or defect of
Tregs and impaired function of IL-10 in FA children.
It was confirmed that the amount of FOXP3 mRNA
expression correlated directly with the percentage of Treg
cells in peripheral blood mononuclear cells (PBMCs)
determined by flow cytometry . More than 95% of
normal human peripheral blood CD3+ T cells that express
FOXP3 are found in the CD4+ population. Thus, we
utilized FOXP3 mRNA as a surrogate marker for relative
Most findings of Tregs activity in allergic disease come
from studies of immunotherapy  rather than spontaneous
tolerance, and the most convincing data suggest a role for
the IL-10 secreting, so-called Tr1 . Based on the mouse
model of FA, it has been shown that TGF-β and FOXP3
may be the aims of further studies on immunotherapy in FA
. Mori et al. demonstrate that CD4+CD25+Foxp3+ T
cells have no significance in oral desensitization in children
with FA but play an important role in achieving natural
tolerance in children with CMA [ 41]. Our study seems to
partly confirm this finding.
In general, the knowledge on the role and suppressive
effect of CD4+CD25+ Tregs in atopic subjects is still
limited and somewhat contradictory. Several studies
suggest that the number or function of Tregs is impaired
or altered in patients with allergies compared with
healthy individuals [4, 6, 20–22, 24–26, 28, 30, 42–47,
49, 50]. Our results are consistent with these independent
lines of evidence. However, there are many other results,
which have shown the opposite [21, 33, 46, 47]. It seems
that the diversity of results in terms of the number or
function of Tregs in the pathogenesis of allergic diseases
may be related to difficulties in proper identification of
Tregs using conventional cell markers, as well as to
different allergic diseases, disease status, patients’ age,
differential severity, differential environmental influences,
or differential methods.
Data concerning the role of Treg in the pathogenesis of
pediatric allergic disease are rare. The study of Hinz et al.
indicates that reduced maternal Treg numbers during
pregnancy might influence the allergy risk of the child
. Haddeland et al. found that neonates at high atopic
risk generated reduced numbers of CD4+CD25+ cells with
diminished capacity .
The role of CD4+CD25+ Tregs in FA is not well
established. The reported frequency of Tregs in peripheral
blood from patients with FA is inconsistent. Summary of
the literature is demonstrated in Table IV [26–30, 42]. Rare
studies evaluate the role of Tregs in FA in parallel with the
expression of FOXP3 mRNA (the method used in our
study) [27, 29, 30, 42]. Differences in comparison with our
study may stem from different samples, different patient
age, and different methods.
The relationship between Tregs still in uterus and risk of
FA at the first year of life was the aim of the study of Smith
et al. . This study demonstrated diminished function of
CD4+CD25+ Tregs in newborns at risk of egg allergy
development. However, it was suggested that although
FOXP3 is a useful marker of neonatal Tregs, it does not
predict function of these cells. In this light, we do not know
Fig. 2 FOXP3, IL10, and TGF-β expression in control and study
group: 1 heated allergen reactive, 2 heated allergen tolerant, 3
outgrown, and 4 control
Table III FOXP3, IL-10, and TGF-β gene expression in the relationship to the grade of reaction in the course of food challenge
Reaction grade in the course of food challenge FOXP3 mean ± SDIL-10 mean ± SD TGF-β mean ± SD
210 J Clin Immunol (2011) 31:205–215
whether the decreased FOXP3 expression in our study can
be explained by the function defect or drop in the frequency
Furthermore, increased levels of CD4+CD25+ Tregs
have been reported to be associated with acquired tolerance
to cow’s milk . Karlsson et al. found that PBMCs from
children who outgrown CMA had higher percentages of
total CD4+CD25+ and were less proliferative to milk
allergen than their counterparts who still had allergy. A
recent study by Shreffler et al. reported a difference in
antigen-specific but not polyclonal Tregs in children with
CMA . Shreffler et al. showed that children with HM
tolerance more quickly outgrow of allergies than children
with allergies to HM and have simultaneously a higher
Fig. 3 a. FOXP3 expression in correlation with tIgE in study group. b. FOXP3 expression in correlation with tIgE and reaction grade during food
J Clin Immunol (2011) 31:205–215 211
level of allergen-specific Treg cells and better prognosis
. The data of Shreffler et al. suggest that there may be
no functional defect of the CD25+ Treg cell subset in
individuals with allergy but that a higher frequency of these
cells is associated with tolerance. It has been also presented
that children with milk allergy who are strictly avoiding the
antigen have a low frequency of Tregs . We demon-
strated that the FOXP3 gene expression was the highest in
Table IV Summary of literatures concerning the role of Treg in food allergy
et al. 
