Soluble IL-2RA levels in multiple sclerosis subjects and the effect of soluble IL-2RA on immune responses.
ABSTRACT Multiple sclerosis (MS) is an organ-specific autoimmune disorder that is in part genetically determined. The gene encoding the alpha-chain of the IL-2 receptor, IL2RA, harbors alleles associated with risk to MS and other autoimmune diseases. In addition, IL2RA genetic variants correlate with the levels of a soluble form of the IL-2 receptor in subjects with type 1 diabetes and multiple sclerosis. Here, we show that the IL2RA genotypes differentially affects soluble IL-2RA (sIL-2RA) levels in MS cases vs healthy controls; the two variants associated with MS (rs12722489 and rs2104286) account for 15 and 18% of the total variance in log(10)-transformed sIL-2RA concentration in control subjects but less so in subjects with MS (2 and 5%), suggesting that perturbations associated with disease or treatment may influence sIL-2RA levels in subjects with MS. Whereas analyses demonstrate that sIL-2RA serum concentrations are a remarkably stable phenotype in both healthy controls and untreated MS subjects, a difference is observed between benign and malignant MS. These data indicate that, in addition to specific allelic variants at IL2RA, immunological perturbations associated with aggressive forms of the disease can influence sIL-2RA levels in serum of MS subjects. We also demonstrate, functionally, that sIL-2RA can inhibit IL-2 signaling, yet enhance T cell proliferation and expansion. In summary, we propose that before disease onset, strong genetic factors associated with disease risk dictate sIL-2RA levels that may be further modulated with onset of chronic systemic inflammation associated with MS.
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ABSTRACT: Introduction Adult T-cell leukemia/lymphoma (ATL) responds poorly to conventional chemotherapy, but allogeneic stem cell transplantation (allo-SCT) may improve disease prognosis. Herein, we report a female patient with human T-cell leukemia virus type I (HTLV-I)-associated myelopathy (HAM)-like myelopathy following allo-SCT for ATL. Case report: She developed crural paresis 14 months after allo-SCT. Initially, she was diagnosed with central nervous system (CNS) relapse of ATL and treated with intrathecal injection and whole brain and spine irradiation. Her symptoms recurred 5 months later, when a cerebrospinal fluid (CSF) specimen showed increased CD4 + CXCR3 + CCR4+ cell numbers and levels of neopterin and CXCL10 (IP-10). Discussion These results suggest the possible involvement of a certain immunological mechanism such as HAM in her symptoms, irrespective of the lack of anti-HTLV-I antibody in her CSF. Because a definitive diagnosis of CNS manifestation of ATL is sometimes difficult, multi-modal laboratory data are required for differential diagnosis.SpringerPlus 10/2014; 3:581.
- Molecular Biology 07/2014; 48(4):496-507. · 0.74 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The use of cytokines from the IL-2 family (also called the common γ chain cytokine family) such as Interleukin (IL)-2, IL-7, IL-15, and IL-21 to activate the immune system of cancer patients is one of the most important areas of current cancer immunotherapy research. The infusion of IL-2 at low or high doses for multiple cycles in patients with metastatic melanoma and renal cell carcinoma was the first successful immunotherapy for cancer proving that the immune system could completely eradicate tumor cells under certain conditions. The initial clinical success observed in some IL-2–treated patients encouraged further efforts focused on developing and improving the application of other IL-2 family cytokines (IL-4, IL-7, IL-9, IL-15, and IL-21) that have unique biological effects playing important roles in the development, proliferation, and function of specific subsets of lymphocytes at different stages of differentiation with some overlapping effects with IL-2. IL-7, IL-15, and IL-21, as well as mutant forms or variants of IL-2, are now also being actively pursued in the clinic with some measured early successes. In this review, we summarize the current knowledge on the biology of the IL-2 cytokine family focusing on IL-2, IL-15 and IL-21. We discuss the similarities and differences between the signaling pathways mediated by these cytokines and their immunomodulatory effects on different subsets of immune cells. Current clinical application of IL-2, IL-15 and IL-21 either as single agents or in combination with other biological agents and the limitation and potential drawbacks of these cytokines for cancer immunotherapy are also described. Lastly, we discuss the future direction of research on these cytokines, such as the development of new cytokine mutants and variants for improving cytokine-based immunotherapy through differential binding to specific receptor subunits.Cytokine & Growth Factor Reviews 08/2014; · 6.54 Impact Factor
Soluble IL-2RA Levels in Multiple Sclerosis Subjects and the
Effect of Soluble IL-2RA on Immune Responses1
Lisa M. Maier,*†David E. Anderson,* Christopher A. Severson,* Clare Baecher-Allan,*
Brian Healy,*‡David V. Liu,§K. Dane Wittrup,§¶Philip L. De Jager,*†?and David A. Hafler2*†
Multiple sclerosis (MS) is an organ-specific autoimmune disorder that is in part genetically determined. The gene encoding the
?-chain of the IL-2 receptor, IL2RA, harbors alleles associated with risk to MS and other autoimmune diseases. In addition, IL2RA
