Download Murine Regulatory T Cells Contain Hyperproliferative and Death

The Journal of Immunology
Murine Regulatory T Cells Contain Hyperproliferative and
Death-Prone Subsets with Differential ICOS Expression
Yong Chen,*,†,1 Shudan Shen,*,1 Balachandra K. Gorentla,*,1 Jimin Gao,† and
Xiao-Ping Zhong*,‡
Regulatory T cells (Treg) are crucial for self-tolerance. It has been an enigma that Treg exhibit an anergic phenotype reflected by
hypoproliferation in vitro after TCR stimulation but undergo vigorous proliferation in vivo. We report in this study that murine
Treg are prone to death but hyperproliferative in vitro and in vivo, which is different from conventional CD4+Foxp32 T cells (Tcon).
During in vitro culture, most Treg die with or without TCR stimulation, correlated with constitutive activation of the intrinsic
death pathway. However, a small portion of the Treg population is more sensitive to TCR stimulation, particularly weak stimulation, proliferates more vigorously than CD4+ Tcon, and is resistant to activation-induced cell death. Treg proliferation is
enhanced by IL-2 but is less dependent on CD28-mediated costimulation than that of Tcon. We demonstrate further that the
surviving and proliferative Treg are ICOS+ whereas the death-prone Treg are ICOS2. Moreover, ICOS+ Treg contain much
stronger suppressive activity than that of ICOS2 Treg. Our data indicate that massive death contributes to the anergic phenotype
of Treg in vitro and suggest modulation of Treg survival as a therapeutic strategy for treatment of autoimmune diseases and
cancer. The Journal of Immunology, 2012, 188: 1698–1707.
R
egulatory T cells (Treg) actively suppress autoimmunity
and play critical roles in self-tolerance. Natural Treg are
generated in the thymus and are governed by the forkhead
transcription factor Foxp3. Expression of Foxp3 is critical for
Treg generation, maintenance, and function (1–5). Foxp3+ Treg are
composed of a small portion of T cells with distinct molecular
patterns and properties. Different from naive conventional CD4+
Foxp32 T cells (Tcon), Foxp3+ Treg express signature molecules
such as CD25, GITR, and CTLA4 that are usually expressed by activated Tcon (6–8). However, Treg do not express IL-2 or IFN-g. Instead, they express suppressive cytokines such as IL-10 and TGF-b
(9). Moreover, Treg have been marked as “anergic” in vitro and
in vivo. In vitro, Treg have been found unable to proliferate after
TCR engagement. In vivo, adoptively transferred CD4+CD25+
Treg that recognize OVA display decreased proliferative responses
to Ag challenge in mice (10–13). Although these results point to an
*Division of Allergy and Immunology, Department of Pediatrics, Duke University
Medical Center, Durham, NC 27710; †School of Laboratory Medicine, Wenzhou
Medical College, Wenzhou, Zhejiang 325035, China; and ‡Department of Immunology, Duke University Medical Center, Durham, NC 27710
1
Y.C., S.S., and B.K.G. contributed equally to this work.
Received for publication August 24, 2011. Accepted for publication December 6,
2011.
This work was supported by the National Institutes of Health (Grants R01AI076357,
R01AI079088, and R21AI079873), the American Cancer Society (Grant RSG-08186-01-LIB), the American Heart Association (to X.-P.Z.), and the Chinese National
Science Foundation (31071237).
Address correspondence and reprint requests to Dr. Xiao-Ping Zhong or Dr. Jimin
Gao, Division of Pediatric Allergy and Immunology, Department of Pediatrics, Duke
University Medical Center, Room 133 MSRB-I, Research Drive, Box 2644, Durham,
NC 27710 (X.-P.Z.) or Zhejiang Provincial Key Lab for Technology and Application
of Model Organisms, School of Life Sciences, Wenzhou Medical College, Wenzhou,
Zhejiang 325035, China (J.G.). E-mail addresses: [email protected] (X.-P.Z.)
and [email protected] (J.G.)
