An essential role for IL-2 receptor in regulatory T cell function
Takatoshi Chinen
#1
, Arun K. Kannan
#2
, Andrew G Levine
1
, Xiying Fan
1
, Ulf Klein
3
, Ye
Zheng
4
, Georg Gasteiger
1,5
, Yongqiang Feng
1
, Jason D. Fontenot
6,8
, and Alexander Y.
Rudensky
1,8
1
Howard Hughes Medical Institute and Immunology Program, Memorial Sloan Kettering Cancer
Center, New York, NY, USA
2
Immunology Discovery, Biogen, Cambridge, MA, USA
3
Herbert Irving Comprehensive Cancer Center, Department of Pathology and Cell Biology, and
Department of Microbiology and Immunology, Columbia University, New York, NY, USA
4
Nomis Foundation Laboratories for Immunobiology and Microbial Pathogenesis, The Salk
Institute for Biological Studies, La Jolla, CA, USA
5
Institute for Medical Microbiology and Hygiene, University of Mainz Medical Centre, Mainz,
Germany
6
Exploratory Biology, Juno Therapeutics, Seattle, WA, USA
#
These authors contributed equally to this work.
Abstract
Regulatory T (T
reg
) cells, expressing abundant amounts of the IL-2 receptor (IL-2R), are reliant on
IL-2 produced by activated T cells. This feature implied a key role for a simple network based on
IL-2 consumption by T
reg
cells in their suppressor function. However, congenital deficiency in
IL-2R results in reduced expression of the T
reg
cell lineage specification factor Foxp3,
confounding experimental efforts to understand the role of IL-2R expression and signaling in T
reg
suppressor function. Using genetic gain and loss of function approaches, we demonstrate that IL-2
capture is dispensable for control of CD4
+
T cells, but is important for limiting CD8
+
T cell
activation, and that IL-2R dependent STAT5 transcription factor activation plays an essential role
in T
reg
cell suppressor function separable from T cell receptor signaling.
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8
Corresponding authors. Alexander Y. Rudensky, HHMI and Immunology Program, Memorial Sloan Kettering Cancer Center, 417
East 68
th
Street, New York, NY 10065, USA., rudenska@mskcc.org, Phone: 646-888-3109 FAX: 646-422-0453, Jason D. Fontenot,
Juno Therapeutics, 307 Westlake Avenue North, Seattle, WA 98109, USA, jason.fontenot@junotherapeutics.com, Phone:
206-566-5670.
Author contributions
T.C., J.D.F. and A.Y.R. conceived the project and designed the experiments; T.C., A.K.K., A.G.L., X.F., Y.Z., G.G., and Y.F. conducted
experiments; U.K. generated
Il2rb
fl
allele; J.D.F. generated
Il2ra
fl
allele; T.C., J.D.F. and A.Y.R. wrote and edited the manuscript.
Competing financial interests
The authors declare no competing financial interests.
Accession codes
GSE84553
HHS Public Access
Author manuscript
Nat Immunol
. Author manuscript; available in PMC 2017 March 05.
Published in final edited form as:
Nat Immunol
. 2016 November ; 17(11): 1322–1333. doi:10.1038/ni.3540.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Regulatory T (T
reg
) cells expressing the transcription factor Foxp3 restrain immune
responses to self and foreign antigens
1-3
. T
reg
cells express abundant amounts of the
interleukin 2 receptor α-chain (IL-2Rα; CD25), but are unable to produce IL-2. IL-2 binds
with low affinity to IL-2Rα or the common γ -chain (γ
c
)-IL-2Rβ heterodimers, but
receptor affinity increases ~1,000 fold when these three subunits together interact with
IL-2
4
. IL-2 and STAT5, a key IL-2R downstream target, are indispensable for Foxp3
induction and differentiation of T
reg
cells in the thymus
5-11
. IL-2Rβ and γ
c
are shared with
the IL-15 receptor, whose signaling can also contribute to the induction of Foxp3
12
. IL-2, in
cooperation with the cytokine TGF-β, is also required for extrathymic T
reg
cell
differentiation
13
.