genetic variants correlate with the levels of a soluble form of the IL-2 receptor in subjects with type 1 diabetes and multiple
sclerosis. Here, we show that the IL2RA genotypes differentially affects soluble IL-2RA (sIL-2RA) levels in MS cases vs healthy
controls; the two variants associated with MS (rs12722489 and rs2104286) account for 15 and 18% of the total variance in
log10-transformed sIL-2RA concentration in control subjects but less so in subjects with MS (2 and 5%), suggesting that pertur-
bations associated with disease or treatment may influence sIL-2RA levels in subjects with MS. Whereas analyses demonstrate that
sIL-2RA serum concentrations are a remarkably stable phenotype in both healthy controls and untreated MS subjects, a difference
is observed between benign and malignant MS. These data indicate that, in addition to specific allelic variants at IL2RA, immu-
nological perturbations associated with aggressive forms of the disease can influence sIL-2RA levels in serum of MS subjects. We
also demonstrate, functionally, that sIL-2RA can inhibit IL-2 signaling, yet enhance T cell proliferation and expansion. In sum-
mary, we propose that before disease onset, strong genetic factors associated with disease risk dictate sIL-2RA levels that may be
further modulated with onset of chronic systemic inflammation associated with MS. The Journal of Immunology, 2009, 182:
infiltrates result in demyelination and loss of neurological function
(1). There is systemic activation of the adaptive immune system
characterized by activated T cells and of the innate system with
activated dendritic cells (2). Blocking T cell traffic from the cir-
culation into the CNS with anti-VLA-4 mAbs has a strong effect
on the frequency of acute neurological events (3), which occur
unpredictably in a remitting-relapsing pattern. In the peripheral
circulation, increased levels of cytokine receptors, including the
ultiple sclerosis (MS)3is an organ-specific autoim-
mune disorder characterized by chronic inflammation
of CNS myelin where focal T cell and macrophage
IL-2R ?-chain, have been observed (4, 5). Clinically, resolution of
acute attacks or relapses is often followed by recurrent attacks and,
over time, about one-half of the relapsing-remitting subjects will
enter the progressive stage of the disease. This progressive phase
may involve more degenerative rather than immunological
changes, with a substantial degree of axonal degeneration present
within the CNS (6).
Susceptibility to MS is conferred by genetic as well as largely
unknown environmental factors (1, 7). The sequencing of the hu-
man genome has allowed large-scale whole genome association
scans aiming at identifying risk alleles associated with risk to de-
veloping autoimmune disease. We identified single nucleotide
polymorphisms (SNP) within genes encoding the IL-2R? (IL2RA),
IL-7R? (IL7RA), CD58 (CD58) as well as other nonimmune re-
lated genes (8). Of particular interest is the overlap among auto-
immune diseases at some of these loci; for example, at IL2RA, a
number of SNPs also contribute to risk to type 1 diabetes (T1D) (9)
and Graves’ disease (10), indicating that some pathways are shared
among autoimmune disease. In the IL2RA locus, variants not only
associate with disease risk but also influence the levels of sIL-2RA
in the serum (9).
The IL-2/IL-2RA(CD25) pathway plays an essential role in reg-
ulating immune responses (11). IL-2 is central for both expansion
and apoptosis of T cells and soluble IL-2RA (sIL-2RA) binds IL-2
at a similarly low affinity as full-length IL-2RA (12). Mitogen- and
Ag-activated leukocytes release sIL-2RA into culture supernatants
(13). All or part of this release of sIL-2RA from activated immune
cells involves proteolytic cleavage, and thus far, an mRNA encod-
ing this isoform has not been identified. High concentrations of
soluble IL-2RA chain are found in sera from healthy subjects but
are elevated in subjects with autoimmune disease, inflammation,
and infection (4, 5, 14–17), which is why sIL-2RA is thus con-
sidered to be a biomarker for immune activation in the peripheral
blood (12),. sIL-2RA, like many other soluble cytokine receptors
*Division of Molecular Immunology, Center for Neurologic Diseases, Department of
Neurology, Brigham and Women’s Hospital and Harvard Medical School, Boston,
MA 02115;†Program in Medical and Population Genetics, Broad Institute, Massa-
chusetts Institute of Technology and Harvard University, Cambridge, MA 02139;
‡Biostatistics Center, Massachusetts General Hospital, Boston, MA 02115;§Depart-
ment of Chemical Engineering and¶Department of Biological Engineering, Massa-
chusetts Institute of Technology, Cambridge, MA 02139; and?Partners Center for
Personalized Medicine, Boston, MA 02115
Received for publication July 28, 2008. Accepted for publication November 19, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was funded by the National Multiple Sclerosis Society, the National
Institutes of Health-National Institute of Allergy and Infectious Diseases (P01
AI39671) and the National Institutes of Health (R01 NS049477). Further support was
provided by a JDRF Postdoctoral Fellowship (to L.M.M.), a grant by the American
Cancer Society (to D.E.A.), a Harry Weaver Neuroscience Scholar award by the
National Multiple Sclerosis Society (to P.L.D.), and Jacob Javits Merit Award
NS2427 (to D.A.H.) from the National Institute of Neurological Disorders and Stroke.
