Homeostatic imbalance of regulatory and effector T

cells due to IL-2 deprivation amplifies murine lupus

Jens Y. Humricha,1, Henner Morbachb, Reinmar Undeutscha, Philipp Engharda, Stefan Rosenbergera, Olivia Weigerta,
Lutz Klokea, Juliane Heimanna, Timo Gabera, Susan Brandenburgc, Alexander Scheffoldc, Jochen Huehna,d,
Andreas Radbruchc, Gerd-Rüdiger Burmestera, and Gabriela Riemekastena
a
Department of Rheumatology and Clinical Immunology, University Hospital Charité, 10117 Berlin, Germany; bPediatric Rheumatology, Immunology, and
Infectious Diseases, Children’s Hospital, University of Wuerzburg, 97080 Wuerzburg, Germany; cGerman Rheumatism Research Center, 10117 Berlin, Germany;
and dHelmholtz-Zentrum für Infektionsforschung GmbH, 38124 Braunschweig, Germany

Edited by Christophe Benoist, Harvard Medical School, Boston, MA, and approved November 17, 2009 (received for review March 24, 2009)

The origins and consequences of a regulatory T cell (Treg) disorder
in systemic lupus erythematosus (SLE) are poorly understood. In
the (NZBxNZW) F1 mouse model of lupus, we found that
CD4+Foxp3+ Treg failed to maintain a competitive pool size in
the peripheral lymphoid organs resulting in a progressive homeo-
static imbalance of CD4+Foxp3+ Treg and CD4+Foxp3− convention-
al T cells (Tcon). In addition, Treg acquired phenotypic changes
that are reminiscent of IL-2 deficiency concomitantly to a progres-
sive decline in IL-2-producing Tcon and an increase in activated,
IFN-γ-producing effector Tcon. Nonetheless, Treg from lupus-
prone mice were functionally intact and capable to influence the
course of disease. Systemic reduction of IL-2 levels early in disease
promoted Tcon hyperactivity, induced the imbalance of Treg and
effector Tcon, and strongly accelerated disease progression. In
contrast, administration of IL-2 partially restored the balance of
Treg and effector Tcon by promoting the homeostatic proliferation
of endogenous Treg and impeded the progression of established
disease. Thus, an acquired and self-amplifying disruption of the
Treg-IL-2 axis contributed essentially to Tcon hyperactivity and
the development of murine lupus. The reversibility of this homeo-
static Treg disorder provides promising approaches for the treat-
ment of SLE.
homeostasis | immunotherapy | interleukin-2 | SLE | autoimmunity
egulatory CD4+ T cells (Treg) that express the transcription
R factor Foxp3 are crucial for the maintenance of immuno-
logical tolerance to self (1, 2). Predominantly derived from a
distinct T cell subpopulation in the thymus, CD4+Foxp3+ Treg
principally recognize self-antigens and are required to control
the expansion of self-reactive T cells in the peripheral lymphoid
organs (2, 3). In view of that, there is increasing evidence that
numeric or functional Treg deficiencies are associated with
particular autoimmune diseases, suggesting a contribution of a
Treg dysfunction to disease development (4).
The cytokine IL-2 was initially identified as a potent T cell
growth factor (5). However, more recent data strongly indicate
that IL-2 is essential for immune tolerance (5). Accordingly,
mice deficient in IL-2 or IL-2 receptor components, including
CD25, succumb to a rapidly progressing autoimmune disease
that is caused by an uncontrolled activation of CD4+ T cells and
B cells (6–8). The fundamental function of IL-2 in Treg biology
was recently highlighted with the demonstration that IL-2 was
critically required for the homeostatic maintenance of Treg in
the peripheral lymphoid organs (9–11). Other studies have also
suggested a requirement of IL-2 for the suppressive function and
the thymic development of Treg (12, 13). Therefore, dis-
turbances in the Treg-IL-2 axis can result in autoimmunity or
contribute to the development of immune-mediated diseases.
Systemic lupus erythematosus (SLE) is a prototypic systemic
autoimmune disease with complex genetics and unknown etiol-
ogy. It is characterized by a breakdown of tolerance to ubiquitous
nuclear antigens, including double-stranded DNA (ds-DNA),
that results in an uncontrolled activation of self-reactive B and T
cells and consequent multiorgan inflammation, most importantly
nephritis (14, 15). Despite a few contradictory reports, most
studies have found a low prevalence of CD4+CD25+ T cells in
SLE patients and murine SLE models, suggesting a disturbed
maintenance of the Treg pool size (16–18). Nonetheless, the
interpretation of most of these studies may be restricted because
of the common identification of Treg by CD25, a surface marker
that is also expressed by activated conventional CD4+ T cells
(Tcon) and absent in a large proportion of Treg.
