Research program, Anna Rudin, 611014-4042
Discovery of tolerogenic T cells in infants and factors that induce
these cells to prevent inflammatory disease
Summary:
There is an increasing prevalence of inflammatory disease such as allergy, celiac disease,
inflammatory bowel disease and autoimmune diabetes type 1 in developed countries. The reason
for this is poorly functioning immunologic tolerance mechanisms mainly because of defective
down regulation on T cell activation in young children. CTLA-4 is an essential molecule in
maintaining immunologic tolerance. CTLA-4 is expressed by both FOXP3+ regulatory T cells
and by activated FOXP3- T cells. Recent data show that CTLA-4+ FOXP3- activated T cells and
not only FOXP3+ regulatory T cells play a crucial role in inhibiting tissue destruction and
autoimmune disease in animal models. We want to examine whether CTLA-4+ T cells regardless
of type are important to induce immunologic tolerance in children and how the frequency of these
T cells can be increased. In our prospective birth cohorts we therefore analyze the frequency of
CTLA-4 and FOXP3 expressing T cells, respectively, at seven time points from birth to 5-6
years of age, together with T cell activation and tissue migration markers. Through collaboration
with Agnes Wold and Ingegerd Adlerberth we also analyze the relation between these
immunologic markers and early bacterial gut colonization and development of allergic disease,
respectively. Preliminary results show that CTLA-4 expressing cells seem to play a more
important role than FOXP3 expressing cells in inhibiting activation of T cells at 18 and 36
months of age. High level of CTLA-4 expressing cells seems to be associated with early gut
colonization with E. coli and negatively associated with allergic disease. The results from these
studies may lead to discovery of T cell types that maintain immunologic tolerance and thus
prevent the development of inflammatory disease in children. Moreover, we may find methods to
increase the frequency of these cells in infants.
Specific aims:
A. CTLA-4 and FOXP3 expressing T cells in human infants and their effects on
activation of T cells later in childhood. Using detailed data on cellular development in
prospective birth cohorts that are being followed up to 5-6 years of age we will explore
which tolerogenic cellular mechanisms are most important in suppressing T cell
activation.
B. CTLA-4 and FOXP3 expressing T cells in infancy and effect on later development of
allergic disease. Preliminary data indicate that higher numbers of circulating CTLA-4
expressing T cells in infancy are negatively associated with development of allergic
disease. We are now comparing proportions of CTLA-4 and FOXP3 expressing cells in
infancy with development of allergic disease in larger birth cohorts.
C. Effect of specific bacteria colonizing the gut in early infancy on the expression of
CTLA-4 and FOXP3 in T cells during childhood. We will analyze whether any specific
bacterial strain promotes expression of CTLA-4 and FOXP3 in T cells. We will also
examine if an induced expression of these molecules leads to suppression of T cell
activation.
D. Effects on blocking CTLA-4 in activation of T cells induced by selected bacterial
strains. We will also examine if blocking CTLA-4 on in vitro activated human cord blood
cells results in potentiated activation of the T cells. The cells will be activated both by
polyclonal stimuli and bacterial strains or bacterial components.
1
Research program, Anna Rudin, 611014-4042
Background and hypotheses:
Immunologic tolerance and regulatory T cells
The FOXP3+ CD25+ CD4+ regulatory T cells are essential to maintain peripheral immunological
tolerance to autoantigens and foreign antigens, such as allergens, by controlling T cells, antigenpresenting cells and B cells. Mutations in the FOXP3 gene leads to lack of these regulatory T
cells and to the syndrome X-linked autoimmunity-allergic dysregulation syndrome, which
includes organ-specific autoimmunity, enterocolitis with food allergy, atopic dermatitis, asthma
and high levels of IgE (Chatila, 2000). The natural FOXP3+ regulatory T cells are continuously
produced in the thymus where they are functionally competent also in infants (Wing, 2002),
although the suppressive function is enhanced when the cells have been exported to the peripheral
blood (Wing, 2005). Birch pollen-allergic adult individuals have similar levels of FOXP3+
expression in their CD4+ T cells as non-allergic individuals, but their regulatory T cells do not
seem to be able to suppress allergic cytokine responses (Grindebacke, 2004, 2009). Therefore, it
may be a better option to enhance the regulatory capacity in early childhood when the immune
system is more flexible and before the T cells react for the first time to various exogenous
antigens, such as allergens.