2Address correspondence and reprint requests to Dr. Lisa M. Maier, Center for Neu-
rologic Diseases, New Research Building, 77 Louis Pasteur Ave., Boston, MA 02115.
E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: MS, multiple sclerosis; SNP, single nucleotide
polymorphism; T1D, type 1 diabetes; sIL-2RA, soluble IL-2RA; EDSS, Expanded
Disability Status Scale; AICD, activation-induced cell death.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
(18), can compete with cell surface IL-2RA for IL-2 binding and
thus block IL-2 function, a hypothesis supported by a recent report
that sIL-2RA inhibits the proliferative response of a murine CD8?
T cell line cultured in the presence of high doses of murine IL-2
(19). Although less likely, sIL-2RA might alternately complex
with IL-2 and potentiate signaling, as has been demonstrated for
sIL-6R-IL-6 (20) and sIL-15R-IL-15 complexes (19). The IL-2-
IL-2RA system is also central for the production and function of
conventional and regulatory T cells, which are critical for main-
tenance of immunological self tolerance and prevention of auto-
reactive processes (21). Thus, there are a variety of mechanisms by
which sIL-2RA may influence T cell function, but its impact on the
function of human T cells is presently unclear.
Here, we have characterized the levels of sIL-2RA in the serum
of healthy controls and subjects with different MS subtypes and
have examined the extent to which the sIL-2RA serum levels are
related to the allelic variants that have thus far been associated
with MS susceptibility. In addition, we provide in vitro evidence
that the levels of sIL-2RA affect human T cell function, including
both effector and regulatory T cells.
Materials and Methods
Healthy controls, subject cohorts, and disease definitions
Healthy control and MS subject samples were obtained through the Part-
ners Healthcare MS Center (Boston, MA) as part of its MS Registry
project, which is approved by the Partners Healthcare institutional review
board. All subjects were older than 18 years of age and met criteria of
either MS per the revised McDonald diagnostic criteria (22), or clinically
isolated syndrome as defined by a history of a single episode of inflam-
matory demyelination documented by a neurologist, lack of evidence for
alternative diagnoses, and two or more periventricular or ovoid hyperin-
tense T2 lesions of ?3 mm on magnetic resonance imaging (23). Untreated
subjects are defined as having no disease-modifying treatment or steroids
in the preceding 4 wk (steroids), 12 wk (glatiramer acetate, IFN-?1a or
-?1b, and methotrexate) or 24 wk (Cytoxan and mitoxantrone). Progressive
disease is defined as progressive functional decline with deficits lasting 6
mo or more.
Disease subtype definitions. Primary progressive disease is defined as dis-
ease progression from onset without a history of an initiating clinical at-
tack. Secondary progressive disease is defined as an initially relapsing-
remitting course followed by gradual progression with or without
occasional relapses, minor remission, and plateaus. Relapsing-remitting
disease is defined as disease characterized by clearly defined relapses with
full recovery or with sequelae and residual deficit upon recovery. Periods
between disease relapses are characterized by a lack of disease progression.
Clinically isolated syndrome is defined as a single demyelinating event
with a positive magnetic resonance imaging (two or more lesions ?3 mm
in diameter and characteristic of MS (e.g., periventricular or ovoid).
Definition of benign and malignant disease course. All subjects have a
remitting-relapsing course. Benign subjects are defined as having an Ex-
panded Disability Status Scale (EDSS) of ?1, excluding visual function,
10–15 years after first symptom or EDSS of ?2, excluding visual function,
?15 years after first symptom. Malignant subjects are defined as having an
EDSS of ?6 within 5 years of first symptom.
sIL-2RA measurement using ELISA
ELISA measurement of sIL-2RA was performed according to the manu-
facturer’s recommendations (BD Biosciences). Serum samples were di-
luted 1/20 using PBS supplemented with 10% FBS. Microtiter plates were
read using a Bio-Rad Benchmark microplate reader.
pSTAT5 phosphorylation analysis in ex vivo CD4?FoxP3?