Egg allergy CD4(+)CD25(+)
≡ in CBa
although a ↓
proportion in FA
of perinatal differences
in Treg cellb
function in neonates
who progress to
Children who outgrew
their allergy had
T cellscand decreased
in vitro proliferative
responses to bovine
T regulatory cell-related
genes reflect the
clinical activity of
is not characterized
by activation of
A higher frequency
of milk allergen-
specific Treg cells
correlates with a
phenotype of mild
clinical disease and
regulatory T cellscmay
play a role in the
resolution of milk
expression might be
a marker of allergic risk
et al. 
↓ in PBa
↑ in PBa
↓ in persistent
et al. 
16 active CMA
≡ in PBa
↑ highest in
et al. 
↑ in PBa
↓ in PBain
≡ in PBa
8 HM reactive
18 HM tolerant
et al. 
CMA n.a.n.a. n.a.
et al. 
n.a.n.a. FA n.a.n.a.
increased in the
duodenum of patients
with untreated FA,
but these cells are not
able to suppress the
by the low expression
of Foxp3 transcripts
↓ decreased frequency, ↑ increased frequency, ≡ equal frequency, n.a. data not available, PB peripheral blood, CB cord blood, FOXP3 forkhead
box P3, Treg T regulatory cells, FA food allergy, CMA cow milk allergy
aExpressed as a percentage relative to total CD4+ T cells
212 J Clin Immunol (2011) 31:205–215
children who acquired tolerance to foods, lower in heated
allergen tolerant children, and the lowest among heated
allergen reactive children. Our results, similarly to the
studies of Karlsson et al., Mori et al., and at least partly to
Sheffler et al. [26, 28, 41], indicate the significance of
FOXP3 gene expression in tolerance induction. This
indirectly suggests, in compliance with the others, that
heated milk or heated egg-tolerant children would be more
likely to outgrow FA [29, 48]. It suggests also that mucosal
induction of tolerance against dietary antigens is associated
with the development of CD4+CD25+ Treg cells. In
contrast, Savilahti et al. question the concept that Tregs
activation in peripheral blood indicates induction of
tolerance in FA . Moreover, Sicherer et al. did not
report any evidence of increased regulatory Tcell responses
in food allergic and non-allergic groups of children, as
measured by the levels of FOXP3 and IL-10 expression
. Our results are contrary to the above-mentioned
findings but, in line with the study of Westerholm-Ormio
et al. who demonstrated the decreased ratio of FOXP3
mRNA to FOXP3 cells in patients with FA. The author
reported that FOXP3 cells are increased in the duodenum of
patients with untreated FA; however, these cells are not able
to suppress the harmful immune response, indicated by the
low expression of FOXP3 transcripts .
To our knowledge, the relationship between the number
of Tregs and FA severity has not been emphasized so far.
Our study demonstrated no relationship between FOXP3,
IL-10, and TGF-β expression and the severity of clinical
response after oral food challenge. Opposite to a previous
studies, although concerning asthma or atopic dermatitis
[24, 49], we showed that the lowest, however non-
significant, level of FOXP3 expression affected children
with severe reaction after food challenge, which may
indicate primary or predominant defect of nTregs as
compared with the possible compensatory increase in T
effector cells in these children. Another explanation of this
finding may be associated with a greater defect of Tregs in
severe type of disease, or possible recruitment of Tregs into
the gastrointestinal tract by the presence of antigen.