Impaired IL-2 production by T cells has also been attributed a
critical role in murine and human SLE (19, 20). However, it
remains unclear whether a shortage of IL-2 causes abnormalities
of the Treg population in lupus-prone individuals and how such a
disturbance is causally linked to the pathogenesis of this disease.
To address these fundamental questions, we explored the
origins and consequences of abnormalities in the CD4+Foxp3+
Treg pool during the course of SLE progression using
(NZBxNZW) F1 lupus mice, a spontaneous autoimmune model
that displays many features of human SLE, including fatal
nephritis (21). We found that the lupus-prone mice failed to
sustain a competitive number of CD4+Foxp3+ Treg in the pe-
ripheral lymphoid organs because of an acquired deficiency of
IL-2 and IL-2-producing CD4+ T cells. This homeostatic im-
pairment of Treg boosted Tcon hyperactivity, resulting in a
progressive imbalance of Treg and effector Tcon, and promoted
the progression of disease. Furthermore, we showed that com-
pensation of the IL-2 deficiency in lupus mice by treatment with
rIL-2 impeded the progression of established disease most likely
by re-establishing the homeostatic balance of Treg and effector
Tcon, indicating the reversibility of this acquired Treg disorder.
Results
Progressive Homeostatic Imbalance of Treg and Tcon. The percent-
age and absolute numbers of CD4+Foxp3+ Treg were evaluated
in different organs and at different time points during the de-
velopment of disease in (NZBxNZW) F1 mice by flow cytometry.
A comparison between young animals (young), animals at the
onset of disease (onset), and old, diseased animals (diseased)
showed a progressive deficiency in the number of Treg in the
lymph nodes and the peripheral blood. (Fig. 1 A and B). This
deficiency of Treg was contrasted to progressive increases in the
Author contributions: J.Y.H., A.R., and G.R. designed research; J.Y.H., H.M., R.U., P.E., S.R.,
O.W., L.K., J. Heimann, and S.B. performed research; A.S. and J. Huehn contributed new
reagents/analytic tools; J.Y.H., H.M., T.G., and A.S. analyzed data; and J.Y.H., A.R., G.-R.B.,
and G.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: humrich@drfz.de.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0903158107/DCSupplemental.

204–209 | PNAS | January 5, 2010 | vol. 107 | no. 1 www.pnas.org/cgi/doi/10.1073/pnas.0903158107


percentage and absolute numbers of Treg in the spleens and of
CD4 single positive (SP) Treg in the thymi (Fig. 1 A and B).
Next, we studied the in vivo proliferation of CD4+Foxp3+
Treg and CD4+Foxp3− Tcon by BrdU incorporation. The pro-
liferation rates of Treg and Tcon in lymphoid organs from young
(NZBxNZW) F1 mice were not different from those in aged-
matched BALB/c mice (Fig. S1A). However, this changed in
lupus mice at the onset of disease: the proliferation rate of Treg
was now lower in spleens and lymph nodes (Fig. S1A) compared
to aged-matched BALB/c mice, whereas the proliferation rate of
CD4 SP Treg was increased in the thymus (Fig. S1A). The
changes in Treg proliferation were accompanied by a continuous
increase in the proliferation rate of Tcon in the peripheral
lymphoid organs and in the peripheral blood that was not ob-
served in age-matched BALB/c mice (Fig. S1B). Additionally,
and in contrast to BALB/c mice, the majority of proliferating
CD4+ T cells consisted of CD44+ effector/memory T cells in lupus
mice (Fig. S1C). The calculated ratio between proliferating
CD4+Foxp3+ Treg and CD4+Foxp3− Tcon—representing a
measure of the homeostatic balance between Treg and Tcon—was
therefore progressively reduced in the lymphoid organs from
(NZBxNZW) F1 mice compared to that of age-matched BALB/c
mice (Fig. 1C). Consistent with the increased proliferation of CD4
SP Treg, an increased Treg:Tcon ratio was observed in the thymus
(Fig. 1C). Collectively, these data indicated an acquired impairment
of the homeostatic balance between Treg and effector Tcon in
parallel to a partial deficiency of Treg in the periphery.
