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Diminished CD4+CD25+ Regulatory T Cell
Lowers IL-2 Signaling and Contributes to
Type 1 Diabetes-Associated IL2RA Variation
Wicker and Timothy I. M. Tree
Sarah Nutland, Mark Peakman, John A. Todd, Linda S. Bell,J. Cutler, Kate Downes, Marcin Pekalski, Gwynneth L.
Garima Garg, Jennifer R. Tyler, Jennie H. M. Yang, Antony
http://www.jimmunol.org/content/188/9/4644
doi: 10.4049/jimmunol.1100272
March 2012;
2012; 188:4644-4653; Prepublished online 28J Immunol
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References http://www.jimmunol.org/content/188/9/4644.full#ref-list-1
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The Journal of Immunology
Type 1 Diabetes-Associated IL2RA Variation Lowers IL-2
Signaling and Contributes to Diminished CD4
+
CD25
+
Regulatory T Cell Function
Garima Garg,*
,†,1
Jennifer R. Tyler,*
,1
Jennie H. M. Yang,*
,1
Antony J. Cutler,
‡
Kate Downes,
‡
Marcin Pekalski,
‡
Gwynneth L. Bell,
‡
Sarah Nutland,
‡
Mark Peakman,*
,†
John A. Todd,
‡
Linda S. Wicker,
‡
and Timothy I. M. Tree*
,†
Numerous reports have demonstrated that CD4
+
CD25
+
regulatory T cells (Tregs) from individuals with a range of human
autoimmune diseases, including type 1 diabetes, are deficient in their ability to control autologous proinflammatory responses
when compared with nondiseased, control individuals. Treg dysfunction could be a primary, causal event or may result from
perturbations in the immune system during disease development. Polymorphisms in genes associated with Treg function, such as
IL2RA, confer a higher risk of autoimmune disease. Although this suggests a primary role for defective Tregs in autoimmunity,
a link between IL2RA gene polymorphisms and Treg function has not been examined. We addressed this by examining the impact
of an IL2RA haplotype associated with type 1 diabetes on Treg fitness and suppressive function. Studies were conducted using
healthy human subjects to avoid any confounding effects of disease. We demonstrated that the presence of an autoimmune disease-
associated IL2RA haplotype correlates with diminished IL-2 responsiveness in Ag-experienced CD4
+
T cells, as measured by
phosphorylation of STAT5a, and is associated with lower levels of FOXP3 expression by Tregs and a reduction in their ability to
suppress proliferation of autologous effector T cells. These data offer a rationale that contributes to the molecular and cellular
mechanisms through which polymorphisms in the IL-2RA gene affect immune regulation, and consequently upon susceptibility to
autoimmune and inflammatory diseases. The Journal of Immunology, 2012, 188: 4644–4653.
Type 1 diabetes (T1D) is characterized by autoimmune
destruction of pancreatic bcells, a process in which au-
toreactive T cells play a pivotal role (1–3). There is now
a growing body of evidence to suggest that in T1D, this patho-
logical autoimmunity is the direct result of a failure of immune
regulation (4). This includes the defective function of various
populations of regulatory T cells (Tregs), especially those char-
acterized as CD4
+
CD25
hi
FOXP3
+
. In support of this, we and
others have demonstrated that the suppression of autologous re-
sponder T cells by CD4
+
CD25
hi
Tregs in individuals with newly
diagnosed T1D is reduced significantly compared with that ob-
served in age-matched control subjects (5–8). Importantly, we also
demonstrated in an independent cohort of patients that defective
suppression is not only present close to diagnosis, but also in
individuals who have had T1D for .20 y, suggesting that the
functional defect represents a phenotype that is stable over time
and most likely under genetic control (9).
A candidate gene study identified an association between T1D
and the IL-2RA gene (encoding the IL-2 receptor a-chain, CD25)
(10, 11). There are three protective IL2RA haplotypes, one of
which is marked by the single nucleotide polymorphism (SNP)
rs12722495, where the protective allele confers a relative risk for
T1D of 0.65. Recently, the disease-associated IL2RA rs12722495
haplotype was correlated with several distinct cellular immuno-
phenotypes, most notably the higher expression of CD25 on
memory CD4
+
T cells and higher levels of IL-2 secretion from
these memory T cells (12). IL-2RA is part of the high-affinity IL-2
receptor complex and is constitutively expressed at high levels on
both naturally occurring and peripherally induced FOXP3
+
Tregs
(13, 14). Numerous lines of evidence from both mice and humans
have demonstrated that IL-2 plays a key role in both the genera-
tion and function of FOXP3
+
Tregs (15–20). Studies in vitro have
demonstrated that activation of CD4
+
CD25
+
T cell suppressor
function requires IL-2 (21) and in vivo that IL-2 is required for
peripheral survival and expansion of CD4
+
CD25
+
Tregs (reviewed
*Department of Immunobiology, School of Medicine, King’s College London, Lon-
don SE1 9RT, United Kingdom;
†
National Institutes of Health Research Biomedi-
cal Research Centre at Guy’s and St. Thomas’ National Health Service Foundation
Trust and King’s College London, London SE1 7EH, United Kingdom; and
‡
Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation
Laboratory, Cambridge Institute for Medical Research, University of Cambridge,
Addenbrooke’s Hospital, Cambridge CB2 0XY, United Kingdom
1
G.G., J.R.T., and J.H.M.Y. contributed equally to this study.
Received for publication January 28, 2011. Accepted for publication February 14,
2012.