Phosphorylation-state analysis was performed on human whole blood us-
ing BD Phosflow technology according to the manufacturer’s instructions
(BD Biosciences), and as previously described (24). All human blood sam-
ples were obtained with informed consent and according to the Institutional
Ethics Review Board Protocols. All blood samples were collected in sterile
10-ml lithium-heparin Monoject tubes. Four milliliters of fresh, ex vivo
blood from healthy control donors were used per condition and time point.
Blood samples were incubated with IL-2 (Proleukin; Chiron) or with a
mixture of IL-2 and sIL-2RA (R&D Systems) in 50-ml polypropylene
Falcon conical tubes for 30 min in a 37°C water bath. The neutralizing
anti-IL2 mAb was purchased from R&D Systems (clone 5334). Fixation of
cells and preservation of phosphorylation status were obtained by adding
prewarmed BD Lyse/Fix buffer and incubation in a 37°C water bath. Per-
meabilization of cells was performed by incubation of cells in BD Perm
Buffer III on ice for 30 min. Cells were subsequently washed twice with
2% FBS-PBS and stained using BD Staining Buffer (all reagents from BD
Biosciences). Cells were stained using allophycocyanin mouse anti-human
CD4 (clone RPA-T4; BD Biosciences), PE anti-human FoxP3 (clone
206D; Biolegend), and Alexa Fluor-488 mouse anti-human pSTAT5
(pY694; clone 47; BD Biosciences).
PBMCs and T cell isolation
PBMCs were isolated from heparinized venous blood by centrifugation
over Ficoll-Hypaque (Amersham Pharmacia) according to standard meth-
odologies. CD4?T cells, isolated by negative selection using immuno-
magnetic beads (Miltenyi Biotec) from PBMCs, were FACS sorted on a
FACS ARIA (BD Biosciences) after staining for HLA-DR (PerCP, clone
L243), CD62L (allophycocyanin; clone Dreg 56), CD32 (FITC; clone
3D3), CD14 (FITC; clone M5E2), and CD116 (FITC; clone M5D12), all
from BD Pharmingen; and CD25 (peripheral blood; clone BC96 from Bio-
Legend) to typically ?98% purity in post-sort analysis. The FITC-labeled
mAbs were used as a combined mixture to ensure that no accessory cells
A, Longitudinal sIL-2RA measurements in healthy control subjects. Sam-
ples were obtained over a period of 12 mo. B, Longitudinal sIL-2RA mea-
surements in MS subjects with relapsing-remitting disease course. Samples
were obtained over a maximum period of 3 years. C, sIL-2RA measure-
ment in 68 healthy control subjects vs 284 MS subjects. Difference be-
tween means was tested using a two-sample t test with unequal variance.
A summary of demographic characteristics of study participants and stor-
age duration of samples is given in Table I. The difference remained sig-
nificant (p ? 0.0001) after adjusting for age, gender, and sample storage
duration (data not shown).
sIL-2RA serum levels in healthy controls and MS subjects.
1542sIL-2RA IN MS AND EFFECT ON IMMUNE RESPONSES
were isolated in the target T cell population (CD4?DR?CD25?
Cells were grown in serum-free X-VIVO15 medium (BioWhittaker).
PBMCs, CD4?T cells, CD8?T cells, and monocytes were plated at
100,000 cells/well. CD4?DR?CD25?CD62Lhigh(Tresp) cells were plated
at 2.5 ? 103/well. T cell proliferation was assessed at 72 h by [3H]TdR
incorporation (1 ?Ci/well) on a beta scintillation counter. Activation-in-
duced cell death (AICD) was measured by annexin V staining using the
annexin V-PE apoptosis detection kit following the manufacturer’s instruc-
tions (BD Pharmingen). Control Ig was purchased from R&D Systems.
Differences between means were tested using two-sample t tests or
ANOVA, and adjustment for confound was completed using linear regres-
sion. When appropriate, a paired t test was used. sIL-2RA concentrations
were log10transformed before analysis. Values of p ? 0.05 were consid-
Serum sIL-2RA levels in healthy controls and subjects with MS
We analyzed sIL-2RA levels from 14 healthy control individuals,
for which longitudinal samples taken over a period of 12 mo were
available. As shown in Fig. 1A, sIL-2RA level is a remarkably
stable phenotype in healthy controls. We extended this analysis to
15 untreated MS subjects with a relapsing-remitting disease
course. Between 4 and 10 serum samples were available for this
collection and were obtained over a maximum period of 3 years.
Little fluctuation in sIL-2RA levels was observed in the longitu-
dinal measurements of individual subjects with MS (Fig. 1B).