Abbreviations used in this article: 4E-BP1, translational repressor 4 elongation factor
binding protein 1; LN, lymph node; mTOR, mammalian target of rapamycin;
mTORC1, mTOR complex 1; mTORC2, mTOR complex 2; S6K1, 70-kDa ribosomal
S6 kinase; Tcon, conventional CD4+ Foxp32 T cell; Treg, regulatory T cell; TregFBS, Treg in FBS-containing medium; Treg-PBS, Treg rested in PBS; WT, wild-type.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102448
anergic property of Treg, Treg manifest normal or even stronger
homeostatic proliferation after injection into lymphopenic hosts
or TCR-induced proliferation in vivo (11, 14). Furthermore, Treg
incorporate a higher level of BrdU than that of Tcon after administration of BrdU into mice, suggesting that Treg are more proliferative than Tcon in normal mice (15). The causes of these distinct
observations and conclusions about Treg properties have been unclear. By tracking Treg proliferation and death simultaneously, we
report in this study that CD4+Foxp3+ Treg are hyperproliferative
but prone to death in vitro and in vivo, which differs from CD4+
Foxp32 Tcon. During in vitro culture with a TCR-stimulating Ab,
the majority of Treg die before or during the first division. The
remaining surviving subset of Treg is hyperproliferative to TCR
stimulation, particularly when the stimulation is weak. Treg proliferation is enhanced by IL-2 and is less dependent on CD28 costimulation than that of Tcon. In addition, freshly isolated Treg
show enhanced death and proliferation compared with Tcon.
Whereas proliferative Tcon are sensitive to activation-induced
death, the hyperproliferative Treg are resistant to activation-induced
death. Lastly, we demonstrate that the living hyperproliferative
Treg are ICOS+ and the death-prone Treg are ICOS2 and that
ICOS+ Treg have much stronger suppressive activity than ICOS2
Treg. Together, our data indicate that Treg should not be simply
defined as anergic. Rather, they contain a predominant ICOS2
death-prone subset and a minor ICOS+ hyperproliferative subset.
Our data provide an explanation for the aforementioned contradictory properties of Treg and suggest that promotion of Treg
survival could be used as an effective strategy to enhance immune
suppression and tolerance.
Materials and Methods
Mice
The C57BL6/J, Foxp3GFP (16), CD282/2, and B6.PL-Thy1a/CyJ congenic
mice were all purchased from The Jackson Laboratory. Thy1.1-Foxp3GFP
mice were generated by breeding B6.PL-Thy1a/CyJ mice with Foxp3GFP
mice. All mice were housed in an approved pathogen-free facility, and
experiments were performed in accordance with protocols approved by the
Duke University Institutional Animal Care and Use Committee.
The Journal of Immunology
Flow cytometry
Single-cell suspensions of spleen and lymph nodes (LNs) were stained with
Abs in PBS containing 2% FCS. The Abs include PE–Cy7–anti-CD4
(RM4-5), allophycocyanin–Cy7–anti-CD8a (53-6.7), PE–Cy7–anti-ICOS
(398.4A), PE- or allophycocyanin-conjugated anti-CD90.1 (HIS51), and
PE- or allophycocyanin-conjugated anti-CD90.2 (30-H12) (BD Biosciences). Biotin-conjugated Abs were developed with PE-conjugated, PE–
Cy5–conjugated, or allophycocyanin-conjugated streptavidin. For intracellular staining, cells were permeabilized using the Foxp3 staining kit
(eBioscience) after cell surface staining, followed by PE–anti-Foxp3 (FJK16s; eBioscience) or unconjugated anti-Ki67 (B56; BD Biosciences). An
Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (Invitrogen) was
used to detect anti-Ki67 Ab. Dying cells were identified using 7-aminoactinomycin D or the fixable Violet Live/Dead cell staining kit (Invitrogen). Allophycocyanin-conjugated annexin V (BD Biosciences) was
used to detect apoptotic cells. Stained samples were collected on a Canto
II flow cytometer (BD Biosciences). Data were analyzed using FlowJo
software (Tree Star).
Treg isolation
CD4 T cells were enriched using the APC selection kit (Stemcell Technologies) or MACS sorting with the LD column (Miltenyi Biotec) according
to the manufacturers’ protocols with ∼80% purity. The isolated CD4 cells
were stained for CD4, CD25, ICOS, 7-aminoactinomycin D, Thy1.1, and
Thy1.2 when needed and sorted for live CD4+GFP2 and CD4+GFP+ cells.
The purities of sorting cells was usually .95%.
Proliferation assay
CFSE proliferation assay was performed as previously described (17).
Single-cell suspension was made from spleens and LNs of mice in IMDM
supplemented with 10% FBS, penicillin/streptomycin, and glutamine.