Phenotypic Changes of Treg and Tcon During Disease Development.
Next, we analyzed the phenotype of CD4+Foxp3+ Treg and
CD4+Foxp3− Tcon from lupus-prone (NZBxNZW) F1 mice
during the course of disease. The percentage of CD25+ cells
among Treg decreased with advancing disease in (NZBxNZW)
F1 mice (Fig. 2A and Fig. S2A). This was accompanied by an
increase in the percentage of CD25+ cells among Tcon (Fig. 2A
and Fig. S2A). Compared to age-matched BALB/c mice, young
(NZBxNZW) F1 mice already had higher frequencies of CD69+
and CD44+ cells among both Treg and Tcon that strongly in-
creased with disease progression (Fig. 2 B and D and Fig. S2B
and D). In contrast, the percentage of CD62L+ cells among Treg
and Tcon markedly declined concomitantly with the increase in
CD44+ cells (Fig. 2C and Fig. S2C). Similar phenotypic changes
were present in the parental autoimmune-prone NZB strain that
develops a milder form of lupus. In contrast, these phenotypic
Humrich et al.
Fig. 1. Progressive homeostatic imbalance
of Treg and Tcon. (A and B) Average per-
centage of Foxp3+ cells among CD4+ and
among CD4 SP T cells (A) and absolute
numbers (B) of CD4+Foxp3+ T cells in spleens
(SN), lymph nodes (LN), peripheral blood
(PB), and of CD4 SP Foxp3+ T cells in thymi
(TH) from (NZBxNZW) F1 mice (filled bars) at
the indicated disease stages compared to
age-matched BALB/c mice (open bars) de-
termined by flow cytometry. Data represent
the mean of 6–10 mice per group from two
to three independent experiments with
three to four mice per group. (C) Calculated
ratios of the percentage of BrdU+ cells
among CD4+Foxp3+ and CD4+Foxp3− cells
from (NZBxNZW) F1 (filled bars) compared to
age-matched BALB/c mice (open bars) in the
indicated organs and at the indicated dis-
ease stages. Data represent the mean of
3–10 mice per group from one to three in-
dependent experiments with two to four
mice per group. Error bars indicate SEM (*, P <
0.05; **, P < 0.01; ***, P < 0.001 vs. BALB/c).
changes were not observed in the clinically healthy parental
NZW strain, except for a moderate activation, or the healthy
BALB/c strain (Fig. 2 and Fig. S2). Together, Treg and Tcon
from lupus-prone mice acquire phenotypic changes similar to
those previously described in IL-2-deficient mice (9).
Progressive Decline in IL-2-Producing CD4+ T Cells. To assess whether
lupus-prone mice develop IL-2 deficiency, we determined the
percentage of CD4+ T cells that are capable to produce IL-2 and
IFN-γ during disease development in (NZBxNZW) F1 mice. In
comparing young, healthy (NZBxNZW) F1 and age-matched
BALB/c mice, we did not observe substantial differences in the
percentage of IL-2-producing CD4+ T cells in either spleens or
lymph nodes (Fig. 3 A and B). Consistent with our observation of
the acquired predominance of effector/memory Tcon, CD4+ T
cells acquired the ability to produce IFN-γ as the disease pro-
gressed (Fig. 3 A and B). In parallel, CD4+ T cells lost the ability
to produce IL-2 (Fig. 3 A and B). Moreover, the IFN-
γ-producing CD4+ T cells from (NZBxNZW) F1 mice exhibited
diminished IL-2 production compared to BALB/c mice (Fig. 3 A
and B). To confirm the shortage of IL-2 in lymphoid organs, we
determined the spontaneous IL-2 secretion in 24-h cell cultures
from spleen and lymph node cells at different disease stages. In
addition, we determined IL-2 levels in the plasma during disease
progression. In line with the decline of CD4+ IL-2-producing T
cells, spontaneous IL-2 secretion in lymphoid organs from
(NZBxNZW) F1 mice was also progressively diminished from
the disease onset compared to age-matched BALB/c mice in
parallel to decreasing IL-2 levels in the plasma (Fig. 3 C–E).