Several studies indicate the role of TGF-β and IL-10 in
oral tolerance [1, 3, 12]. Tiemessen et al. suggested that
activated CD4+CD25high T cells might contribute to the
tolerogenic immune response toward an antigen, through
the production of IL-10 . Additionally, it was detected
that proportions of T cells of food-sensitized children
spontaneously secreting IL-10 were lower than in non-
atopic children . It was found that impaired production
of IL-10 in the patches of Peyer et al. favored the
development of food allergy . The potential role of
IL-10 in the resolution of milk (and other) allergy was also
indirectly supported by the observation made by many
investigators that an elevation of IgG4 (and ratio of IgG4/
IgE) is associated with tolerance and that in vitro IL-10 can
modulate TH2 cytokine-induced B cell class switching in
favor of IgG4 over IgE [50, 51]. We have demonstrated
significantly lower IL-10 gene expression in children with
IgE-dependent FA than in the control group. It is consistent
with other studies [10, 13]. The lack of differences
between IL-10 and TGF-β expression in our study group
and the course of food allergy related to tolerance
achievement may suggest that the FOXP3 expression is
more significant in tolerance induction as compared to IL-
10 and TGF-β expression. It is difficult to say whether
such a result is due to a Treg defect in cytokine secretion
or to the methods used in our study. Unfortunately, we
have not evaluated the function of definite cells but the
total genes expression of these cytokines, and additionally,
we determined it at one point of time, in stable conditions,
theoretically without any allergen influence. Perhaps the
analysis of gene expression carried out at different points
of time might indicate some differences.
Our study does not reveal differences in TGF-β expres-
sion. Beyer et al. showed no expression of TGF-β and IL-10
in specific cells of duodenal mucosa in children with
gastroenteropathy in the course of CMA . According
to recent studies, the role of TGF-β remains controversial
. Nevertheless, previous studies demonstrated decreased
potential for TGF-β production in food allergic children [3,
Opposite to Lee et al.  and in line with Jartti et al.
 and Hinz et al. , we show significant inverse
correlation between the FOXP3 and tIgE level. In addition,
Matsumoto et al., using a mouse model, revealed that the
frequency of Foxp3+CD4+CD25+ T cells was inversely
correlated with BHR and allergen-specific IgE levels in the
In our study, all children were on the elimination diet, in
a stable condition, and out of season in the case of seasonal
allergy, so fundamentally children were not exposed to
allergens. Therefore, we suggest that our finding results
from a possible inherent defect in suppression.
Our study has some limitations that deserve a comment.
First, we studied the FOXP3 expression within the PBMC
cell population and only in peripheral blood samples of
food allergic patients and non-atopic controls. Certainly an
evaluation of FOXP3 expression will be more appropriate
within the CD4+CD25high T cell population, but since
recent studies indicate the presence of FOXP3 in the
different cells, it seems that the evaluation only in CD4
+CD25+, in turn, does not encompass all the cells
expressing FOXP3. This is the FOXP3 gene that is the
most responsible for regulatory functions that are charac-
teristic not only for CD4+CD25+ T but also for other cells.
Secondly, however, as Tregs can differ between the
peripheral blood and the site of inflammation, it would be
J Clin Immunol (2011) 31:205–215 213
interesting to study FOXP3 expression in intestinal biopsies
or target organs in food allergic patients. Finally, our study
population was rather small and heterogeneous. It is
therefore possible that due to low statistical power, some
effects could not be reliably detected or excluded. Thus,
although some effects concerning FOXP3 expression have
been confirmed by the authors, the study cannot provide
definite proof on the differences of the control and study
groups in regard of FOXP3 and IL10 expression, but it does
elucidate the involvement of both factors in the process of
food allergy. Another shortcoming of the study is post hoc
analysis and to draw definitive conclusions would carry out
Possibility of new therapeutic approaches based on the
modulation of Tregs is certainly a hope for the future in the
treatment of food allergy. Thus, our findings warrant
longitudinal studies to see how the studied values change
as patients may outgrow their allergy.
1. Children with food allergy showed statistically signif-
icant lower level of the FOXP3 and IL-10 gene
expression than healthy children.
2. Children acquiring tolerance to the food show signif-
icantly higher levels of the FOXP3 gene expression
than children with active food allergy.
3. The correlation between the level of FOXP3 and total
IgE was detected.
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