Cells were stained with 10 mM CFSE for 9 min at room temperature in
PBS–0.5% BSA. Labeled cells were left unstimulated or stimulated with
an anti-CD3ε Ab (2C-11) at the indicated concentrations at 37˚C for different times in a 5% CO2 incubator. Where indicated, IL-2 (1000 U/ml;
Peprotech) or CTLA4Ig (10 mg/ml; BioXcell) were added at the beginning
of the culture. When cultured with IL-2, CD8+ cells were depleted using
anti-CD8 microbeads and LD columns according to the manufacturer’s
protocol (Miltenyi Biotec).
Treg contact inhibition assay
Sorted Thy1.1+Thy1.2+ Tcon were labeled with CFSE. Splenocytes from
TCR b2/2d2/2 mice were treated with 25 mg/ml mitomycin C for 30 min
and used as APCs. In a 96-well U-bottom plate, 1.5 3 105 CFSE-labeled
Tcon, the same number of APCs, and different numbers of sorted Thy1.2+
ICOS+ or ICOS2 Treg according to the indicated ratios were added to each
well. The cells were left unstimulated or stimulated with an anti-CD3ε
(2C11) Ab (1 mg/ml). After 72-h cultures, the cells were stained with PE–
Cy7–anti-Thy1.1, allophycocyanin–anti-Thy1.2, and allophycocyanin–
Cy7–anti-CD4 and analyzed by flow cytometry.
Western blot
CD4+GFP2 and CD4+GFP+ cells were sorted into IMDM–10% FBS. Cell
pellets of 0.5 to 1 million cells were lysed in 1% Nonidet P-40 buffer (1%
Nonidet P-40, 50 mM Tris, 150 mM NaCl) supplemented with protease and
phosphatase inhibitors. Protein lysates were boiled for 5 min in denaturing
sample buffer, separated by SDS-PAGE, and were transferred to a TransBlot Nitrocellulose membrane (Bio-Rad Laboratories). After blocking in
TBST (10 mM Tris pH 8, 150 mM NaCl, 2% Tween 20) supplemented with
3% (w/v) dried skim milk powder, the membrane was probed with indicated primary Abs in 5% (w/v) BSA–TBST solution followed by HRPconjugated secondary Abs in TBST supplemented with 3% (w/v) dried
skim milk. Proteins on the membrane were detected by ECL. Abs for pPLC-g1 (no. 2821), p-RSK1 T359/S363 (no. 9344), p-Foxo1 (no. 9461S),
p-ERK1/2 (no. 91015), p-p70S6K1 (no. 9204S), p70S6K1 (no. 9202), p-S6
(no. 4838), S6 (no. 2217), p-4E-BP1 (no. 2855S), 4E-BP1 (no. 9644),
cleaved caspase-3 (no. 9661), cleaved caspase 9 (no. 9509), and p-Akt
S473 (no. 9271S) were purchased from Cell Signaling Technology. AntiGFP (no. 632376) Ab was purchased from Clontech, and anti–b-actin Ab
was from Sigma-Aldrich (A1978).
Real-time PCR
CD4+ ICOS+Foxp3GFP+ and ICOS2Foxp3GFP+ Treg were isolated by
MACS purification and FACS sorting. Total RNA from sorted Treg was
prepared with the TRIzol reagent. First-strand cDNA was synthesized
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using the Superscript III First-Strand Synthesis System (Invitrogen). Realtime PCR was prepared using the RealMasterMix (Eppendorf) and performed on the Mastercycler ep realplex2 system (Eppendorf). Expressed
levels of target mRNAs were normalized with b-actin calculated using the
2–DDCT method. Primers used were Bax forward, 59-TGTTTGCTGATGGCAACTTC-39; Bax reverse, 59-GATCAGCTCGGGCACTTTAG-39; Bad
forward, 59-GCACACGCCCTAGGCTTGAGG-39; Bad reverse, 5-GGAACATACTCTGGGCTGGTC-39; Actin forward, 59-TGTCCACCTTCCAGCAGATGT-39, Actin reverse, 59-TGTCCACCTTCCAGCAGATGT-39.