This work was supported by Juvenile Diabetes Research Foundation U.K. Centre for
Diabetes Genes, Autoimmunity, and Prevention Grant 4-2007-1003, the Juvenile
Diabetes Research Foundation International, Wellcome Trust Grant WT061858, the
National Institute for Health Research Cambridge Biomedical Research Centre, the
National Institute for Health Research Biomedical Research Centre at Guy’s and St.
Thomas’ National Health Service Foundation Trust and King’s College London, and
the Medical Research Council Cusrow Wadia Fund. The research leading to these
results received funding from European Union’s Seventh Framework Programme
Grant FP7/2007-2013 under Grant Agreement 241447 (Natural Immunomodulators
as Novel Immunotherapies for Type 1 Diabetes). J.R.T. is the recipient of a Diabetes
U.K. Ph.D. studentship. The Cambridge Institute for Medical Research is the recip-
ient of Wellcome Trust Strategic Award 079895.
Address correspondence and reprint requests to Dr. Timothy Tree, Department of
Immunobiology, King’s College London, School of Medicine, 2nd Floor, New Guy’s
House, Guy’s Hospital, London SE1 9RT, United Kingdom. E-mail address: timothy.
tree@kcl.ac.uk
The online version of this article contains supplemental material.
Abbreviations used in this article: aTreg, activated regulatory T cell; iTreg, induced
regulatory T cell; MFI, median fluorescence intensity; mTconv, conventional mem-
ory T cell; mTreg, memory regulatory T cell; nTconv, conventional naive T cell;
rTreg, resting regulatory T cell; SNP, single nucleotide polymorphism; Tconv, con-
ventional T cell; T1D, type 1 diabetes; Treg, regulatory T cell.
Copyright Ó2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100272
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in Ref. 13). Furthermore, signaling via common g-chain cyto-
kines, of which IL-2 is a key member, is required for the main-
tained expression of FOXP3 by Tregs, which is essential for their
suppressive function (19, 22).
These findings invoke the hypothesis that gene polymorphisms
in the IL-2/IL-2RA pathway exert their influence on T1D risk via
effects on the number or functional ability of FOXP3
+
Tregs. In
support of this, recent studies show that Tregs from individuals
with T1D are more prone to apoptosis and more easily lose ex-
pression of FOXP3 and that these phenotypes may be linked to
a relative defect in signaling via the IL-2 pathway (7, 23, 24). To
date, however, direct evidence linking polymorphisms in IL2RA
with altered Treg function is lacking. To address this knowledge
gap, we examined whether the T1D susceptibility allele defined by
the IL2RA SNP rs12722495 is associated with a reduction in the
functional capacity of Tregs. To avoid any potential confounding
effects of disease on Treg phenotype, we conducted these studies
in individuals without disease. Our studies show that the T1D-
susceptibility IL2RA haplotype identified by rs12722495 is asso-
ciated with decreased signaling via the IL-2 pathway in both
memory T cells and Tregs and that this is linked to diminished
Treg function.
Materials and Methods
Subjects and study design
Individuals homozygous for the T1D protective rs12722495 IL2RA
haplotype and the fully susceptible IL2RA haplotype (denoted as P1P1
and SS, respectively) were recruited for the present study. The levels of
CD25 expression on CD4
+
T cell subsets have previously been studied in
these individuals, and pairs of P1P1 and SS individuals were preselected
who showed typical haplotype-specific patterns of CD25 expression on
the conventional memory CD4
+
T cell (Tconv) subset (i.e., a relatively
higher level of CD25 expression in P1P1 individuals compared with SS).
For PBMC isolation, blood samples were collected in vacutainers con-
taining sodium heparin anti-coagulant, diluted 1:1 with RPMI 1640 sup-
plemented with 100 mg/ml penicillin/streptomycin (Invitrogen, Paisley,
U.K.) and stored overnight under constant rotation. The following day,
PBMCs were isolated from whole blood by density gradient centrifu-
gation (Lymphoprep; Axis-Shield, Oslo, Norway). For whole blood
staining, blood was collected into vacutainers containing sodium heparin
anti-coagulant and used on the day of collection. Because of the day-
to-day variation inherent in intracellular staining protocols, it was not
possible to normalize pSTAT5a and CD25 median fluorescence intensity
(MFI) values through time as we were able to do previously using a cell
surface staining protocol (12). Therefore, all analyses were performed in
a paired manner with one P1P1 and one SS individual analyzed on a
single day. Ethical approval for this study was granted by the local
Ethics Committee and informed consent was obtained. All experiments
were performed in a blinded manner without knowledge of genotype of
the individual.
mAbs and reagents
The following Abs were used in these studies as indicated: PE-conjugated
monoclonal anti-CD25 (clones M-A251 and 2A3), FITC-conjugated anti-
CD4 (clone SK3), Alexa Fluor 647-labeled anti-STAT5a (pY694) and
Alexa Fluor 488-labeled anti-STAT5a (pY694) were obtained from BD
Biosciences (Oxford, U.K.); eFluor 450-labeled anti-CD4 (clone SK3),
anti-CD45RA (clone HI100), and PerCP-Cy5.5-conjugated anti-CD127
(clone eBioRDR5) were obtained from eBioscience (Hatfield, U.K.); and
Alexa Fluor 700-labeled anti-CD45RA (clone HI100), allophycocyanin-
Cy7-conjugated anti-CD14 (clone HCD14), FITC-conjugated anti-Helios
(clone 22F6), Alexa Fluor 647-labeled anti-FOXP3 (clone 259D), Alexa
Fluor 700-labeled anti-CD4 (clone RPA-T4), Pacific Blue-conjugated anti-
CD45RA (clone H100), and PE-conjugated anti-FOXP3 (clone 259D) were
obtained from BioLegend (Cambridge, U.K.). Ab concentrations used were
based on manufacturers’ recommendations and optimization studies.