However, some of the subjects with MS experienced marked tran-
sient elevations in sIL-2RA levels. Some of these elevations cor-
related with clinical or radiological evidence of disease exacerba-
tions (data not shown). However, given the small number of such
events, it is impossible to draw any conclusions as to the signifi-
cance of these correlations. Furthermore, the subjects with relaps-
ing-remitting disease course tested in this longitudinal study ex-
hibited active disease, with multiple relapses a year.
Next, in a collection of 68 healthy controls and 264 MS subjects,
we quantified serum concentrations of sIL-2RA. Consistent with
previous findings (4, 5, 17), we found that sIL-2RA levels are
increased in MS subjects compared with healthy controls (mean
sIL-2RA concentration in healthy controls, 2.022 ng/ml and 95%
confidence interval, 1.852–2.192; mean sIL-2RA concentrations in
MS subjects, 2.345 ng/ml and 95% confidence interval, 2.256–
2.435; p ? 0.9 ? 10?4; Fig. 1C). We determined the percentage
of the observed variance in sIL-2RA levels that is due to the ge-
notypes we have discovered in subjects with MS. For the two
ease subtype. A, Mean levels and 95% confidence intervals of sIL-2RA
levels measured in subjects with primary progressive (PP), secondary pro-
gressive (SP), and untreated relapsing-remitting (RR) disease course as
well as in untreated subjects with clinically isolated syndrome (CIS). Dif-
ferences among groups were tested using an F test (p ? 0.05). After ad-
justing for age, gender, and sample storage duration, similar results were
obtained (p ? 0.084). B, Mean levels and 95% confidence intervals of
sIL-2RA levels measured in MS subjects with benign and malignant dis-
ease. Difference between means was tested using a two-sample t test with
unequal variance. The difference remained significant (p ? 0.03) after ad-
justing for age, gender, and sample storage duration. Disease definitions are
provided in Materials and Methods. A summary of demographic charac-
teristics of study participants and storage duration of samples is given in
Table I. There is no overlap between the MS subjects tested in A and B.
sIL-2RA serum levels in MS subjects categorized by dis-
els for treatment is the average of repeat measures (up to nine repeat sam-
pling points) over a period of up to 4 years. A paired t test was used to test
for a difference between the untreated value and the average treatment
value. Treatment definitions are provided in Materials and Methods.
MS treatment does not affect sIL-2RA levels. sIL-2RA lev-
Table I. Summary of demographic characteristics of study participants and storage duration of serum samples
Study Population Females (%)Males (%) Mean Age (yr) at Time of SampleMean Sample Storage Duration (yr)
Benign MS subjects
Malignant MS subjects
aNumbers in parentheses, range.
bCIS, clinically isolated syndrome.
1543The Journal of Immunology
genetic variants that we have previously correlated with sIL-2RA
levels, between 2 and 5% of the variance observed in the MS cases
was due to IL-2RA chain genotype, whereas in the healthy control
subjects, between 15 and 18% of the variance observed was due to
the genotype. This led us to hypothesize that perturbations asso-
ciated with disease or treatment may influence sIL-2RA levels in
the MS cases because less of the variance in sIL-2RA levels could
be explained by IL2RA variants in the MS subject collection than
in the healthy control collection.
Severe MS disease course influences sIL-2RA levels, but
treatment does not
To explore the role of clinical events in sIL-2RA expression, we
studied a new set of subjects with MS; these individuals were not
used in our prior genetic analysis of sIL-2RA levels. Specifically,
sIL-2RA levels were quantified in 22 MS subjects with primary
progressive disease, 44 MS subjects with secondary progressive
disease, and 83 untreated MS subjects with a relapsing-remit-
ting disease course along with 60 untreated subjects with clin-
ically isolated demyelinating syndrome (subjects with a single
episode of demyelination; Table I). No significant differences
among the four groups were observed (p ? 0.13; Fig. 2A). We
then studied additional subjects with MS representing extreme
forms of the disease, namely, 115 subjects with benign MS and
60 subjects with malignant MS. sIL-2RA levels were increased
in subjects with the malignant form of remitting-relapsing MS
(p ? 0.027; Fig. 2B).