Importance of CTLA-4
CTLA-4 has long been known to be an extremely important cellular molecule in maintaining
tolerance in animal models of autoimmune disease (Tivol 1995). CTLA-4 is expressed by both
FOXP3+ regulatory T cells as well as by highly activated FOXP3- T cells. Recently, it was
shown in animal models that CTLA-4 is the central molecule in the mechanism of suppression of
the FOXP3+ regulatory T cells (Wing 2008). It was then thought that the tolerogenic effects of
CTLA-4 were mediated mainly by the FOXP3+ regulatory T cells. However, this year it has been
convincingly shown that CTLA-4 in activated FOXP3- T cells also play a crucial role in
inhibiting tissue destruction and autoimmune disease in animal models (Jain 2010, Ise 2010). In
fact, CTLA-4 expressing regulatory T cells and FOXP3- T cells induce immunologic tolerance
and reduce damage caused by autoimmune disease by different levels of action. In animal models
of autoimmunity CTLA-4+ FOXP3+ regulatory T cells inhibit activation of naive T cells, while
CTLA-4 positive activated FOXP3- T cells reduce the infiltration of T cells into non-lymphoid
tissue thereby reducing immune pathology (Jain 2010, Ise 2010). Using prospective birth cohorts
started in 2006 our group has repetitively analyzed both FOXP3 and CTLA-4 expression in T
cells, naive and memory T cell markers and homing receptors. Thus, we now have the unique
opportunity to answer the question whether FOXP3- or CTLA-4 expressing T cells are more
important in inhibiting naive T cell activation and homing receptor switch early in life.
Mechanisms of tissue homing of T cells
The cells reach different tissues by expressing homing receptors that bind to tissue-specific
addressins on the endothelial cells or that are attracted to chemokines produced in the tissue. The
homing receptor 47 is expressed by lymphocytes that home to the intestinal lymphoid tissue.
When the cells are activated in the lymphoid tissue they switch homing receptors to be able to
enter peripheral tissue where they can exert their actions, for example causing inflammation (Lim
2006). One of these tissue homing receptors is the chemokine receptor CCR4 that has been
shown to direct T cells both to the skin and the lung. The expression of homing receptors on T
cells (naive, activated and regulatory, respectively) and homing receptor switch in growing
infants has not been explored until we showed in that tissue-specific homing receptor switch was
closely correlated to activation and memory development of the cells (switch from CD45RA to
CD45RO) and happened after 18 months of age on a group level (Grindebacke, 2009). However
there was a large variation in fraction of cells expressing these markers at each age. Thus,
2
Research program, Anna Rudin, 611014-4042
children vary in the extent of maturation of their T cells and this is very likely explained by
internal factors such as FOXP3 or CTLA-4 expressing T cells and external factors such as gut
bacterial colonization. Although it is known that gut bacteria are needed for effective mucosal
tolerance development it is unknown what microbes are most efficient in children and also by
which immunological mechanisms they induce tolerance. Moreover, it is not known if induction
of tolerogenic molecules on T cells is associated with prevention of inflammatory disease.
Project description and preliminary data:
A. CTLA-4 and FOXP3 expressing T cells in human infants and their effects on activation
of T cells later in childhood. Using detailed data on cellular development in prospective birth
cohorts that are being followed up to 5-6 years of age we will explore which tolerogenic cellular
mechanisms are most important in suppressing T cell activation.
Design and methods:
We analyse blood samples collected from a prospective birth cohort started in 2006 and in which
124 newborns from Göteborg and also the Västra Götaland region have been included. The vast
majority of the children have now reached 3 years of age. Blood samples are drawn from cord
blood and at the ages 3-5 days, 1 month, 4 months, 18 months, 36 months, and 5-6 years of age
and flow cytometric analyses are being performed. We stain for FOXP3 and CD25 to identify the
regulatory T cells, and CTLA-4 and CD25 to identify all cells with regulatory action, whether
FOXP3 positive or negative. We also stain for the receptors CD45RA and CD45RO to determine
the conversion to memory T cells, and the homing receptors 47 and CCR4 to identify homing
receptor switch. The large amount of data that we obtain is being analysed by multivariate factor
analysis using the program SIMCA P+ software (Umetrics, Umeå, Sweden) followed by
univariate analyses using Spearmans rank correlation test. In the multivariate analysis principal
component analysis (PCA) is used to get an overview of the variation in the data to detect
groupings. Projection to latent structures by means of partial least squares (PLS) is implemented
to correlate the data matrices, X (expression of homing receptors and memory markers) and Y
(CTLA-4+ CD25+T-cells or FOXP3+CD25+ Tregs) to each other by a linear multivariate model.