Complete media for functional studies was X-Vivo 15 media (Lonza,
Wokingham, U.K.) supplemented with 5% human pooled AB
+
sera (PAA
Laboratories, Lutterworth, U.K.) and 100 mg/ml penicillin/streptomycin
(Invitrogen). Proleukin (Chiron, Emeryville, CA) was used as a source
of human rIL-2.
Flow cytometric analysis for pSTAT5a
Analysis of pSTAT5a in PBMCs was performed using BD Phosflow
reagents (BD Biosciences) according to the manufacturer’s instructions.
Briefly, 1 310
6
PBMCs were incubated with various concentrations of
IL-2 for 10 min at 37˚C, fixed with Phosflow buffer I, permabilized with
Perm Buffer III, stained with anti–CD4-FITC, anti–CD25-PE, anti–CD127-
PerCP-Cy5.5, anti–CD45RA-eFluor 450, and anti–STAT5a-pY694-Alexa
Fluor 647 and analyzed in a BD FACSCanto II (BD Biosciences). Be-
cause of a technical failure in one sample, only nine pairs of PBMC samples
were analyzed for expression of pSTAT5a. For simultaneous detection of
pSTAT5a and FOXP3, 500 ml fresh blood was incubated with 500 mlX-
Vivo media containing various concentrations of IL-2 for 10 min at 37˚C,
fixed with warm BD Lyse/Fix buffer (BD Biosciences), and permeabilized
with 100% methanol for 20 min on ice. After extensive washing with PBS
containing 0.2% BSA, cells were stained with anti–CD4-Alexa Fluor 700,
anti–CD25-allophycocyanin, anti–CD45RA-Pacific Blue, anti–FOXP3-PE,
and anti–STAT5a-pY694-Alexa Fluor 488 and analyzed using a BD For-
tessa (BD Biosciences). Results are expressed as the MFI of pSTAT5a
staining for all cells within a particular T cell subset.
Isolation and analysis of cell populations for functional studies
PBMCs were stained with anti–CD4-eFluor 450, anti–CD25-PE, anti–
CD127-PerCP-Cy5.5, anti–CD45RA-Alexa Fluor 700, and anti–CD14-
allophycocyanin-Cy7 and control populations stained with anti–CD4-
eFluor 450, IgG1-PE, IgG1-PerCP-Cy5.5, anti–CD45RA-Alexa Fluor
700, and anti–CD14-allophycocyanin-Cy7. Lymphocytes were identified
based on forward and side scatter parameters and populations isolated for
functional analysis using a BD FACSAria II flow cytometer and FACSDiva
software (BD Biosciences). Flow cytometry data were analyzed using
FlowJo software (Tree Star, Ashland, OR).
Flow cytometric analysis for FOXP3 and Helios
FOXP3 and Helios staining was performed on cells immediately postsorting
and after 48 h culture with various concentrations of IL-2 using the Bio-
Legend FOXP3 Fix/Perm buffer set according to the manufacturer’s
instructions. For analysis of FOXP3 and Helios in cultured cells, 10
4
sorted
Tregs were incubated in complete media supplemented with IL-2 for 48 h
prior to analysis.
In vitro coculture suppression assays
Suppression assays were performed by culturing memory or naive Tconv
populations (2.5 310
3
/well) in the presence or absence of either autologous
Tregs or a third party Treg cell line at the ratios indicated. Cells were activated
by the addition of Dynabeads Human T-Activator anti-CD3/anti-CD28
beads (Invitrogen) at a bead/conventional cell ratio of 1:1. All conditions
were conducted in triplicate. The third party Treg cell line was generated by
expanding FACS sorted Tregs from a single donor for 14 d in 600 U/ml IL-2.
Expanded Tregs were cryopreserved and a single aliquot was thawed for use
with each pair of samples analyzed. After 5 d culture, 100 ml supernatant was
removed and stored at 280˚C for later cytokine analysis. Proliferation was
assessed by the addition of 0.5 mCi/well [
3
H]thymidine (PerkinElmer, Wal-
tham, MA) for the final 18 h coculture. The percentage of suppression was
calculated using the following formula: % suppression = 100 2[(cpm in the
presence of Tregs 4cpm in the absence of Tregs) 3100].
Statistics
The normality of all datasets was tested using the D’Agostino–Pearson
omnibus normality test. Where data did not significantly deviate from the
normal distribution, either an independent or paired Student ttest was used
to test for significance as indicated. Where one or more datasets were
found to significantly deviate from the normal distribution, statistical
significance was determined using Wilcoxon matched-pairs signed rank
test for paired data. All statistical analyses were performed using Graph-
Pad Prism (GraphPad Software, La Jolla, CA).