We hypothesized that treatment of MS subjects may lower
sIL-2RA levels. We thus measured sIL-2RA levels in a cohort
of 15 subjects with MS for which pretreatment sera and up to 9
posttreatment serum samples obtained over a period of up to 4
years were available. No significant, prolonged effect of treat-
ment was observed in this longitudinal study (Fig. 3 and Sup-
plemental Fig. 1).4Several patients experienced treatment
changes over the course of the study period and/or received com-
bination therapies. The effect of any specific treatment regimen on
serum sIL-2RA levels will need to be investigated using larger
sample sizes. In contrast to healthy controls or untreated MS pa-
tients, there was somewhat greater variability in sIL-2RA levels
over time, which may be due to transient perturbation, disease
activity, or a combination of these factors.
sIL-2RA inhibits IL-2-mediated signaling
We then explored a potential functional role for the sIL-2RA
molecule given that little is known regarding the relevance of
increased sIL-2RA levels in the pathogenesis of autoimmunity.
sIL-2RA, like many other soluble cytokine receptors (18),
might compete with cell surface IL-2RA for IL-2 binding and
thus block IL-2 function. Alternately, sIL-2RA might complex
with IL-2 and potentiate signaling, as has been demonstrated for
sIL-6R-IL-6 (20) and sIL-15R-IL-15 complexes (19). However,
the reported inhibition of proliferation of a murine CD8?T cell
line cultured in the presence of high doses of murine IL-2 and
human sIL-2RA (19) makes this alternative hypothesis less
4The online version of this article contains supplemental material.
CD4?FoxP3?T cells obtained from fresh ex vivo blood from a healthy human subject. Background levels (black histogram), induction of phosphorylation
following stimulation with 100 IU of IL-2/ml (white histogram), as well as the effect of sIL-2RA addition (gray histogram) are shown. Stimulation with
IL-2 occurred for 30 min at 37°C. Results are representative of three independent experiments. C, sIL-2RA, complexed with an IL-2 antagonist, can reverse
inhibition of IL-2 signaling by an IL-2 antagonist (mutated IL-2). FACS histograms showing pSTAT5 (Y694) phosphorylation in CD4?FoxP3?in fresh
ex vivo blood obtained from a healthy human subject. Background levels (black histogram) and induction of phosphorylation following stimulation with
10 IU of IL-2/ml (4 pM, open histogram) are shown as well as various experimental conditions with sIL-2RA and an IL-2 antagonist (mutated IL-2 V91R5).
Mean fluorescence intensity values are: 10 IU IL-2 (39.6); IL-2 plus 1? IL-2 V91R (8.5); IL-2 plus 1:1 sIL-2RA-IL-2 V891R (30.8); unstimulated (5.9).
Stimulation with IL-2 occurred for 30 min at 37°C. A 1:1 sIL-2RA:IL-2 (V91R) ratio means that sIL-2RA and IL-2 (V91R) were added in equimolar
amounts (400 pM) and preincubated for 30 min at 37°C before addition to whole blood with IL-2. The addition of 1? IL-2 (V91) equals to the addition
of 400 pM. Results are representative of two independent experiments. D, For comparison with the effect of sIL-2RA on pSTAT5 signaling, the inhibition
of pSTAT5 signaling by a neutralizing anti (?)-IL-2 mAb is shown.
sIL-2RA inhibits IL-2-mediated signaling. A and B, FACS histograms showing pSTAT5 (Y694) phosphorylation in CD4?FoxP3?and
1544sIL-2RA IN MS AND EFFECT ON IMMUNE RESPONSES
We studied the effect of sIL-2RA on IL-2 signaling in human
T cells. Because phosphorylation of signal transducer and ac-
tivator of transcription 5 (STAT5) is strongly induced upon T
cell activation with IL-2 (25), we measured STAT5 phosphor-
ylation upon stimulation with IL-2 in ex vivo human CD4?T
cells. We used a flow cytometry-based phosphorylation assay
(24) to detect STAT5 phosphorylation in response to IL-2 stim-
ulation of T cells. As shown in Fig. 4A, the addition of sIL-2RA
did not induce signaling in CD4?FoxP3?T responder cells or
CD4?FoxP3?regulatory T cells, which are characterized by
high levels of IL-2RA cell surface expression. This indicated
that sIL-2RA by itself does not exert agonistic function. In con-
trast, the addition of sIL-2RA strongly inhibited STAT5 phos-
phorylation in CD4?FoxP3?T cells and moderately in
CD4?FoxP3?T cells (Fig. 4B). This result suggests that sIL-
2RA is an IL-2 antagonist.
To examine whether sIL-2RA antagonism of STAT5 phos-
phorylation induced by IL-2 is due to the specific interaction of
sIL-2RA, we used an IL-2 analog with a valine to arginine
substitution at position 91. This analog has been shown to bind
the IL-2 receptor without transducing signaling, effectively act-
ing as an antagonist (34). We reasoned that by preincubating
sIL-2RA with the engineered IL-2 analog at equimolar concen-
trations, we could selectively neutralize the IL-2 binding ca-
pacity of sIL-2RA. In Fig. 4C, we demonstrate that the ability
of sIL-2RA to antagonize STAT5 phosphorylation (Fig. 4B) is
lost when it is preincubated with the IL-2 analog. Furthermore,
the analog by itself antagonizes STAT5 phosphorylation (Fig.