Preliminary data:
Having analysed all data up to three years of age for the first 50 children in the cohort we found
the following results:
First, CTLA-4 expressing T cells
projected together with naive
RA+ CD4 4m
0,30
RA CD25
CD45RA and 47-expressing
0,25
cells, while CD45RO and CCR4RO CD25 3y RO CD43y
RA CD25 4m
0,20
RO CD25 3y
expressing activated T cells
CCR4 CD25 18m CCR4 CD4 18m
0,15
RO CD25 18m
projected on the opposite side.
0,10
FOXP3 4m
RO CD4 18m
RA CD25 18m CTLA-4 18m
FOXP318m
CTLA-4 4m
CCR4 CD25 18m
CCR4 CD4 4m
0,05
The FOXP3+ T cells projected in
α4β7
CD25
4m
RA CD4 18m
CCR4 CD25 4m CCR4 CD25 3y
α4β7 CD25 4m
0,00 RA CD25 18m
α4β7 CD25 18m
the centre of the PCA plot,
RO CD25 18m
α4β7 CD25 18m
α4β7 CD4 4m α4β7 CD4
CCR4 CD25 4m
18m
-0,05
CCR4 CD4 3y
indicating no or less association
CCR4 CD25 3y
-0,10
with the expression of the
-0,15 RA CD4 3y RA CD25 3y
RA CD25 3y
RO CD25 4m
memory markers, homing
-0,20
RO CD25 4m
α4β7
CD25
3y
RO
CD4
4m
receptors or CTLA-4+ T cells.
-0,25
-0,30
These data suggest that CTLA-4
α4β7 CD25 3y
-0,35
expression on T cells is the most
α4β7 CD4 3y
-0,40
important factor for inhibiting T
-0,20
-0,15
-0,10
-0,05
-0,00
0,05
0,10
0,15
0,20
0,25
p[1]
cell maturation.
neg
neg
pos
pos
neg
neg
pos
pos
neg
p[2]
pos
neg
pos
pos
neg
pos
neg
pos
neg
neg
pos
pos
neg
pos
neg
R2X[1] = 0,310998 R2X[2] = 0,131942
3
Research program, Anna Rudin, 611014-4042
Secondly, these data were analyzed by univariate analyses and indeed we found positive
correlations between high percentage of CTLA-4 but not FOXP3 expressing T cells at 4 months
of age and naïve T cells at 18 months of age from the individual child and negative correlations
between CTLA-4 expressing T cells at 4 months and memory and tissue-homing T cells later in
childhood.
6
4
2
0
8
6
4
2
20
40
+
60
80
+
100
+
% CD45RA of CD25 CD4 T cells
at 18m
8
6
4
2
0
0
0
r= -0.51
P= 0.009
10
% CTLA-4+CD25 +
of CD4 + T cells at 4m
% CTLA-4+CD25 +
of CD4 + T cells at 4m
8
r= -0.41
P= 0.007
10
% CTLA-4+CD25 +
of CD4 + T cells at 4m
r= 0.46
P= 0.002
10
0
20
+
40
60
+
80
+
% CD45RO of CD25 CD4 T cells
at 18m
0
20
40
60
80
% CCR4+ of CD25+CD4+ T cells
at 3y
Further work in the project:
We will continue to analyse the cells in the birth cohort and follow the children up to 5-6 years of
age. In this study we also isolate blood mononuclear cells from all the children at 4, 18 and 36
months of age and stimulate them in vitro with the allergens beta-lactoglobulin, ovalbumin, birch
and grass allergen extract as well as with the polyclonal stimulant PHA. The production of Th1,
Th2 and Th17 cytokines are being measured in the supernatants by ELISA or CBA kit. The
proportion of CTLA-4+ T cells and FOXP3+ T cells will be correlated to the cytokine production
thereby examining which type of cell is the better suppressor of different T helper cell types ex
vivo.