Treg from Lupus-Prone Mice Are Functionally Intact and Influence
IMMUNOLOGY
Disease Course. Loss of suppressive function of Treg results in the
appearance of systemic autoimmune syndromes (1, 2). We found
that Treg of both young and diseased (NZBxNZW) F1 mice
displayed similar suppressive functions in vitro (Fig. 4A) and
similar Foxp3 mRNA expression levels compared to age-
matched BALB/c mice (Fig. S3A).
To gain insights into the role of Treg in preventing disease
development, we reduced the numbers of Treg in young
(NZBxNZW) F1 mice with a single injection of depleting anti-
bodies against CD25. This resulted in a transient (≈2 weeks)
contraction of the CD4+Foxp3+ Treg pool in the peripheral blood
(Fig. S3B), was followed by higher frequencies of CD4+CD44+
Tcon in the peripheral blood (Fig. S3B), and resulted in an
PNAS | January 5, 2010 | vol. 107 | no. 1 | 205

Fig. 2. Phenotypic changes of Treg and Tcon during disease progression.
Average expression of CD25 (A), CD69 (B), CD62L (C), and CD44 (D) on
CD4+Foxp3+- and CD4+Foxp3−-gated cells from spleens of BALB/c (white
bars), NZW (light gray bars), NZB (black bars), and (NZBxNZW) F1 mice (dark
gray bars) at the age of 8–10 weeks (young) or 6–9 months (aged), re-
spectively. Data were determined by flow cytometry and represent the mean
of 3–10 mice per group from one to three independent experiments with
three to four mice per group. Error bars indicate SEM (*, P < 0.05; **, P <
0.01; ***, P < 0.001 vs. BALB/c).
acceleration of disease progression indicated by the development
of nephritis ≈4–6 weeks earlier than controls (Fig. 4B). Consistent
with these observations, the Treg-depleted mice also had a reduced
survival rate (Fig. 4B). However, no acceleration of anti-ds-DNA-
antibody titers was observed (Fig. S3B).
To test whether increasing the size of the peripheral Treg
pool could influence already established disease, purified
CD4+Foxp3+CD25+ Treg were adoptively transferred into mice
with active disease. Because the IL-2-deficient environment in
diseased animals would affect the survival and metabolic fitness
of the transferred Treg, Treg were activated for 24 h in the
presence of high doses of IL-2 before transfer (Fig. S3C).
Adoptive transfer of Treg delayed disease progression and sig-
nificantly increased the survival time (Fig. 4C). Again, there was
no significant change observed in the anti-ds-DNA IgG antibody
titers at any time point analyzed (Fig. S3D). Thus, CD4+Foxp3+
Treg from lupus-prone mice were functionally intact and phys-
iologically relevant inhibitors of disease progression. Moreover,
these data suggested that efficient regulation of CD4+ Tcon
activation and disease progression in lupus-prone mice de-
pended on the presence of sufficient numbers of Treg.
IL-2 Is Required to Retain Treg:Tcon Balance and to Impede Disease
Progression. To explore the role of IL-2 in the pathogenesis of
murine lupus, we reduced systemic IL-2 levels with neutralizing
antibodies in young and clinically healthy (NZBxNZW) F1 mice.
This induced a strong and persistent reduction in the percentage of
CD4+Foxp3+CD25+ Treg in the peripheral blood (Fig. S4A), in
the lymph nodes (Fig. 5A), and later also in the spleen (Fig. 5A). In
contrast, the percentage of CD4+Foxp3+ Treg that lacked ex-
206 | www.pnas.org/cgi/doi/10.1073/pnas.0903158107
Fig. 3. Decline in IL-2-producing CD4+ T cells. Representative dot plots show
the percentage of IL-2- and IFN-γ-producing cells among CD4+-gated spleen
(A) and lymph node (B) cells from 6- to 9-month-old BALB/c and diseased
(NZBxNZW) F1 mice (NZBW). Numbers in quadrants indicate the percentage
of the designated population. The respective bar diagrams show the average
percentage of IL-2+ and IFN-γ+ cells among CD4+ T cells from (NZBxNZW) F1
mice (filled bars) during disease progression compared to age-matched
BALB/c mice (open bars). Data represent the mean of three to six mice per
group from one to two independent experiments with three mice per group.