4C) Taken together, these data support the theory that sIL-2RA
acts as an antagonist of IL-2 signaling through its ability to bind
IL-2. A neutralizing anti-IL-2 mAb inhibited pSTAT5 signaling
in a similar fashion to IL-2RA (Fig. 4D).
sIL-2RA promotes T cell activation and expansion
We have confirmed the observation by others that many cell types
in the peripheral blood (T cells, monocytes, B cells) produce sIL-
2RA and that the production of sIL-2RA is positively correlated
with activation and proliferative responses in vitro (data not
shown). To study the influence of sIL-2RA on T cell activation and
expansion, sIL-2RA was added to anti-CD3-stimulated PBMCs,
and T cell proliferation was measured after 72 h. We observed
increases in proliferation in the presence of sIL-2RA at all doses of
anti-CD3 stimulation (Fig. 5A). Analysis of eight healthy donors at
an optimal amount of anti-CD3 mAb (0.5 ?g/ml) demonstrated a
modest yet consistent increase in proliferation (p ? 0.01 using a
paired, two-tailed t test). Elevated levels of IL-2 can potentiate
AICD (26), and we speculated that increased sIL-2RA levels might
inhibit AICD by reducing levels of IL-2. To test this hypothesis,
weused FACSto obtain
(CD4?DR?CD25?CD62Lhigh) T cells and then evaluated the in-
fluence of sIL-2RA on T cell expansion. Consistent with short-
term (3 day) effects of sIL-2RA on proliferative response, we
found that T cell numbers were markedly elevated at day 14 in the
presence of sIL-2RA (Fig. 5, B and C), although we did not ob-
serve a significant difference in AICD as measured by annexin V
staining (Fig. 5D).
Here, we characterized the levels of a biomarker of peripheral
inflammation, sIL-2RA, in healthy controls and subjects with MS.
It has been previously shown that genetic variants in the IL2RA
locus correlate with the levels of sIL-2RA (9). Here we show that
the IL2RA genotype more strongly affects sIL-2RA levels in
human T cell function. A, Total
PBMCs were stimulated for 72 h with
a range of plate-bound anti (?)-CD3
concentrations. sIL-2RA was added
at a concentration of 5 ng/ml. Error
bars represent SD in proliferation.
Results are representative of eight in-
dependent experiments. B, FACS-
sorted DR?CD25?CD62LhighT cells
under light microscope on day 14 of
culture. ?20. Cells were stimulated
beads and received either no treat-
ment or 10 ng/ml sIL-2RA. C, FACS-
T cells grown in culture for 14 days.
Cells were stimulated with anti-CD3-
anti-CD28-conjugated beads. Error
bars represent SEM in cell number.
Results are representative of three in-
dependent experiments. D, The addi-
tion of sIL-2RA does not reduce ac-
tivation-induced cell death compared
with addition of a control Ig (R&D
Systems), as measured by the per-
centage of annexin V?CD4?CD25?
T cells. CD4?CD25?T cells were
negatively isolated using magnetic
beads (Miltenyi Biotec) from PBMCs
and cultured for up to 11 days. Data
are representative of two independent
Effect of sIL-2RA on
1545 The Journal of Immunology
healthy control subjects than in subjects with MS. Although lon-
gitudinal analyses demonstrated that sIL-2RA serum concentra-
tions were a remarkably stable phenotype in both healthy controls
and untreated MS subjects, a difference was observed between
benign and malignant MS, indicating that in addition to specific
allelic variants at IL2RA, immunological perturbations associated
with extreme disease course can influence sIL-2RA levels.
Whereas we observed that sIL-2RA at concentrations observed in
sera can enhance the expansion and proliferation of CD4?T cells
cultured exvivo, sIL-2RA
CD4?FoxP3?T cells as measured by STAT5 phosphorylation.
These data indicate that before disease onset, strong genetic factors
associated with disease risk dictate sIL-2RA levels that are likely
to modulate disease onset, potentially affecting the function of
multiple lymphocyte subsets.
Our data agree with previous reports of increased levels of sIL-
2RA in MS subjects compared with controls (4, 5, 17). In our
longitudinal analyses, sIL-2RA serum concentrations were found
to be a remarkably stable phenotype in both healthy controls and
untreated MS subjects with relapsing-remitting disease course.
These data are contrary to what was previously observed in a small
study of 60 MS subjects with relapsing-remitting MS and 33
healthy controls, which showed fluctuations over time (27). Al-
though no differences were observed in our collection of serum
samples from subjects classified into the more commonly used
categories of primary progressive, secondary progressive, and re-
lapsing-remitting MS and clinically isolated syndrome, future
studies with larger sample sizes might show subtle differences. We
extended our study to subjects with different forms of MS and
show a difference between the two extreme forms of MS, benign
and malignant. These data indicate that in addition to specific al-
lelic variants at IL2RA, extreme disease course may influence sIL-
2RA levels in the serum of MS subjects.