B. CTLA-4 and FOXP3 expressing T cells in infancy and effect on later development of
allergic disease. Preliminary data indicate that higher numbers of circulating CTLA-4 expressing
T cells in infancy are negatively associated with development of allergic disease. We are now
comparing proportions of CTLA-4 and FOXP3 expressing cells in infancy with development of
allergic disease in larger birth cohorts.
Design and methods:
We analyse the blood samples collected from part of another prospective birth cohort
(Allergyflora) in which 64 newborns from Mölndal have been included. All children reached 3
years of age in 2006. Blood samples were drawn from cord blood and at the ages 3-5 days, 4
months, 18 months, 36 months and flow cytometric analyses were performed staining for CTLA4 and CD25 to indentify all CD4+ T cells with regulatory action whether FOXP3 positive or
FOXP3 negative. At 18 months of age all children are examined for signs and symptoms of
allergy, including standardised questionnaire, clinical examination and measurement of specific
IgE. When food allergy is suspected food provocations are performed. Clinical analyses are
performed by collaborating paediatric allergists in Göteborg. In the second birth cohort, which is
analyzed more in detail regarding T cell development as in part A, the children are examined
clinically according to the protocol above by collaborating paediatricians in Falköping,
Mariestad, Lidköping or Vara, depending on where the children live. The data is being analysed
by multivariate factor analysis as described above. Projection to latent structures by means of
partial least squares (PLS) is implemented to correlate the data matrices, X (development of
allergy and environmental factors) and Y (CTLA-4+ CD25+ T cells) to each other by a linear
multivariate model.
4
Research program, Anna Rudin, 611014-4042
1,6
1,4
1,2
1,0
0,8
0,6
0,4
0,2
-0,0
-0,2
-0,4
-0,6
-0,8
-1,0
CTLA-4pos T cells
CTLA-4negT cells
*
0.12
0.5
0.03
0.02
0.3
0.2
0.00
0.0
18
y
er
g
all
No
Al
ler
gy
y
er
g
all
18
0.1
18
0.01
18
0.04
0.4
Al
ler
gy
Counts 106/ml blood
(CTLA-4 neg T cells)
0.06
0.05
No
Counts 106/ml blood
(CTLA-4 pos T cells)
Food allergy 18m
Allergy 18m
Breastfed 4m
Asthma 18m
Caesarean
Antibiotics ≤18m
Eczema 18m
Dog dander ng/g
Asthma 36m
No allergy 18m
0.09
CTLA-4 pos 18m
pq[1]
Preliminary data:
Multivariate analysis of the data in this cohort of children using projection to latent structures by
means of PLS showed that CTLA-4 expressing T cells were negatively associated with the
presence of allergy at 18 months of age. In univariate analyses, higher counts of CTLA-4 positive
cells were significantly associated with a lower risk of allergy, while CTLA-4-negative T cells
showed no relation to allergy development.
Further work in the project:
This type of analysis including all data in project A as well as clinical evaluation as described
above will be performed in the more recently started birth cohorts comprising 124 children. A
database on the clinical data on allergic disease and specific IgE at 18 and 36 months of age is
now being compiled. We will then be able to analyse the detailed T cell data as described in part
A in relation to allergy development to confirm and extend the results from Allergyflora. In these
cohorts we will be able to compare the relative significance of early T cell expression of CTLA-4
and FOXP3 in relation to allergic disease later in childhood as well as the relation of maturation
and tissue homing molecule expression with the development of allergic disease. We will also be
able to analyse the levels of different tissue specific chemokines in serum/plasma of the children
at the different ages as we have frozen plasma from all time points.
C. Effect of specific bacteria colonizing the gut in early infancy on the expression of CTLA4 and FOXP3 in T cells during childhood. We will analyze whether any specific bacterial strain
promotes expression of CTLA-4 and FOXP3 in T cells. We will also examine if an induced
expression of these molecules leads to suppression of T cell activation.
Design and methods:
In both birth cohorts intestinal commensal bacterial colonization has been determined by a rectal
swab at 3-5 days of age and a feacal sample at 1, 2, 4 and 8 weeks of age by Ingegerd Adlerberth
and Agnes Wold, Dept of Bacteriology. Briefly, the faecal samples are cultured quantitatively for
all major groups of aerobic and anaerobic bacteria. The presence of colonizing specific bacterial
strains at different time points after birth was analyzed in both the prospective birth cohorts and
these data are analyzed in the multivariate factor analysis as described above.