(C–E) Bar diagrams show IL-2 concentrations determined by ELISA from su-
pernatants of spleen (C) and lymph node cells (D) cultured for 24 h and from
plasma (E) of (NZBxNZW) F1 mice (filled bars) at the indicated disease stages
compared to age-matched BALB/c mice (open bars). Data represent the
mean of four to five mice per group. Error bars indicate SEM (*, P < 0.05; **,
P < 0.01 vs. BALB/c).
pression of CD25 remained either unaffected 1–3 weeks after IL-2
neutralization or even considerably increased in the spleen and in
the peripheral blood at later time points (Fig. S4 B–D). Thus, IL-2
deficiency affected nearly exclusively the CD25+ subpopulation of
Foxp3+ Treg with differing kinetics and impacts in the peripheral
blood, in the lymph nodes, and in the spleen, respectively. In
parallel, we observed a continuous increase of CD69+ and CD44+
cells among both the Treg and Tcon in the lymphoid organs (Fig.
5B) and of CD44+ Tcon in the peripheral blood (Fig. S4E), but we
did not observe an increase in anti-ds-DNA-IgG titers (Fig. S4F).
Thus, IL-2 deficiency induced similar abnormalities in Treg and
Tcon that are present in untreated mice with advanced disease.
Neutralization of IL-2 also resulted in a strong acceleration of
nephritis (Fig. 5C) and significantly increased mortality compared
to isotype-treated control mice (Fig. 5D). Together, IL-2 was
necessary to maintain a pool size of Treg in the periphery that was
competitive enough to prevent Tcon hyperactivity and disease
progression in lupus-prone mice.
IL-2 Partially Restores Treg:Tcon Balance and Ameliorates Established
Disease. We next examined whether IL-2 was capable of influ-
encing the endogenous Treg pool in (NZBxNZW) F1 mice.
Treatment of onset and diseased lupus mice with recombinant
IL-2 (rIL-2) strongly promoted the expansion of endogenous
CD4+Foxp3+ Treg in spleens (1.5-fold; Fig. 6A), lymph nodes
(1.5-fold; Fig. S5A), and peripheral blood (2-fold; Fig. S5A)
compared to the PBS-treated control mice. In parallel, Treg
displayed a remarkable increase in the surface expression of
CD25 (Fig. 6A) and an increase in the expression of Foxp3 on a
per cell basis in rIL-2-treated mice (Fig. S5B). Analysis of BrdU
incorporation in vivo revealed a 2- to 3-fold increase in the
proliferative activity of Treg in rIL-2-treated versus untreated
mice (Fig. 6B). Although an increased proliferation of Tcon was
Humrich et al.
Fig. 4. Treg are functionally intact and influence disease progression. (A) In
vitro suppression assays compare the suppressive capacity of CD4+CD25+
Treg from (NZBxNZW) F1 and age-matched BALB/c mice. The proliferation of
CFDA-SE-labeled responder cells in the presence of titrated numbers of Treg
was analyzed by flow cytometry and is presented in percent of the pro-
liferation of responder cells without Treg (taken as 100%). Error bars in-
dicate SD from triplicate cultures. One representative of two to three
independent experiments is shown. (B) Nephritis development, presented as
average proteinuria score, and survival, presented as Kaplan–Meier curves
(P = 0.116; n = 10 per group), after injection of depleting antibodies against
CD25 (anti-CD25) into 8- to 10-week-old, clinically healthy (NZBxNZW) F1
mice compared to rat IgG1-treated controls (Control). (D) Average protei-
nuria score and survival (P = 0.0439) after adoptive transfer of 5 × 105 acti-
vated CD4+CD25+Foxp3+ cells into diseased (NZBxNZW) F1 mice at the age of
6.5 months (Foxp3+) compared to a PBS-treated control group (Control). One
of two independent experiments is shown (n = 5 per group). (B and C) Error
bars indicate SEM.
also induced by administration of rIL-2, proliferation of Treg was
favored as shown by the higher ratio between BrdU+ Treg and
BrdU+ Tcon (Fig. 6B). Short-term rIL-2-treatment (3× 2 μg/24 h)
of diseased lupus mice already delayed disease progression (Fig.
S5C) and significantly decreased mortality (Fig. S5D); however, it
did not lead to a persistent expansion of the Treg pool (Fig. S5E).
In contrast, repetition of rIL-2 injections in intervals of 5 days
after the initial short-term treatment for a total of four times re-
sulted in a much more stable Treg expansion, especially of CD25+
Treg, which was still detectable >10 days after the last rIL-2 in-
jection (Fig. S5 F–H). In addition, the repetitive rIL-2 regimen
efficiently impeded disease progression (Fig. 6C) and considerably
prolonged the survival of diseased mice (Fig. 6D). Together, these
data indicated that the acquired IL-2 deficiency could be com-
pensated with exogenous rIL-2 and that the resulting re-estab-
lishment of the Treg:Tcon balance was associated with a beneficial
outcome in murine lupus.