While the role for IL-2R signaling in the induction of Foxp3 expression and T
reg
cell
differentiation in the thymus has been well established by previous studies, the significance
of IL-2R expression in mature T
reg
cells is not well understood. Although the deficiency in
STAT5 abolishes Foxp3 expression, it can be rescued by increased amounts of the anti-
apoptotic molecule Bcl2. This finding raised a possibility that a primary role for IL-2 is in
the survival of differentiating T
reg
cells or their precursors
14
. It was also reported that
ablation of the proapoptotic protein Bim can rescue T
reg
cells or their precursors from
apoptosis associated with IL-2 or IL-2R deficiency and restore T
reg
cell numbers, but it did
not prevent fatal autoimmunity
15
. However, a profound effect of a congenital deficiency in
IL-2, Bcl2 and Bim on differentiation and selection of T
reg
and self-reactive effector T (T
eff
)
cells confounds interpretation of this observation. Antibody-mediated neutralization of IL-2
in thymectomized mice reduces T
reg
cell numbers and Foxp3 expression in T
reg
cells
16, 17
.
Thus, IL-2 supports T
reg
cell lineage stability after differentiation
18, 19
. However, expression
of a transgene encoding IL-2Rβ chain exclusively in thymocytes was reported to rescue the
lethal autoimmune disease in
Il2rb
−/−
mice, suggesting that IL-2R expression is dispensable
in peripheral T
reg
cells
7, 11
. Thus, a role for IL-2R expression and signaling in peripheral
T
reg
cells remains uncertain. Hypothetically, a role for IL-2R in peripheral T
reg
cells could
be threefold: 1) guidance for T
reg
cells to sense their targets – activated self-reactive T cells,
which serve as a source of IL-2; 2) T
reg
cell-mediated deprivation of IL-2 as a mechanism of
suppression, and 3) cell-intrinsic IL-2 signaling in differentiated T
reg
cells to support their
maintenance, proliferation, or function due to triggering of JAK–STAT5, PI3K–Akt, or Ras–
ERK signaling pathways. Previous studies primarily focused on the induction or
maintenance of Foxp3, while other aspects of IL-2R function have not been firmly
established due to aforementioned limitations.
Despite their high reliance on IL-2 for the maintenance of Foxp3 expression, T
reg
cells are
unable to produce IL-2. The reason for the inhibition of autologous activation of STAT5 in
T
reg
cells, and potential biological significance of this IL-2-based T
reg
-T
eff
cell regulatory
loop, also remain unknown. It has been suggested that repression of IL-2 is required to
maintain the ‘unbound’ state of high affinity IL-2R on T
reg
cells, and unbound IL-2R serves
a key role in T
reg
cell-mediated suppression by depriving T
eff
cells of IL-2
20-24
, however,
whether this mechanism has a non-redundant role in suppression
in vivo
is unknown.
To address the role of IL-2R and downstream signaling pathways in differentiated T
reg
cells,
we ablated IL-2Rα, IL-2Rβ, and STAT5 in Foxp3-expressing cells. By simultaneously
Chinen et al. Page 2
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. Author manuscript; available in PMC 2017 March 05.
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inducing expression of an active form of STAT5, we assessed the differential requirements
for IL-2R expression and IL-2 signaling for T
reg
cell homeostasis vs. suppressor activity.
Results
IL-2R is indispensable for T
reg
cell function
To definitively establish a role for IL-2R in T
reg
cell function
in vivo
, we generated a T
reg
cell-specific IL-2Rβ conditional knockout mice using Cre recombinase driven by the
endogenous
Foxp3
locus (
Foxp3
Cre
), in which a loxP-flanked
Il2rb
allele (
Il2rb
fl/fl
) was
deleted in T
reg
cells after Foxp3 was expressed.