Preliminary data:
Analysis of the data from the first cohort of children, Allergyflora, using projection to latent
structures by means of PLS showed that high number CTLA-4 expressing T cells in the blood at
18 months of age was positively associated with the presence of Escherichia coli in the gut
during the first month of life. Univariate analyses showed significant positive associations
between high number of CTLA-4 expressing T cells and presence of E. coli in the gut during the
5
Research program, Anna Rudin, 611014-4042
first two weeks of life. The presence of Clostridia in the gut early in life was negatively
associated with the number of CTLA-4 expressing T cells.
1,2
CTLA-4pos 18m
0.10
8w
ns
ns
0.05
0.05
0.04
0.03
0.02
0.01
2w
0.15
E.
co
E. li co
li +
When analyzing children having CTLA-4 positive T cell counts above median or below median
at 4, 18 and 36 months of age, 90% of those having high CTLA-4 expression at all time points
were colonized by E. coli, whereas 10% of those having low CTLA-4 expression at all time
points were colonized by E. coli. This strongly suggests that E. coli in the infant gut stimulates
the expression of CTLA-4, which may lead to development of tolerance.
Further work in the project:
Based on the preliminary data from the first birth cohort described above we will continue to
analyze the tolerogenic mechanisms in relation to the gut colonization pattern in more detail as
described in part A. In this larger study we will not only be able to confirm or refute our
preliminary results but also compare the effects on the CTLA-4 and the FOXP3 expression as
well as the effects on the maturation and homing receptor expression of the T cells. Moreover, in
this cohort the T cells will be analyzed also at 5-6 years of age.
D. Effects on blocking CTLA-4 in activation of T cells induced by selected bacterial strains.
We will also examine if blocking CTLA-4 on in vitro activated human cord blood cells results in
potentiated activation of the T cells. The cells will be activated both by polyclonal stimuli and
bacterial strains or bacterial components.
Design and methods:
We obtain cord blood samples from unselected healthy children born at the maternity ward of
Sahlgrenska University Hospital/Mölndal, who have provided us with such cells for several
studies in the past years. We isolate the peripheral blood mononuclear cells and stimulate these
cells with polyclonal stimuli (PHA), selected UV-killed bacterial strains or bacterial components.
We select strains that have shown either positive or negative associations with level of CTLA-4
or FOXP3 expressing T cells in the children from the birth cohorts. The expression of CTLA-4
and FOXP3, CD45RA and RO and the expression of a range of homing receptors on the
CD4+CD25+ T cells are analyzed before and after stimulation by flow cytometry. If we find
specific effects of certain bacterial strains we also stimulate with components of these bacteria,
such as endotoxins, exotoxins or membrane components to evaluate whether parts of the bacteria
might be used for tolerance induction and prevention of inflammatory disease. To confirm the
importance of CTLA-4 we will add anti-CTLA-4 antibodies to the cell cultures and compare the
memory cell conversion and homing receptor switch to cultures with antibody controls.
6
*
0.10
0.05
0.05
0.04
0.03
0.02
0.01
0.00
0.00
E.
co
E. li co
li +
Enteroba cteria 2w
Clostridia 8w
Clostridia 2w
Enterococci 4w
S. a ureus 8w
S. a ureus 2w
La ctoba cilli 4w
La ctoba cilli 8w
Ba cteroides 8w
Ba cteroides 1w
E. coli 8w
E. coli 3d
E. coli 4w
E. coli 2w
E. coli 1w
-0,4
4w
*
E.
co
E. li co
li +
-0,2
2w
**
E.
co
E. li co
li +
0,2
-0,0
1w
Cl
os
tr
Cl idia
os
tri dia
+
0,4
3d
**
Counts 10 6/ml blood
CTLA-4pos T cells
0.15
Counts 10 6/ml blood
CTLA-4pos T cells
0,6
CTLA-4 pos 18m
pq[1]
0,8
E.
co
E. li co
li +
1,0
Research program, Anna Rudin, 611014-4042
Significance:
In developed countries there is an increasing prevalence of inflammatory disease such as allergy,
celiac disease, inflammatory bowel disease and autoimmune diabetes type 1. Importantly, the
debut of inflammatory disease also occurs at lower ages than before. The reason for this is poorly
functioning tolerance mechanisms mainly because of defective down regulation on T cell
activation and on change in tissue homing properties in children younger than 2 years of age
when the immune system is still highly flexible. Therefore, environmental factors acting on the
immune system early are possible future agents of intervention. This projects aims at discovering
the most important immunologic mechanisms responsible for tolerance in children and using
these as markers for both disease risk and targets for prevention of disease.