Results
We have previously reported that the IL2RA rs12722495 pro-
tective haplotype is associated with significantly higher levels
of expression of CD25 on conventional memory CD4
+
Tcells
(mTconv) and we observed a similar trend for FOXP3
+
Tregs
(12). Therefore, we first sought to investigate whether increased
levels of CD25 expression from individuals with the P1P1 IL2RA
haplotype resulted in altered responsiveness to IL-2 signaling,
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measured via phosphorylation of STAT5a in all T cell popula-
tions after brief in vitro exposure to IL-2. Because fixation pre-
cluded the use of CD127 as a surface marker of Tregs and FOXP3
costaining was found to be unreproducible using the pSTAT5a
staining protocol for PBMC samples, Tregs were initially identi-
fied based on a high level of CD25 staining and reduced CD4
staining as previously described by other investigators and as il-
lustrated in Fig. 1A (the frequency of CD4
lo
CD25
+
Tregs defined
using this method and via CD25
+
CD127
lo
expression was highly
correlated; R
2
= 0.87, p,0.0001; Supplemental Fig. 1). Addi-
tionally, a more stringent definition was applied to identify Tregs
expressing very high levels of CD25 (CD25
hi
Tregs) by gating on
the top 1% of CD25-staining CD4
+
T cells as previously described
by other investigators (23, 25) (gating on an identical population in
unfixed cells confirmed that .98% of these cells were CD127
lo/2
in all individuals examined). Tconv were subdivided based on ex-
FIGURE 1. Example of the gating used for the identification of T cell populations for STAT5a phosphorylation studies using PBMCs. (A) Lymphocytes
identified by their forward and side scatter properties were gated for CD4
+
expression and then examined for expression of CD25. Tregs were identified
using two different gating strategies: 1) based on a high level of CD25 staining and reduced CD4 staining (CD4
lo
CD25
+
Tregs), and 2) gating on the top 1%
of CD25-staining CD4
+
cells (CD25
hi
Tregs). Tconv were identified by low/intermediate levels of CD25 staining. (B–D) All populations were analyzed for
expression of CD45RA to delineate populations of CD45RA
+
and CD45RA
2
Tconv and Tregs.
FIGURE 2. Relationship between IL2RA haplotype and CD25 expression on CD4
+
Tconv and Treg populations from PBMCs. PBMCs from donors with
the protective rs12722495 IL2RA haplotype (P1P1) and donors with the susceptible IL2RA haplotype (SS) were isolated and analyzed as matched pairs by
flow cytometry as shown in Fig. 1. Each symbol type represents a pair of individuals analyzed on the same day and are joined by a horizontal line in paired
analysis. MFI is shown of CD25 staining on (A) mTconv, (B) CD4
lo
CD25
+
Tregs, (C) CD4
lo
CD25
+
CD45RA
2
Tregs, and (D) CD4
lo
CD25
+
CD45RA
+
Tregs.
Statistical significance was determined using a two-tailed paired Student ttest.
4646 IL2RA HAPLOTYPE AFFECTS CD4
+
CD25
+
TREG FUNCTION
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pression of CD45RA to delineate memory (CD45RA
2
,mTconv)
and naive (CD45RA
+
, nTconv) cells (Fig. 1B). Similarly, Tregs
were subdivided into CD45RA
2
Tregs and CD45RA
+
(Fig. 1C).
Because CD25
hi
Tregs were .90% CD45RA
2
(Fig. 1D; i.e., con-
sisted mainly of Ag-experienced Tregs), this population was not
subdivided for analysis. In all data presented, a pair of subjects dif-
fering at the rs12722495 SNP has been studied in a discrete, simul-
taneous experiment; each pair is denoted by a distinct symbol that is
consistent throughout all graphs in the results section. Details of
the gender and age bands of subjects and the symbols used to denote
the results are shown in Supplemental Table I.
Relationship between IL2RA haplotype and expression of
CD25 on CD4
+
Tconv and Treg populations
Analysis of isolated PBMCs indicated that individuals with the
P1P1 IL2RA haplotype express significantly more CD25 on mTconv
(p= 0.0002) and Treg (p= 0.01) compared with individuals with
the SS haplotype (Fig. 2A, 2B). The higher levels of CD25 were
FIGURE 3. Example of pSTAT5a staining in Tconv and Treg populations from PBMCs. PBMCs were incubated with various concentrations of IL-2 for
10 min, fixed, and stained for CD4, CD25, CD45RA, and pSTAT5a (Y694). (Aand B) Representative plot of CD4
+
cells from one individual incubated with
(A) 0 U/ml and (B) 10 U/ml IL-2. (C) Representative example of a dose response curve in Tconv and Treg populations from one individual: 4, nTconv; :,
mTconv; n, CD4
lo
CD25
+
CD45RA
+
Tregs; d, CD4
lo
CD25
+
CD45RA
2
Tregs; N, represent CD25
hi
Tregs.
FIGURE 4. Relationship between IL2RA haplotype and STAT5a phosphorylation in Tconv and Treg populations from PBMCs. PBMCs from donors with
the protective rs12722495 IL2RA haplotype (P1P1) and donors with the susceptible IL2RA haplotype (SS) were isolated and analyzed as described in Fig. 3.
Populations of conventional and regulatory cells were defined as described in Fig. 1. The percentage of cells positive for pSTAT5a in nTconv (A,B),
mTconv (C,D), CD4
lo
CD25
+
Tregs (E), CD4
lo
CD25
+
CD45RA
+
Tregs (F), CD4
lo
CD25
+
CD45RA
2
Tregs (G), and CD4
+
CD25
hi
Tregs (H) following
exposure to IL-2 as indicated. Each symbol type represents a pair of individuals analyzed on the same day, and individuals are joined by a horizontal line.
Results are expressed as the MFI of pSTAT5a staining for all cells within a given population following exposure to IL-2 as indicated. Statistical significance
was determined using a two-tailed paired Student ttest.