Il2rb
fl/fl
Foxp3
Cre
mice developed systemic
fatal autoimmune inflammatory lesions and lymphoproliferation, albeit somewhat milder
than that observed in
Foxp3
−
mice (Fig. 1a–c). IL-2Rα expression was diminished in
peripheral IL-2Rβ-deficient T
reg
cells (Fig. 1d), and tyrosine phosphorylation of STAT5 in
response to IL-2 was lacking (Fig. 1e). The frequency of Foxp3
+
cells among CD4
+
T cells
and the expression of Foxp3 on a per-cell basis were both diminished (Fig. 1f). In healthy
heterozygous
Il2rb
fl/fl
Foxp3
Cre/wt
females, where both IL-2Rβ-sufficient (YFP
−
) and -
deficient (YFP
+
) T
reg
cells co-exist due to random X-chromosome inactivation, IL-2Rβ-
deficient T
reg
cells were underrepresented (Fig. 1g, h). It has been suggested that IL-2 is
selectively required for the maintenance of CD62L
hi
CD44
lo
Treg cells, but is dispensable for
CD62L
lo
CD44
hi
T
reg
cells
25
. However, we found both CD62L
hi
CD44
lo
and CD62L
lo
CD44
hi
T
reg
cells to be significantly reduced in the absence of IL-2Rβ in healthy heterozygous
females. In these mice, IL-2Rβ-deficient T
reg
cells expressed reduced amounts of Foxp3 and
T
reg
-cell ‘signature’ molecules IL-2Rα chain (CD25), CTLA-4, GITR, and CD103
regardless of CD62L and CD44 expression (Fig. 1i, j and Supplementary Fig. 1a).
Although in diseased
Il2rb
fl/fl
Foxp3
Cre
mice, a majority of T
reg
cells were CD62L
lo
CD44
hi
,
this was likely a consequence of severe inflammation, because T
reg
cell frequencies were
also markedly reduced at sites where CD62L
lo
CD44
hi
cells were prevalent, i.e., the small
and large intestines (Supplementary Fig. 1b). Accordingly, many characteristic T
reg
cell
markers, except for CD25 and Foxp3, were upregulated as the result of T
reg
cell activation in
Il2rb
fl/fl
Foxp3
Cre
mice (Supplementary Fig. 1c). These observations suggested that both
CD62L
hi
CD44
lo
and CD62L
lo
CD44
hi
T
reg
cell subsets, including those residing in the non-
lymphoid tissues, are dependent on IL-2, though under inflammatory conditions the latter
can be sustained to some extent by IL-2R-independent signals. Despite the upregulation of
CTLA-4, GITR, ICOS, and CD103, the ‘activated’ IL-2Rβ-deficient T
reg
cells from
Il2rb
fl/fl
Foxp3
Cre
mice were still incapable of controlling inflammation in the diseased mice
and were not suppressive when co-transferred with T
eff
cells into lymphopenic recipients
(data not shown).
Our findings raised the question whether ablation of IL-2Rα, which, in addition to
facilitating IL-2 signaling, enables its sequestration from T
eff
cells, would result in a similar
T
reg
cell deficiency and disease compared to those in
Il2rb
fl/fl
Foxp3
Cre
mice. Thus, we
generated a loxP-flanked
Il2ra
allele (J.D.F. manuscript in preparation) and similarly
induced its conditional ablation in T
reg
cells. We found that T
reg
cell-specific IL-2Rα
deficiency resulted in a disease with comparable early onset and severity to those observed
upon IL-2Rβ ablation (Supplementary Fig. 1d–f). Of note, germ-line deficiency of either
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Il2ra
or
Il2rb
in mice on the same C57BL6/J as our conditional knockout mice resulted in a
considerably less aggressive disease with a delayed onset, likely due to a role for IL-2R
signaling in T
eff
cells (data not shown). Our findings also indicate that IL-15 was unable to
effectively compensate for the loss of IL-2 signaling in differentiated T
reg
cells because in
Foxp3
Cre
Il2ra
fl/fl
mice, T
reg
cells lacked only IL-2 signaling, whereas in
Il2rb
fl/fl
Foxp3
Cre
mice, they lacked both IL-2 and IL-15 signaling yet were similarly affected. This was in
contrast to T
reg
cell differentiation in the thymus where IL-15 can contribute in part to Foxp3
induction
12
. Since IL-2R activates PI3K–Akt, MAPK, and JAK–STAT5 signaling pathways,
we next sought to assess a role for STAT5 activation downstream of IL-2R signaling in T
reg
cells. We found that STAT5 ablation similarly impaired T
reg
cell function and
Foxp3
Cre
Stat5a/b
fl/fl
mice were similarly affected by fatal autoimmunity as were mice
harboring IL-2R deficient T
reg
cells (Supplementary Fig. 1g–k). Thus, in agreement with
IL-2 neutralization studies, IL-2R signaling is required for T
reg
cell fitness in a cell-intrinsic
manner.