Collaborations and role of the applicant:
This translational project is possible due to longstanding collaboration (since 2001) between
different groups of researchers at three different Institutions at The Sahlgrenska Academy with
each group contributing with essential competence and research infrastructure. The project would
not have been possible if one of these groups had not been able to perform their part in the
project. The role of the group of this applicant is to handle all blood samples and immunological
analyses of the children and to analyze these data in relation to microbiological and clinical data
as shown in the preliminary results of this application. Our specific area of expertise is
immunoregulatory mechanisms in humans and the applicant is consultant in Rheumatology and
professor in Rheumatology and Inflammation Research. The applicant is co-inventor of a
European patent granted in 2010 together with Agnes Wold and Ingegerd Adlerberth, which is a
result of our joint studies and is a project in Gothenburg International Bioscience Business School
(GIBBS) under development. The applicant is currently member of the steering committé of the
project.
7
Research program, Anna Rudin, 611014-4042
Equipment:
All equipment that is needed for this project is available at the Dept of Rheumatology and
Inflammation Research which hosts many groups excellent in immunology research.
Ethical considerations:
All birth cohort studies as well as studies performed on anonymous cord blood cells have been
approved by the Ethics Committee for human studies (Approval Dnr: Ö 342-00, Ö 446-00, 36205, 363-05). The studies are dependent on infants being sampled for peripheral blood, which may
cause pain. However, it is of great importance to study what tolerogenic mechanisms inhibit the
development of inflammatory disease in humans and not only in mice. This cannot be done in
adults since the tolerance mechanisms develop early in life. Each infant is given a study idnumber that is used throughout the study to protect the personal integrity of the individual.
References:
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Chatila TA, Blaeser AF, Ho N, Lederman HM, Voulgaropoulos C, Helms C, Bowcock
AM. J Clin Invest 2000;106:R75.
Grindebacke H, Wing K, Andersson A-C, Suri-Payer E, Rak S, Rudin A. Defective
suppression of Th2 cytokines by CD4+CD25+ regulatory T cells in birch allergics during
birch pollen season. Clin Exp All 2004; 34:1364.
Grindebacke H, Larsson P, Wing K, Rak S, Rudin A. Specific immunotherapy to birch
allergen does not enhance suppression of Th2 cells by CD4+CD25+ regulatory T cells
during pollen season. J Clin Immunol, 2009;29:752-60.
Grindebacke H, Stenstad, H, Quiding-Järbrink M, Waldenström J, Adlerberth I, Wold AE,
Rudin A. Dynamic development of homing receptor expression and memory cell
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Ise W, Kohyama M, Nutsch KM, Le HM, Suri A, Unanue ER, Murphy TL, Murphy K.
CTLA-4 suppress the pathogenicity of self antigen-specific T cells by cell-intrinsic and
cell-extrinsic mechanisms. Nature Immunology, 2010;11, 129-135.
Jain N, Nguyen H, Chambers C, Kang J. Dual function of CTLA-4 in regulatory T cells
and conventional T cells to prevent multiorgan immunity. PNAS, 2010;107, 1524-1528.
Lim HW, Broxmeyer HE, Kim CH. Regulation of trafficking receptor expression in
human forkhead box P3+ regulatory T cells. J Immunol, 2006;177:4488-94.
Tivol EA, Borriello F, Schweitzer AN, Lynch WP, Bluestone JA, Sharpe AH. Loss of
CTLA-4 leadsa to massive lymfoproliferation and fatal multiorgan tissue destruction,
revealing a critical negative regulatory role of CTLA-4. Immunity, 1995;3,541-47
Wing K, Ekmark A, Karlsson H, Rudin A, Suri-Payer E. Characterization of
humanCD25+CD4+ T cells in thymus, cord and adult blood. Immunology 2002;106:190.
Wing K, Larsson P, Sandström K, Lundin SB, Suri-Payer E, Rudin A. CD4+CD25+
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T cells responses. Immunology 2005;115:516.
Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, Fehervari Z, Nomura T,
Sakaguchi S. CTLA4 control over Foxp3+ regulatory T cell function. Science,
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