Discussion
Perturbations in Treg biology can elicit autoimmune syndromes
and are associated with certain autoimmune diseases. Here we
analyzed CD4+Foxp3+ Treg in the (NZBxNZW) F1 model of
SLE to identify and characterize disturbances in Treg biology
and investigate how they contributed to the pathogenesis of this
systemic autoimmune disease.
Identification of Treg by expression of Foxp3 revealed a par-
tial deficiency in the numbers of Treg in (NZBxNZW) F1 lupus
mice. This deficiency affected lymph nodes and the peripheral
blood, and was progressive during disease development. Along
with the partial deficiency, we found a diminished homeostatic
Humrich et al.
Fig. 5. IL-2 neutralization induces Treg:Tcon imbalance and accelerates
disease. (A and B) Flow cytometry of CD4+-gated T cells 21 and 42 days after
injection of anti-IL-2 antibodies (anti-IL-2; filled bars) into 9- to 10-week-old,
clinically healthy (NZBxNZW) F1 mice compared to rat IgG-treated controls
(Control; open bars). Representative dot plots show the percentage of CD25+
(A) and of CD69+ and CD44+ (B) cells among Foxp3+ and Foxp3− cells in spleens
42 days after IL-2 neutralization. Numbers in quadrants indicate the per-
centage of the designated population. Bar diagrams show the average per-
centage of Foxp3+CD25+ cells among CD4+ T cells in spleens (SN) and lymph
nodes (LN) and of CD69+ and CD44+ cells among CD4+Foxp3+ and CD4+Foxp3−
cells in spleens. Data represent the mean + SD (n = 3 per group and time
point). (C and D) Nephritis development (C) and survival (D) after injection of
1 mg of neutralizing anti-IL-2 antibodies (anti-IL-2) into young, clinically
healthy (NZBxNZW) F1 mice compared to rat IgG-treated controls (Control);
nephritis development is presented as average proteinuria score and survival
as Kaplan–Meier curves (P = 0.0197). One representative of two independent
experiments is shown (n = 5 per group). Error bars indicate SEM.
proliferation of Treg in lupus-prone mice at the onset of disease.
This contrasted with a progressive increase in the proliferation of
effector Tcon starting from the onset of disease, indicating a
progressive homeostatic imbalance between Treg and effector
Tcon. These data also suggested that the peripheral pool of Treg
failed to expand adequately enough to compete with the en-
hanced proliferation of Tcon, a condition similar to that recently
IMMUNOLOGY
described in IL-2-deficient mice (9, 10). Phenotypic analyses
revealed further similarities between IL-2-deficient and diseased
(NZBxNZW) F1 lupus mice, including a progressive reduction in
the expression of CD25 in Treg, the activated, memory-like phe-
notype of Treg, and the progressive increase in CD44+CD62L−
effector Tcon that predominately belong to the Th1 lineage (7, 9).
Indeed, we could confirm a state of IL-2 deficiency in lupus-prone
mice by the detection of a progressive deficiency of IL-2 and IL-
2-producing Tcon in the lymphoid tissues. This deficiency was
characterized by a decline in single IL-2-producing Tcon, which
predominantly belong to the naïve Tcon repertoire (5), and a di-
minished IL-2 production by IFN-γ+ effector Tcon. The decline in
PNAS | January 5, 2010 | vol. 107 | no. 1 | 207

Fig. 6. rIL-2 restores Treg:Tcon balance and impedes disease progression.