Statistical analysis
For statistical analysis, two-tailed Student t test was performed (*p , 0.05,
**p , 0.01, ***p , 0.001).
Results
Live Foxp3+ Treg are hyperproliferative after TCR stimulation
in vitro
We compared proliferative responses between CD4+Foxp32 Tcon
and CD4+Foxp3+ Treg to different strengths of TCR stimulation
in vitro using a CFSE dilution assay. CFSE-labeled splenocytes
from wild-type (WT) mice were left unstimulated or stimulated
with different concentrations of an anti-CD3 Ab for 72 h. After
cell surface staining for CD4 and CD8 as well as fixable Live/
Dead staining for dead cells, cultured cells were intracellularly
stained for Foxp3. Comparison between total CD4+Foxp3+ Treg
and CD4+Foxp32 Tcon revealed much weaker proliferation of
Treg than Tcon (Fig. 1A), which is consistent with previously
published observations that Treg are “anergic” in vitro. However,
if gated on live CD4+Foxp3+ Treg and CD4+Foxp32 Tcon (Fig.
1B, 1C), we found that Treg had undergone more cell divisions
than Tcon, particularly when the stimulation was weak (Fig. 1D).
Differential sensitivity to activation-induced death between
proliferating Treg and Tcon
The differences of Treg proliferation between total Treg and live
Treg prompted us to examine survival and death of Treg during
in vitro culture. We analyzed the relationship of cell death versus
proliferation of total CD4+Foxp3+ and CD4+Foxp32 cells from the
same experiments in Fig. 1A–D. As shown in Fig. 1E, most CD4+
Foxp32 died after multiple divisions, and live CD4+Foxp32 T cells
had gone through less divisions than dead cells, particularly when
the stimulation was strong. In contrast, most of the CD4+Foxp3+
Treg died before the first cell division, and the hyperproliferating
CD4+Foxp3+ Treg were mostly live cells. Together, these observations suggest that proliferative CD4+Foxp32 Tcon are sensitive
to activation-induced cell death and that most Treg are death prone,
but proliferating Treg are resistant to activation-induced cell death
in vitro.
Hyperproliferative Treg are not converted from Tcon
Because some Tcon can convert to Foxp3+ after TCR stimulation,
this small population of hyperproliferative Foxp3+ cells could be
derived from Foxp32 Tcon during in vitro TCR stimulation. To
rule out such a possibility, we performed a mixed culture experiment. In this experiment, we used the Foxp3GFP mice in which
an IRES-GFP cassette is knocked into the 39 untranslated region
of the Foxp3 gene so that GFP expression can be used to identify
Foxp3+ cells (16). Sorted Thy1.1+CD4+Foxp3GFP2 Tcon from
Thy1.1+Foxp3GFP mice were mixed with Thy1.2+ splenocytes in
an amount so that the ratio of Thy1.1+ to Thy1.2+ Tcon was close
to 1:1. The mixed cells were labeled with CFSE and stimulated
with anti-CD3. While Thy1.1+ and Thy1.2+ CD4+Foxp32 contribute almost equally, more than 95% of live and proliferating
CD4+Foxp3+ cells were Thy1.2+ (Fig. 2). These observations indicate that while there was a low rate of conversion of CD4+
Foxp3GFP2 to CD4+Foxp3+ during in vitro TCR stimulation, the
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ICOS DEFINES PROLIFERATIVE VERSUS DEATH-PRONE Treg
FIGURE 1. Comparison of proliferation
and death between Treg and Tcon in vitro.
CFSE-labeled splenocytes were left unstimulated or stimulated with indicated concentrations of anti-CD3 for 72 h. Cells
were stained with anti-CD4, anti-CD8, and
the fixable Live/Dead dye followed by intracellular staining for Foxp3. A, Histogram of CFSE dilution of gated total
CD4+CD82Foxp3+ Treg and CD4+CD82
Foxp32 Tcon. B–D, Gating strategy for
live cells (B, C) and histograms of CFSE
dilution of gated live CD4+CD82Foxp3+
and CD4+CD82Foxp32 cells (D). E, Simultaneous analysis of proliferation and
death of Tcon and Treg after in vitro TCR
stimulation. Cell death staining and CFSE
dilution of gated total CD4+Foxp32 Tcon
and CD4+Foxp3+ Treg in the same experiments as in A. Data shown represent five
experiments.
contribution of such conversion to the proliferating Treg detected
in response to TCR stimulation was minimal and that most of the
hyperproliferating CD4+Foxp3+ cells were natural Treg.