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observed both in the CD45RA
+
(p= 0.005) and CD45RA
2
CD4
lo
CD25
+
Tregs ( p= 0.008) (Fig. 2C, 2D). We observed no difference
in the frequency of mTconv between the P1P1 and SS individuals
(mean, 51.5 and 42.7%, respectively), the proportion of CD4
+
T cells classified as Tregs (mean, 4.28 and 4.38%, respectively), or
the percentage of CD45RA
2
Tregs (mean, 69.2 and 65.2%, re-
spectively). These observations are all in keeping with our previous
report (12), even though CD25 levels were quantified using an in-
tracellular staining protocol in the present study.
mTconv and Tregs from individuals with the protective P1P1
IL2RA haplotype show increased sensitivity to IL-2
Sensitivity to IL-2 was assessed by measuring phosphorylation of
STAT5a in all T cell populations after brief in vitro exposure to IL-2
(Fig. 3A, 3B). As might be predicted from their CD25 levels,
sensitivity to IL-2 in this assay was lowest for nTconv, with
mTconv, CD4
lo
CD25
+
CD45RA
+
Treg, CD4
lo
CD25
+
CD45RA
2
Treg, and CD25
hi
Treg showing successively higher sensitivities,
especially at very low concentrations of IL-2 (Fig. 3C).
mTconv from individuals with the P1P1 IL2RA haplotype were
more responsive to IL-2 at both 10 U/ml (p= 0.01) and 100 U/ml
(p= 0.007) compared with individuals with the SS haplotype;
however, no such difference was observed in nTconv (Fig. 4A–D).
Additionally, we observed that at low concentrations of IL-2,
CD4
lo
CD25
+
Tregs from P1P1 individuals were more responsive
to IL-2 compared to individuals with the SS haplotype (Fig. 4E;
0.1 U/ml, p= 0.01). This difference was observed in both the
CD45RA
+
and CD45RA
2
populations (Fig. 4F, 4G; p= 0.005 and
p= 0.03, respectively). Interestingly, these significant differences
were only observed at the lowest concentration of IL-2 but not at
higher concentrations of 1, 10, or 100 U/ml (data not shown).
Similarly, we observed that CD25
hi
Tregs from P1P1 individuals
were more responsive to IL-2 compared to individuals with the SS
haplotype (Fig. 4H). Again, this was true at low concentrations of
IL-2 (0.1 U/ml, p= 0.02) but not at higher concentrations (data not
shown).
To confirm and extend these findings, we developed a whole
blood assay incorporating additional bona fide markers of Tregs,
that is, the transcription factor FOXP3, to examine IL-2 respon-
siveness (Supplemental Fig. 2). In addition to providing a more
definitive marker for Tregs, examination of the expression level of
FOXP3 combined with expression of CD45RA also allowed the
delineation of three different populations of FOXP3
+
T cells as
described by Mayara and colleagues (26). As an example of the
gating strategy used to identify FOXP3
+
Tregs, the division of this
population into resting Tregs (rTregs, FOXP3
+
CD45RA
+
), mem-
ory Tregs (mTregs, FOXP3
+
CD45RA
2
), and activated Tregs
(aTregs, FOXP3
hi
CD45RA
2
) and the relative levels of CD25
expression on these populations are shown in Fig. 5A–C. Again,
sensitivity to IL-2 in this assay was related to the relative ex-
pression levels of CD25 within each T cell population, especially
at very low concentrations of IL-2 (Fig. 5D, Supplemental Fig. 2).
This assay was then deployed on a fresh cohort of 13 pairs
of individuals with the P1P1 or SS IL2RA haplotype consisting of
6 of the original pairs studied above and 7 new pairs. Consis-
tent with the results in isolated PBMCs, staining in whole blood
demonstrated that P1P1 individuals express significantly more
CD25 on mTconv (p= 0.001) and Ag-experienced FOXP3
+
mTregs (p= 0.045) and FOXP3
hi
aTregs (p= 0.0004) compared
with individuals with the SS haplotype; however, no such dif-
ference was observed in rTregs (Fig. 6A–D). Similarly, results
from the whole blood assay again demonstrated that mTconv and
FOXP3
hi
aTregs from individuals with the P1P1 IL2RA haplotype
were more responsive to IL-2 at nonsaturating doses (mTconv,
4 U/ml, p= 0.04; aTregs, 0.3 U/ml, p= 0.02) compared with
individuals with the SS haplotype (Fig. 6E and 6H, respectively).
However, no significant difference was observed in these pop-
ulations at higher concentrations of IL-2 (mTconv, 10 or 100 U/
ml; aTregs, 1–100 U/ml; data not shown). The lack of a significant
difference between the P1P1 and SS donors in rTregs and mTegs
(Fig. 6F and 6G, respectively) reflects the fact that in three pairs
the SS cells were more responsive to IL-2 than were the P1P1
FIGURE 5. Example of FOXP3-pSTAT5a staining
in Tconv and Treg populations from whole blood.
Whole blood was incubated with various concen-
trations of IL-2 for 10 min, fixed, permeabilized, and
stained for CD4, CD25, CD45RA, FOXP3, and
pSTAT5a (Y694). (Aand B) Example of the gating
strategy to identify Tconv and FOXP3
+
Treg subsets.
Lymphocytes identified by their forward and side
scatter properties were gated for CD4
+
expression and
then examined for expression of CD25 and FOXP3.
FOXP3
+
Tregs were identified by coexpression of
CD25 and FOXP3 (A) and then divided into rTregs
(FOXP3
+
CD45RA
+
), mTregs (FOXP3
+
CD45RA
2
),
and aTregs (FOXP3
hi
CD45RA
2
)(B). (C) Analysis of
CD25 expression levels from whole blood staining in
mTconv (dotted line), FOXP3
+
rTregs (dashed line),
FOXP3
+
mTregs (solid line), and FOXP3
hi
aTregs
(filled histogram). (D) Representative example of
a dose response curve in mTconv and FOXP3
+
Treg
populations from one individual: d,mTconv;n,
FOXP3
+
rTregs; half-open/half-closed square, FOXP3
+
mTregs; N, FOXP3
hi
aTregs.