(A) Flow cytometry of CD4+ T cells from spleens of 5-month-old (NZBxNZW)
F1 mice 12 h after three injections of 2 μg of rIL-2 per mouse within 24 h (rIL-
2) compared to PBS-treated mice (Control). Representative dot plots show
the percentage of CD25+ and Foxp3+ cells among CD4+-gated T cells. Bar
diagrams show the average percentage of the indicated parameters within
the respective population (mean + SD; n = 3 per group). (B) Representative
dot plots and bar diagrams show the percentage of BrdU+ cells among
CD4+Foxp3+ T cells in spleens from 7.5 month old (NZBxNZW) F1 mice (filled
bars) compared to age-matched BALB/c mice (open bars) 4 days after a 24-h
treatment with rIL-2 and daily injections of BrdU. The calculated ratio of the
percentage BrdU+ cells among CD4+Foxp3+ and CD4+Foxp3− cells from
controls (open bar) and rIL-2-treated (NZBxNZW) F1 mice (filled bar) is
compared in a bar diagram. Data represent the mean of three to eight mice
per group from one to three independent experiments (*, P < 0.05; **, P <
0.01 vs. control). Numbers in quadrants indicate the percentage in the des-
ignated population. (C and D) Nephritis development, presented as average
proteinuria score (C), and survival, presented as Kaplan–Meier curves (D) (P =
0.0035) after repetitive treatment of 6.3-month-old, diseased (NZBxNZW) F1
mice with 2 μg of rIL-2 per mouse every 5 days for a total of four times (until
day 20) after an initial short-term treatment with three injections of 2 μg of
rIL-2 per mouse within 24 h (rIL-2) compared to an aged-matched PBS-
treated control group (Control) (n = 8 per group). Error bars indicate SEM.
IL-2 levels and IL-2-producing Tcon was associated with pro-
gressive Treg abnormalities and disease progression, suggesting
that an acquired IL-2 deficiency contributes to disease develop-
ment. This was proven by several experiments. First, neutralization
of IL-2 in young, clinically healthy animals induced the homeostatic
imbalance of Treg and effector Tcon, a characteristic of advanced
disease, and strongly accelerated lupus. Second, treatment of dis-
eased lupus mice with rIL-2 partially restored the balance of Treg
and effector Tcon by preferentially increasing the proliferation of
Treg, and considerably impeded disease progression, and the ef-
ficacy was further enhanced by a repetitive regimen.
Nonetheless, treatment with rIL-2 also resulted in an increased
activation and proliferation of CD4+Foxp3− Tcon. Although this
may carry the risk of increasing disease activity (22, 23), our data
show that in the active phase of the disease and in an IL-2-deprived
environment, IL-2 acted as a Treg adjuvant that outweighed its
simultaneous fueling of the Tcon pool. Apart from that, it was
recently shown in a model of type 1 diabetes that the potency of
IL-2 to increase the activation of Tcon and NK T cells depended on
the administered dosage (23). In line with our data, recovery from
autoimmunity was also observed in the MRL/lpr lupus mouse
208 | www.pnas.org/cgi/doi/10.1073/pnas.0903158107
model after treatment with IL-2-expressing recombinant vectors
(24, 25). Therefore, therapeutic strategies that target the Treg-
IL-2 axis as a treatment for SLE are worth exploring in more detail.
Further research should aim to improve the selectivity of IL-2
treatments to favor exclusively Treg proliferation.
The pathogenic relevance of the impaired Treg-IL-2 axis in
murine lupus, shown here, raises the question of the origin of the
IL-2 deficiency. Both genetic and acquired alterations have been
considered important for the IL-2 deficiency observed in lupus
and other autoimmune diseases (26–28). In our study, the defi-
ciency of IL-2 and IL-2-producing CD4+ Tcon in the
(NZBxNZW) F1 lupus model was not evident before the onset of
disease and did not precede Tcon hyperactivity and Treg defi-
ciency in general. The low prevalence of CD4+Foxp3+ Treg in
young, clinically healthy lupus mice also suggests that factors
other than IL-2 may be involved in regulating the size of the
peripheral Treg pool. This is supported by recent studies in
congenic mouse strains related to the (NZBxNZW) F1 strain
that indicated that the low prevalence of CD25+ Treg was linked
to a disease-related locus, the so-called Sle1a locus (29). Al-
though the responsible genes have not been determined so far, it
is known that the Sle1a locus does not encode for genes involved
in IL-2 transcription or IL-2 signaling (29). Alternatively, the
peripheral Treg deficiency may be caused by an impaired thymic
Treg generation. Instead, we found that the numbers and pro-
liferation rates of thymic Treg were normal in young
(NZBxNZW) F1 mice and even increased during disease pro-
gression pointing to a mechanism that attempts to compensate
for the Treg deficiency in the peripheral lymphoid organs. To-
gether, we propose that IL-2 deficiency in lupus is secondary,
acquired mainly because of the displacement of naïve, IL-2-
producing Tcon by effector Tcon. The IL-2 deficiency arises
because effector Tcon do not contribute sufficiently to overall
IL-2 production. Thus, the IL-2 deficiency in lupus may be the
result of an uncontrolled Tcon hyperactivity.