STAT5b-CA partially rescues IL-2R deficient T
reg
function
The above findings implied that STAT5 activation downstream of IL-2R is continuously
required for T
reg
cell function. However, a marked decrease in IL-2R observed in STAT5-
deficient T
reg
cells (Supplementary Fig. 1g) made it impossible to separate a loss of STAT5
from impairment in all IL-2R functions, i.e., detection of IL-2, transduction of STAT5-
dependent and -independent signals, and consumption and deprivation of IL-2, as a key
contributor to the observed severe T
reg
cell dysfunction.
To address this major caveat and to understand a role for STAT5 vs. IL-2R, we asked
whether expression of a gain-of-function form of STAT5b can rescue T
reg
cell function in
the absence of IL-2R. A previous study using a transgene encoding a constitutively active
form of STAT5b (STAT5b-CA) driven by the proximal
lck
promoter in the absence of
IL-2Rβ showed rescue of T
reg
cell differentiation in the thymus, but not lymphoproliferative
syndrome
9
. However, the expression of this transgene early during thymopoiesis leads to
leukemic lymphoproliferation, which complicated the interpretation of these findings. In
addition, both the activity of the proximal
lck
promoter and the expression of the transgene
diminish in peripheral T cells in these mice
9
. Therefore, we generated a gene-targeted mouse
strain utilizing the
Rosa26
‘gene trap’ locus
26
, where a ‘CAG’ promoter driven STAT5b-
CA
27
is preceded by a loxP-flanked STOP cassette (Supplementary Fig. 2a). In the
resulting
Rosa26
Stat5bCA
mice, STAT5b-CA is expressed only when the loxP sites undergo
Cre mediated recombination. Introduction of the
Rosa26
Stat5bCA
allele into
Il2rb
fl/fl
Foxp3
Cre
mice and the consequent expression of STAT5b-CA in IL-2Rβ-deficient T
reg
cells rescued
the systemic inflammation and early fatal disease (Supplementary Fig. 2b). In these mice,
T
reg
cell frequencies and numbers were comparable to or even surpassed their levels in
IL-2R sufficient
Foxp3
Cre
mice (Fig. 2a). Notably, the expression of IL-2Rα chain was
increased despite the absence of IL-2Rβ chain (Fig. 2a), suggesting the expression of
IL-2Rα on T
reg
cells is primarily controlled by STAT5-dependent, but not by STAT5-
independent signaling. Importantly, these IL-2Rβ-deficient T
reg
cells with heightened
IL-2Rα expression remained unresponsive to IL-2 (Fig. 2b).