4648 IL2RA HAPLOTYPE AFFECTS CD4
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cells. This reflects the fact that in addition to IL2RA, many other
genes, including PTPN2 (27), contribute to variation in the sig-
naling pathways that mediate the phosphorylation and dephos-
phorylation of STAT5a in response to IL-2.
Collectively, our results show that Ag-experienced CD4
+
Tconv
and Tregs from individuals with the protective P1P1 IL2RA hap-
lotype showed increased sensitivity to IL-2.
Tregs from individuals with the protective P1P1 IL2RA haplotype
maintained higher levels of FOXP3 in the presence of IL-2
Lymphocytes were gated for CD4
+
CD14
2
expression, examined
for expression of CD25 and CD127, and sorted into regulatory
(CD4
+
CD14
2
CD25
+
CD127
2/lo
) and Tconv populations (non-
Treg gate) as previously described (28) and shown in Fig. 7A.
We then measured the expression of FOXP3 in isolated Tregs both
immediately after flow cytometric sorting and after culture for
48 h in various concentrations of IL-2. Tregs were also stained for
Helios, a member of the Ikaros family of zinc finger transcription
factors that is expressed at high levels in thymic-derived Treg cells
but not peripherally induced Tregs (iTregs) or activated Tconv
(29). An example of the staining is shown in Fig. 7B. Experi-
mental conditions were selected that represent nonsaturating (2 U/
ml) and saturating concentrations (20 U/ml) of IL-2 as determined
in preliminary experiments (Fig. 8A–D). Immediately postsort
FIGURE 6. Relationship between IL2RA haplotype, CD25 expression, and STAT5a phosphorylation in response to IL-2 in T cell subsets identified by
FOXP3-pSTAT5a staining in whole blood. Whole blood from donors with the protective rs12722495 IL2RA haplotype (P1P1) and donors with the sus-
ceptible IL2RA haplotype (SS) was stimulated and analyzed as described in Fig. 5. (A–D) Relationship between IL2RA haplotype and CD25 expression. MFI
of CD25 staining on (A) mTconv, (B) FOXP3
+
rTregs, (C) FOXP3
+
mTregs, and (D) FOXP3
hi
aTregs. (E–H) Relationship between IL2RA haplotype and
pSTAT5a expression. MFI of pSTAT5a on (E) mTconv, (F) FOXP3
+
rTregs, (G) FOXP3
+
mTregs, and (H) FOXP3
hi
aTregs following exposure to IL-2 as
indicated. Each symbol type represents a pair of individuals analyzed on the same day, and individuals are joined by a horizontal line. Results are expressed
as the MFI of pSTAT5a staining for all cells within a given population. Statistical significance was determined using a two-tailed paired Student ttest.
FIGURE 7. Example of the gating used for the iso-
lation of CD4
+
T cell populations for functional studies
and staining of isolated Treg populations to investigate
maintenance of expression of FOXP3 and Helios. (A)
Lymphocytes identified by their forward and side
scatter properties were gated for CD4
+
CD14
2
expres-
sion and then examined for expression of CD25 and
CD127 (inset plot shows isotype control staining).
Tregs were isolated based on CD4
+
CD14
2
CD25
+
CD127
2/lo
.(B) Isolated Tregs were fixed, permea-
bilized, and stained for expression of the transcrip-
tion factors FOXP3 and Helios (dot plot) or relevant
isotype control (density plot).
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there was no significant difference in the proportion of Tregs that
expressed FOXP3 (mean, 80.1 67.8% SD and 79.6 67.9% SD,
respectively) or those that expressed Helios (mean, 73.3 67.4%
SD and 77.6 66.1% SD, respectively) between individuals with
the P1P1 or SS IL2RA haplotype (data not shown). As previously
reported, culture of Tregs in the absence of IL-2 resulted in a de-
crease in both the percentage of cells expressing FOXP3 and the
level of expression. FOXP3 expression can be “rescued” by ad-
dition of IL-2 (22, 24). Our results indicated that FOXP3 main-
tenance under these conditions is significantly dependent on
IL2RA haplotype. We show that Helios
+
Tregs from individuals
with the protective P1P1 IL2RA haplotype expressed significantly
higher levels of FOXP3 under conditions of limiting IL-2 (0 U/ml,
p= 0.03 and 2 U/ml, p= 0.04; Fig. 8E–H). Taken together, these
data indicate that Tregs from individuals with the protective P1P1
IL2RA haplotype maintained higher levels of FOXP3 in the
presence of limiting concentrations of IL-2.
Tregs from individuals with the protective P1P1 IL2RA
haplotype show increased suppression of autologous mTconv
In conventional “Shevach” suppression assays, Tregs from indi-
viduals with the protective P1P1 IL2RA haplotype displayed
higher levels of suppression of autologous mTconv compared with
the SS IL2RA haplotype (Fig. 9A). A similar difference in sup-
pression was also observed at lower ratios of Treg/mTconv in pairs
of individuals who demonstrated a high degree of difference at the
1:1 ratio (Supplemental Fig. 3); however, the difference between
the two groups of pairs did not reach significance at lower ratios
(data not shown). Importantly, the difference in suppression be-
tween P1P1 and SS groups only seen in cocultures containing
mTconv and autologous Tregs but was not seen when a standard
third party population of Tregs was used in the place of autologous
Tregs (Fig. 9B) or when nTconv were used in the place of mTconv
(Fig. 9C). Using data from the present study and from Dendrou
et al. (12), we propose a model in which the rs12722495 IL2RA
susceptibility genotype may influence two distinct immunophe-
notypes: less IL-2 production from mTconv and a reduced sensi-
tivity to IL-2 signaling in Tregs resulting in lower levels of
pSTAT5a and reduced expression levels of FOXP3 (Fig. 10).