Increased death and proliferation of freshly isolated Treg
To determine whether the aforementioned in vitro observations
reflect properties of Treg in vivo, we examined cell death and
proliferative status of freshly isolated T cells. CD4+Foxp3GFP+
Treg stained positive for annexin V ∼3–4 times more frequently
than CD4+Foxp3GFP2 Tcon (Fig. 3A, 3B), suggesting more apoptosis of Treg than Tcon in vivo. About 1.5- to 2-fold more Treg
than Tcon stained positive for Ki67 (Fig. 3C, 3D), a marker of cell
proliferation. These data are consistent with the observation that
Treg incorporate more BrdU than do Tcon in vivo (15). Together,
these data are consistent with the aforementioned in vitro data and
suggest that Treg are hyperproliferative and prone to death in vivo.
Treg proliferation is less dependent on CD28 costimulation
than that of Tcon
T cell activation requires signals from both the TCR and costimulatory receptors such as CD28. CD28-deficient mice contain
fewer Treg than that of WT controls. CD28-mediated costimulation
has been found to promote human CD4+CD25+ T cell expansion
in vitro (18–20). We examined the role of CD28 costimulation for
Treg proliferation using the same experimental system as in Fig. 1
except that CTLA4-Ig was added to the culture. CTLA4-Ig has
high affinities to B7-1 and B7-2 costimulatory ligands and thus
blocks CD28-mediated costimulation. As expected, CD4+Foxp32
Tcon proliferated less in the presence of CTLA4-Ig than in the
absence of this molecule. However, the inhibitory effect of CTLA4Ig on TCR-induced Treg proliferation was much weaker than that
on TCR-induced Tcon proliferation (Fig. 4A).
To investigate further the importance of CD28-mediated costimulation on Treg proliferation, we analyzed proliferative responses of CD282/2 Tcon and Treg to TCR stimulation. As shown
in Fig. 4B, CD282/2 CD4+Foxp32 Tcon proliferated much more
weakly than WT CD4+Foxp32 Tcon. However, CD282/2 CD4+
Foxp3+ Treg proliferation was only slightly decreased compared with that of WT CD4+Foxp3+ Treg. Together, these observations indicate that Treg are less dependent on CD28 costimulatory signal for their proliferation than are Tcon.
The Journal of Immunology
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FIGURE 2. Hyperproliferative Treg are not derived from Tcon. Sorted Thy1.1+CD4+Foxp3GFP2
Tcon were mixed with Thy1.2+ splenocytes from
mice carrying the Foxp3GFP transgene in a ratio
in which Thy1.1+CD4+Foxp3GFP2 Tcon were almost equal to Thy1.2+CD4+Foxp3GFP2 Tcon in
the mixture. The mixed cells were labeled with
CFSE, and proliferation of Tcon and Treg were
similarly examined as for Fig. 1. A, Histograms
show CFSE dilution of gated live CD4+Foxp32
Tcon. Dot plots show Thy1.1 and Thy1.2 staining
of gated live CD4+Foxp32 Tcon. B, Histograms
show CFSE dilution of live gated CD4+Foxp3+
Treg. Dot plots show Thy1.1 and Thy1.2 staining
of live gated CD4+Foxp3+ Treg. Data shown represent two experiments.
Altered mammalian target of rapamycin signaling and
constitutive activation of the intrinsic death pathway in Treg
To investigate the mechanisms that may control Treg proliferation
and survival, we compared signaling events between Treg and Tcon.
CD4+Foxp3+ Treg and CD4+Foxp32 Tcon were sorted into serumcontaining medium and lysed immediately after sorting. As shown
in Fig. 5A, Treg contain higher levels of ERK1/2 phosphorylation
than those of Tcon. ERK1/2 usually functions downstream of Ras
to promote cell proliferation. The increased ERK1/2 activation correlates with increased proliferation of Treg.
The mammalian target of rapamycin (mTOR) integrates environmental stimuli including growth factors, nutrients, and stress-ac-
tivated signals to regulate cell metabolism, survival, growth, and
proliferation. mTOR signaling is mediated through two signaling
complexes: mTOR complex 1 (mTORC1) and mTOR complex 2
(mTORC2). mTORC1 is sensitive to rapamycin inhibition, whereas
mTORC2 signaling is insensitive to acute rapamycin treatment.
mTORC1 promotes cell growth and proliferation through phosphorylating two major substrates: the 70-kDa ribosomal S6 kinase
(S6K1) and the translational repressor 4 elongation factor binding
protein 1 (4E-BP1). Activated S6K1 phosphorylates S6 to promote
ribosome biogenesis. Phosphorylated 4E-BP1 dissociates from eukaryotic initiation factor 4E to promote protein translation. mTORC2
phosphorylates Akt at serine 473, leading to an increase of Akt
activity (21–23).