In contrast to the progressive deficiency observed in the
peripheral blood and lymph nodes, a continuous increase of the
Treg subpopulation that lacked expression of CD25 was observed
in the spleen, leading to a >4-fold increase in the absolute numbers
in diseased (NZBxNZW) F1 mice compared to young (NZBxNZW)
F1 and aged-matched BALB/c mice. At present, it is not entirely
clear why there are such differences in the distribution of Treg in this
lupus model. However, the diminished or moderate in vivo pro-
liferation rates observed in splenic Treg suggest that an accumu-
lation of Treg in this highly inflamed tissue is more likely than an
expansion in the organ itself. Therefore, the partial deficiency of
Treg found in lymph nodes and in the peripheral blood may also be,
in part, the result of an enhanced attraction of Treg to the spleen.
Interestingly, an increase of splenic Foxp3+ Treg that lacked ex-
pression of CD25 was induced in healthy lupus mice by neutrali-
zation of IL-2, suggesting that IL-2 deficiency plays an important
role in this phenomenon.
Taking all our results into consideration, we propose a sim-
plified model that may explain the gradually acquired and self-
amplifying failure of the CD4+Foxp3+ Treg pool to compete in
systemic autoimmunity (Fig. S6). In this model, several initial
events lead to Tcon hyperactivity. Early in the disease, at least
partial compensation is provided by a qualitatively intact yet
quantitatively already restricted Treg system. The origins of
spontaneous Tcon hyperactivity in lupus are currently speculative
but could include genetic alterations in antigen-presenting cells
that lead to an increase in proinflammatory cytokine production or
an altered context of self-antigen presentation to autoreactive
Tcon (30). This may also promote resistance of autoreactive Tcon
to Treg-mediated suppression, additionally facilitating their acti-
vation, differentiation, and consecutive expansion (31). With dis-
ease progression, the ongoing conversion of naive Tcon into
effector Tcon and their displacement results in a progressive
Humrich et al.
decline in IL-2 production. This acquired IL-2 deficiency further
impedes the peripheral Treg pool to expand sufficiently to com-
pete with the hyperactivity of Tcon. The resulting vicious cycle is
characterized by a progressive homeostatic imbalance between
Treg and effector Tcon. Once the Treg pool declines below a
critical size, the remaining compensatory mechanisms collapse
and the disease manifests itself.
In summary, an acquired and self-amplifying impairment of
the Treg-IL-2 axis induced a progressive homeostatic imbalance
of Treg and effector Tcon, and promoted the development of
disease. The reversibility of this homeostatic impairment, shown
here, provides rationales and approaches for a Treg- and IL-2-
based immunotherapy of SLE.
Materials and Methods
See SI Materials and Methods for details.
Mice. Female (NZBxNZW) F1, NZW, NZB, and BALB/c mice were bred and
maintained under specific-pathogen-free conditions in the Deutsches Rheu-
maforschungszentrum (DRFZ) and were used for experiments at between 6
weeks and 12 months of age in accordance with institutional and federal
guidelines. Mice were divided into groups according to age and disease ac-
tivity, as determined by the degree of nephritis (young, onset, and diseased).
Proteinuria was determined with Multistix 10 Visual (Bayer Diagnostics).
Flow Cytometry. Multicolor flow cytometry was performed with a FACSCa-
libur cytometer (BD Biosciences). Results were processed by using CellQuest
software (BD Biosciences). FACSDiva (BD Biosciences) and FACSAria (BD
Biosciences) cell sorters were used for cell sorting.
In Vivo Proliferation Analysis. Mice of different age groups were injected i.p.
every 24 h for 4 days with 40 mg/kg bodyweight BrdU (BD Biosciences). BrdU
incorporation was analyzed by flow cytometry (BrdU Flow kit; BD Biosciences).
IL-2 Production and ELISA. IL-2 concentrations in cell culture supernatants
from spleens and lymph nodes and in the plasma of (NZBxNZW) F1 and BALB/c
mice at different ages were determined by ELISA (IL-2 ELISA kit; BD Bio-
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Suppression Assays. CD4+CD25+ and CD4−CD25− cells were obtained from
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ACKNOWLEDGMENTS. We thank Hyun-Dong Chang for critically reading
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meinschaft (SFB 650) and grants from the University Hospital Charité Berlin.
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