A significant fraction of the variance in sIL-2RA is explained by
the IL2RA variants tested thus far in healthy controls: the two
rs2104286) account for 15 and 18% of the total variance in log10-
transformed sIL-2RA concentration in our healthy control collec-
tion, whereas only 2 and 5% of the variance are explained in sub-
jects with MS. This is comparable with findings in a sample of
1351 T1D case plasma samples, where the 2 IL2RA variants
(rs41295061 and rs11594656) accounted for 2.3 and 6.6%, respec-
tively (9). In this large sample of T1D case samples, the effect of
various covariates (including subject age, disease duration, collec-
tion month of sample) on sIL-2RA plasma concentration was also
assessed. The studied covariates were found to account for 11.3%
of the total variance in log10sIL-2RA concentrations (9). Thus, the
contribution of other factors, including additional genetic variants
in immune response genes, will need to be determined.
Upon activation and entry into cell cycle the IL-2RA molecule
is proteolytically cleaved off the surface of many cell types, in-
cluding T cells, B cells, and monocytes (13, 28–30). However, it
is as yet unclear how different cell types respond to human sIL-
2RA and what its potential immunomodulatory role may be. Our
group firstdemonstrated that
CD4?CD25highregulatory T cells is normal, the cells are dysfunc-
tional in subjects with MS (31). Given the importance of IL-2 to
regulatory T cell function and the potential for sIL-2RA to neu-
tralize available IL-2, we hypothesized that genetic variants at
IL2RA that correlate with sIL-2RA may act by modulating levels
of IL-2 and thereby affect suppressor cell function and thereby
inflammatory disease. We have demonstrated that sIL-2RA can
enhance the proliferation/expansion of responder CD4?T cells
(Fig. 5, A–C). Recent findings suggest an autoregulatory feedback
althoughthe frequency of
loop for IL-2, in which IL-2 inhibits its own production (32, 33).
Thus, the level of sIL-2RA may affect this autoinhibitory loop and
hence IL-2 production. Moreover, it should be recognized that al-
terations in concentration or kinetics of production of sIL-2RA
might affect both responder and regulatory T cells in the context of
an autoimmune disease. In this regard, we observed an inhibitory
effect of sIL-2RA on IL-2 signaling by CD4?FoxP3?T cells as
measured by STAT5 phosphorylation.
As yet, three associations for T1D susceptibility that are inde-
pendent of each other and two independent associations for MS
susceptibility have been discovered. Despite clear associations and
correlations between genetic variants in IL2RA and both autoim-
mune disease susceptibility (T1D, MS and Graves’ disease; Refs.
8–10) and sIL-2RA levels (9), correlating disease susceptibility
and sIL-2RA levels is not trivial. In the original report of corre-
lations between sIL-2RA levels and T1D risk alleles (9), the SNP
with the strongest association to T1D risk was not the SNP with
the strongest correlation to sIL-2RA. Moreover, it was suggested
that T1D risk alleles correlate with reduced sIL-2RA levels (9). In
contrast, we have shown that the MS risk alleles correlate with
increased levels.6Indeed, a combined genetic and phenotypic anal-
ysis of T1D and MS alleles indicates that a novel IL2RA associ-
ation with T1D harbors a risk allele that correlates with increased
rather than decreased sIL-2RA levels).6These data also emphasize
the value of comparative analysis of susceptibility loci in multiple
autoimmune diseases. Thus, although many autoimmune diseases
may share common susceptibility loci, it is apparent now that there
may be both allelic and phenotypic heterogeneity within these loci
among these diseases, including IL2RA (35). These data indicate
that the net level of sIL-2RA could be determined by several vari-
ants at IL2RA and that susceptibility to autoimmunity at the IL2RA
locus will be defined by a complex interplay of many variants.
Significantly, although IL2RA genotype is associated with several
autoimmune diseases, the extent to which sIL-2RA influences, and
is influenced by, these disease processes may differ.
Future large-scale studies will need to address the overlap and
relationship between variants associated with disease risk and cor-
related with sIL-2RA levels. Even though we demonstrate a func-
tional role of sIL-2RA on immune responses, sIL-2RA may not be
the primary causal factor regulated by IL2RA genetic variants:
given that sIL-2RA is a cleavage product of surface IL-2RA, an
individual’s sIL-2RA level may simply reflect of the individual’s
surface expression of IL-2RA or ability to up-regulate IL-2RA
Sample collection in the MS Registry project was supported by a collab-
oration with Millenium Pharmaceuticals. We thank Ms. Mira Weiner for
her work in subject recruitment and sample collection.
The authors have no financial conflict of interest.
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