FIGURE 3. Assessment of proliferative and surviving status of freshly
isolated Treg. A and B, Increased apoptosis of Treg. Freshly isolated splenocytes and LN cells from Foxp3GFP
mice were stained with CD4, CD8,
and annexin V. Annexin V staining
of CD4+Foxp32 , CD4+Foxp3+, and
CD8+ cells of mice was determined by
FACS. A, Representative contour plots.
B, Mean 6 SEM presentation of data
from multiple mice (n = 9). C and D,
Ki67 staining of Treg, Tcon, and CD8
T cells. Freshly isolated splenocytes
from C57BL6/J mice were stained with
CD4 and CD8 followed by intracellular
staining for Ki67 and Foxp3. C, Representative dot plot of Ki67 expression
in Treg, Tcon, and CD8 T cells. D,
Mean 6 SEM presentation of Ki67+
Treg, Tcon, and CD8+ cells in the
spleen and LN from multiple mice (n =
5).
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ICOS DEFINES PROLIFERATIVE VERSUS DEATH-PRONE Treg
FIGURE 4. Differential requirement
of CD28-mediated costimulation for
TCR-induced Treg and Tcon proliferation. A, Treg proliferation is less sensitive to CTLA4-Ig than Tcon. B, Treg
proliferation is less affected by CD28
deficiency than Tcon. Experiments were
performed similar to those of Fig. 1
except that CTLA4-Ig was added to
block costimulation (A) or CD282/2
T cells were compared with WT T cells
(B). Data shown are representative of
three experiments.
Both S6K1 and S6 phosphorylation was lower in CD4+Foxp3+
Treg than in Tcon. In contrast, 4E-BP1 phosphorylation was
higher in Treg than in Tcon (Fig. 5B). These observations revealed
that dissociation of phosphorylation of S6K1 and 4E-BP1 in Treg.
Akt phosphorylation at serine 473 was lower in Treg than in Tcon.
In addition, phosphorylation of Foxo1 and GSK3b, events mediated by Akt, was decreased in Treg compared with that in Tcon.
Thus, mTORC2 and Akt activities are decreased in Treg (Fig. 5C).
Consistent with the decreased Akt activity, cleaved active caspase
9 and caspase 3 were both increased in Treg, suggesting enhanced
activation of the intrinsic death pathway in Treg (Fig. 5D).
TCR signaling in Treg
It has been reported that Treg display defective signaling transduction after TCR engagement (11). For example, decreased calcium influx and ERK1/2 phosphorylation were reported in TCRstimulated CD4+CD25+ Treg (24). To compare TCR signaling
between Treg and Tcon, we rested sorted CD4+Foxp3+ Treg and
CD4+Foxp32 Tcon in PBS and then stimulated them with antiCD3. As shown in Fig. 5E, phosphorylation of PLCg1, ERK1/2,
IkBa, and NF-kB was obviously decreased in Treg compared with
that in Tcon after TCR stimulation, suggesting that proximal TCR
signaling and downstream signaling cascades are impaired in Treg.
Thus, the aforementioned increase of ERK1/2 phosphorylation in
Treg in Fig. 5A is likely induced by cytokines or other growth
factors existing in serum-containing medium.
Akt phosphorylation at S473 and Foxo1a phosphorylation were
diminished in Treg before and after TCR stimulation (Fig. 6B),
suggesting that TCR-induced mTORC2 activation is impaired in
Treg. Whereas S6K1 phosphorylation was decreased before and after
TCR stimulation in Treg, 4E-BP1 phosphorylation in Treg was
higher than that in Tcon before and after TCR stimulation, suggesting
again that Treg are distinctive from other cells in mTORC1 signaling.
Promoting Treg proliferation by IL-2
IL-2 plays important roles for Treg homeostasis. Deficiency of IL-2
causes decreased Treg numbers in mice, which can lead to autoimmune diseases (25–27). In contrast, injection of IL-2 plus an
anti–IL-2 Ab increases Treg numbers in mice, which can promote immune suppression (28). We tested how IL-2 may affect
the hyperproliferative and death-prone properties of Treg. IL-2
can significantly increase proliferation of Tcon (Fig. 6A, 6C).