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The observed restoration of the suppressor function of IL-2Rβ-deficient T
reg
cells and rescue
of the early fatal disease upon STAT5b-CA expression raised the possibility that the
reintroduced high IL-2Rα levels were responsible for these effects. However, the expression
of STAT5b-CA similarly rescued the early fatal disease in
Foxp3
Cre
Il2ra
fl/fl
mice
(Supplementary Fig. 2c–h). Importantly, although the impaired capacity of T
reg
cells in
both
Il2rb
fl/fl
Foxp3
Cre
and
Foxp3
Cre
Il2ra
fl/fl
mice to capture and consume IL-2 was not
rescued upon STAT5b-CA expression (Fig. 2c), CD4
+
T cell reactivity was fully controlled
in these mice (Fig. 2d and Supplementary Fig. 2d–h). These results suggested that the
ability to capture and compete for IL-2 is dispensable for T
reg
cell mediated suppression of
CD4
+
T cell responses. To the contrary, however, expansion of CD8
+
T cells, in particular,
of activated CD62L
hi
CD44
hi
CD8
+
T cells, was only marginally restrained in these mice
(Fig. 2d and Supplementary Fig. 2f, h). Although the expansion of CD8
+
CD62L
lo
CD44
hi
subset was relatively well, albeit not perfectly, controlled in neonatal mice (Fig. 2d and
Supplementary Fig. 2f), this subset also gradually started to expand in these mice as early
as 3 wks after birth (Supplementary Fig. 2i). Although both
Il2rb
fl/fl
Foxp3
Cre
Rosa26
Stat5bCA
and
Il2ra
fl/fl
Foxp3
Cre
Rosa26
Stat5bCA
mice were rescued
from premature death and showed significantly improved clinical status comparable to
healthy controls, they gradually failed to thrive and started to succumb to disease
accompanied by massively expanded activated CD62L
hi
CD44
hi
and CD62L
lo
CD44
hi
CD8
+
T cell subsets in LNs and tissues by approximately 12 wks of age (Supplementary Fig. 2i,
j). These findings raised a possibility that IL-2 consumption by T
reg
cells, while dispensable
for control of CD4
+
T cells, is important for the restraint of CD8
+
T cells.
T
reg
cells suppress CD8
+
T cell responses via IL-2 depletion
To test if the impairment in consumption of IL-2 by T
reg
cells can account for the
proliferation of CD8
+
T cells in
Foxp3
Cre
Il2rb
fl/fl
Rosa26
Stat5bCA
mice, we administered IL-2
neutralizing antibodies to these and control mice starting from 5–7 days of age (Fig. 2e and
Supplementary Fig. 3a). As IL-2 supports the differentiation of T
reg
cells in the thymus,
IL-2 neutralization reduced the frequencies of T
reg
cells in all groups of mice and induced
immunoactivation in control
Il2rb
fl/wt
Foxp3
Cre
mice. In
Il2rb
fl/fl
Foxp3
Cre
mice, which
spontaneously develop disease, the production of T
H
2 cytokines IL-4 and IL-13 by CD4
+
T
cells was significantly reduced by IL-2 neutralization; however, the activation of CD4
+
and
CD8
+
T cells was at best only marginally reduced or unaffected. In contrast, the activation
and proliferation of CD8
+
T cells observed in
Il2rb
fl/fl
Foxp3
Cre
Rosa26
Stat5bCA
mice were
almost completely suppressed by the treatment.
The relative reduction in CD8
+
CD62L
lo
CD44
hi
and more pronounced proliferation of
CD8
+
CD62L
hi
CD44
hi
T cell subset in
Il2rb
fl/fl
Foxp3
Cre
Rosa26
Stat5bCA
and
Il2ra
fl/fl
Foxp3
Cre
Rosa26
Stat5bCA
mice raised a possibility that a loss of IL-2-consumption by
T
reg
cells might selectively impair their suppression for memory CD8
+
T cell expansion, but
not the recruitment of naive CD8
+
T cells into the effector cell pool. We tested this idea by
adoptive transfer of CD4
+
and CD8
+
cell subsets into lymphopenic recipients (Fig. 2f).
Consistent with the observation in
Foxp3
Cre
mice, the impaired suppression of CD4
+
T cell
expansion and activation by IL-2R-deficient T
reg
cells was completely rescued by STAT5b-
CA; in contrast, their ability to suppress memory CD8
+
T cells was not restored, whereas
Chinen et al. Page 5
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. Author manuscript; available in PMC 2017 March 05.
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