Discussion
In the present study we addressed the impact of an IL2RA hap-
lotype associated with autoimmune T1D on Treg fitness and
suppressive function. We showed that the presence of a disease-
associated (SS) IL2RA haplotype leads to diminished IL-2 re-
sponsiveness, resulting in lower levels of FOXP3 expression by
Tregs and a reduction in their ability to suppress proliferation of
autologous mTconv cells. These data offer a rationale that po-
tentially accounts for the molecular and cellular mechanisms
through which polymorphisms in the IL-2RA gene affect immune
regulation, and consequently affect susceptibility to autoimmune
and inflammatory diseases.
Numerous reports have demonstrated that CD4
+
CD25
+
Tregs
from individuals with a range of human autoimmune diseases,
including T1D, are deficient either in their frequency or in their
ability to control autologous proinflammatory responses when
FIGURE 8. Helios and FOXP3 staining in isolated Treg populations cultured with limiting concentrations of IL-2. (A–D) Example of Helios and FOXP3
staining in freshly isolated and cultured Tregs. Tregs were isolated by flow cytometry sorting as described in Fig. 7 and following fixation were stained for
expression of the transcription factors FOXP3 and Helios either immediately postsorting (A) or following culture for 48 h in 0 U/ml IL-2 (B), 2 U/ml IL-2
(C), or 20 U/ml IL-2 (D). Quadrant gates were set based on staining with the relevant isotype controls (99th centile), and FOXP3 MFI reflects FOXP3
staining in the Helios
+
Tregs. (E–H) FOXP3 expression in Helios
+
Tregs under suboptimal IL-2 concentrations. Tregs were isolated from donors with the
protective rs12722495 IL2RA haplotype (P1P1) and donors with the susceptible IL2RA haplotype (SS) and analyzed for expression of FOXP3 and Helios
either immediately postsorting (E) or following culture for 48 h in 0 U/ml IL-2 (F), 2 U/ml IL-2 (G), or 20 U/ml IL-2 (H) as described above. Each symbol
type represents a pair of individuals analyzed on the same day, and individuals are joined by a horizontal line. Statistical significance was determined using
a one-tailed Wilcoxon matched-pairs signed rank test.
4650 IL2RA HAPLOTYPE AFFECTS CD4
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compared with nondiseased, control individuals (5, 6, 30–36).
However, there is a major gap in present knowledge as to how
CD4
+
CD25
+
Treg function relates to the development of human
autoimmune disease in terms of causality. Namely, is Treg dys-
function a primary, causal event or is it a result of alterations in the
immune system due to the disease process? We hypothesized that
if a diabetes susceptibility haplotype could be linked to altered
(decreased) Treg function in individuals with no history of auto-
immune disease, this would support the proposal that Treg dys-
function is causal in T1D. Several genes within the IL-2/IL-2RA
pathway have been identified that influence susceptibility to hu-
man autoimmune diseases (e.g., IL2,IL2RA,IL2RB, and PTPN2)
(11, 37–42), and given the vital importance of IL-2 and IL-2
signaling to the generation and function of CD4
+
CD25
+
FOXP3
+
Tregs, it is likely that that these genes exert their effects by al-
tering Treg frequency or functional ability. In this and our pre-
vious report (12), the diabetes-susceptible IL2RA haplotype
(rs12722495) is shown to confer reduced CD25 expression and
IL-2 secretion by mTconv cells. Although the frequency of Tregs
detectable in the peripheral blood is not influenced by rs12722495,
the ability of Tregs to signal via IL-2 is altered by this polymor-
phism and function is thereby impaired. This constellation of
findings is resonant with observations made in patients with T1D:
the frequency of Treg populations is normal, but Treg function is
reduced (5, 6, 9). Additionally, a number of recent studies show
that patients with T1D have impaired IL-2 signaling and increased
Treg apoptosis (7, 24). This implies that impairment of the IL-2
pathway, through its effect on Treg fitness and immune regulation,
is a key pathway in autoimmune disease pathogenesis.
Binding of IL-2 to its high-affinity receptor complex leads to
a cascade of signaling events, including activation of the Ras/
MAPK, JAK/STAT, and PI3K/Akt pathways. In Tregs a major
cellular consequence of IL-2 signaling is the phosphorylation and
activation of STAT5a, which binds to the FOXP3 promoter, leading
to sustained FOXP3 expression and enhanced suppressive capacity
(19, 21, 43). IL-2 signaling and STAT5a phosphorylation are also
vital for the generation of induced FOXP3
+
Tregs (22). In our
studies, mTconv from individuals with the susceptible IL2RA
FIGURE 9. Percentage suppression of mTconv proliferation by autologous or third party Tregs. Tregs and Tconv were isolated from donors with the
protective rs12722495 IL2RA haplotype (P1P1) and donors with the susceptible IL2RA haplotype (SS). The suppression of proliferation of Tconv by Tregs
was measured by in vitro coculture. Plots shows (A) suppression of mTconv by autologous Tregs, (B) suppression of mTconv by standard third party Tregs,
and (C) suppression of nTconv by autologous Tregs. Each symbol type represents a pair of individuals analyzed on the same day, and individuals are joined
by a horizontal line. Statistical significance was determined using a one-tailed Wilcoxon matched-pairs signed rank test.
FIGURE 10. A model to explain the link be-
tween IL-2 pathway gene polymorphisms and the
development of autoimmunity. Data from the
present study and from Dendrou et al. (12) sug-
gest that the rs12722495 IL2RA susceptibility
genotype may influence two distinct immuno-
phenotypes: less IL-2 production from mTconv
and a reduced sensitivity to IL-2 signaling in
Tregs resulting in lower levels of pSTAT5a and
reduced expression levels of FOXP3. We propose
that that these two immunophenotypes could act
synergistically to reduce Treg function.
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haplotype had a lower level of pSTAT5a in response to IL-2
stimulation. A key question is the extent to which this might
also affect the ability of an individual to respond to immune
regulatory cues and generate iTregs. nTconv (which express lower
levels of CD25 and in whom most IL-2–induced STAT5a phos-
phorylation will occur in a CD25-independent manner) from
IL2RA-susceptible individuals did not show a reduced pSTAT5a
response. However, our in vitro studies did not take into account
the fact that priming of naive T cells in vivo requires TCR ligation,
a process that alters CD25 expression and hence IL-2 respon-
siveness. It remains to be established, therefore, whether lower
levels of IL-2 sensitivity can result in reduced ability to generate
iTregs from naive T cells.
The definitive identification of Tregs by flow cytometry is
challenging, as activated T cells share many of the phenotypic
characteristics of Tregs. Because fixation precluded the use of
CD127 when measuring pSTAT5a in PBMC samples, we adopted
a highly conservative approach to identify CD25
hi
Tregs by gating
on the top 1% of CD25-staining CD4
+
T cells, an approach
adopted by other researchers (7, 25). Gating on an analogous
population in the unfixed samples demonstrated that this pop-
ulation consisted of almost exclusively CD127
2
cells, and we
were therefore confident that the observed difference in STAT5a
phosphorylation is due to a difference in sensitivity to IL-2–me-
diated signaling in Tregs rather than in any contaminating Tconv.
These findings were then confirmed in a separate cohort of indi-
viduals using a whole blood assay that facilitated the simultaneous
detection of FOXP3 and pSTAT5a to allow for more definitive
gating of bona fide Tregs based on expression of FOXP3. Im-
portantly, in both groups of individuals tested, this difference was
only observed under nonsaturating concentrations of IL-2, sug-
gesting that there was not an inherent difference in the ability of
STAT5a to be phosphorylated in individuals with the diabetes-
susceptible IL2RA haplotype, but rather a reduced sensitivity to
IL-2 signaling that is only apparent under conditions of limiting
IL-2.
The decreased STAT5a phosphorylation observed in individ-
uals with the susceptible IL2RA haplotype also translated to a rela-
tive inability to maintain high levels of FOXP3 expression under
limiting conditions of IL-2. Maintained expression of high levels
of FOXP3 are a requirement for sustained suppressive function,
and downregulation of FOXP3 has been associated with a rapid
loss of regulatory function (44, 45). This difference was only
observed in Tregs coexpressing Helios, recently identified as a
transcription factor that is proposed to identify thymic-derived
FOXP3
+
Tregs (nTregs) and distinguish them from those that are
generated in the periphery (iTregs) (29). It is possible that nTregs
and iTregs have different requirements for sustained IL-2 signal-
ing to maintain high levels of FOXP3 and that, at the concen-
trations of IL-2 chosen for this study, a difference could only be
observed in the nTregs.
In the light of these and other studies, a model can be elaborated
to explain the link between the IL-2 pathway gene polymorphisms
and the development of human autoimmunity. This proposes that
diminished Treg function is influenced by the IL-2/IL-2RA sig-
naling pathway via two distinct immunophenotypes: lower levels
of IL-2 production by Tconv and a lower level of responsiveness
to signaling via IL-2 in Tregs, the combined result of which is
impaired immune regulation (Fig. 10). This dual effect may not
grossly affect Treg populations under steady-state conditions (e.g.,
as measured in peripheral blood), but it may only be revealed
under conditions of inflammation, such as those present in the
pancreas and pancreatic lymph nodes during development of T1D
(46). The net effect would be an inability to sustain the suppres-
sive action of activated Tregs and a subsequent failure to suppress
pathogenic autoimmunity.
In summary, we have demonstrated that a disease-susceptible
IL2RA haplotype is associated with reduced Treg fitness and sup-
pressive function in vitro. To our knowledge, this represents the
first demonstration that a gene polymorphism that increases sus-
ceptibility to autoimmune diseases such as T1D is associated
with altered Treg function. Further studies will be required to
determine whether other susceptibility genes also contribute to the
defective Treg function that characterizes individuals with auto-
immunity.
Acknowledgments
We gratefully acknowledge the participation of all Cambridge BioResource
(CBR) volunteers. We thank the staff of the CBR recruitment team for as-
sistance with volunteer recruitment and K. Beer, J. Rice, P. Tagart, and
M. Wiesner for blood sample collection. We thank M. Woodburn and T. Att-
wood for contributing to sample management. We thank members of the
Cambridge BioResource Scientific Advisory Board and management com-
mittee for support and the National Institute for Health Research Cambridge
Biomedical Research Center for funding. We also thank Richard Ellis and
Thomas Hayday for performing flow cytometry cell sorting.
Disclosures
The authors have no financial conflicts of interest.
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