IL-2 showed different effects on Tcon survival dependent on the
strengths of TCR stimulation. Without TCR stimulation, IL-2 promotes Tcon survival. When TCR stimulation was weak, IL-2
drastically promoted Tcon proliferation as well as death. When
TCR stimulation was strong, IL-2 could inhibit Tcon death (Fig.
6A, 6B). IL-2 also enhanced Treg proliferation. However, the effect of IL-2 to promote Treg survival was not obvious and could
only be observed when stimulated with a high concentration (1
mg/ml) of anti-CD3.
Distinguishing hyperproliferative and death-prone Treg subsets
by ICOS expression
An important issue that has arisen from our aforementioned data
are whether the hyperproliferative Treg and death-prone Treg represent different subsets of Treg. To address this question, it is
critical to identify markers that can be used to define Treg with
these distinct properties. It has recently been reported that human
Treg can be divided into ICOS+ and ICOS2 subsets and that the
suppressive activity of the ICOS+ Treg appears to be superior to
that of the ICOS2 Treg (29–31). ICOS has also been found to be
able to promote NKT cell survival (32). We examined the death
and proliferative properties of ICOS+ and ICOS2 Treg. Freshly
isolated ICOS2 Treg displayed higher death rates than those of
ICOS+ Treg (Fig. 7A). Moreover, ICOS+ Treg stained more intensively for Ki67 than that for ICOS2 Treg, suggesting that more
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FIGURE 5. Distinct signaling between Treg and
Tcon. A–D, Assessment of signaling in serumcontaining medium. T cells from WT spleens and
LNs were isolated by using magnetic beads. CD4+
Foxp3GFP2 Tcon and CD4+Foxp3GFP+ Treg
were sorted into FBS-containing medium and were
lysed immediately after sorting. Cell lysates were
examined for signaling events by immunoblot analysis with the indicated Abs. A, ERK1/2 phosphorylation. B, mTORC1 signaling. C, mTORC2–Akt
signaling. D, Cleaved caspase 9 and 3. E and F,
Comparison of TCR signaling between Treg and
Tcon. Sorted Tcon and Treg as described in A–D
were washed twice with PBS, rested in PBS at 37˚C
for 30 min, and then left unstimulated or stimulated
with an anti-CD3 Ab (500A2) for 5 min. Cell lysates were subjected to immunoblot analysis with
the indicated Abs. Data shown represent three
experiments.
ICOS+ Treg and Tcon than ICOS2 Treg and Tcon are cycling
in vivo (Fig. 7B). After in vitro TCR stimulation, the majority of
ICOS2 Treg died and did not proliferate. However, most ICOS+
Treg were alive and proliferated (Fig. 7C). ICOS expression on
Treg may alleviate the requirement of CD28 for TCR-induced
proliferation. ICOS+ Treg expressed a higher level of the proapoptotic molecule Bad than that of ICOS2 Treg, although they
express similar levels of Bax (Fig. 7D). Further studies should
determine whether differential Bad expression controls survival of
these two Treg subsets. Using a contact inhibition assay, we further compared the suppressive activities between these two subsets
of Treg. As shown in Fig. 7E, ICOS+ Treg displayed much
stronger suppressive activity than that of ICOS2 Treg. The inhibitory effect of ICOS+ Treg at 16:1 Tcon to Treg ratio was
similar to that of ICOS2 Treg at 1:1 Tcon to Treg ratio. Together,
these data indicate that ICOS expression defines two subsets of
Treg with distinct survival and proliferating properties as well as
obvious differences of suppressive activities.
Discussion
In this report, we simultaneously analyzed Treg proliferation and
death using fixable Live/Dead staining and the CFSE dilution assay.
We demonstrated that Treg contain two subsets with distinct death
and proliferation properties. The predominant subset is ICOS2 and
is prone to death, defective in proliferation, and weak in suppression. The rare subset is ICOS+, which is characterized by superior
survival, hyperproliferative, and highly suppressive properties. The
massive death of ICOS2 Treg after in vitro TCR stimulation may
be due to the activation of the intrinsic death pathway and contribute to the hypoproliferative phenotype of Treg during in vitro
TCR stimulation. Many studies on Treg proliferation and survival
have been performed with CD4+CD25+ cells. Because most Treg
die in vitro, our data suggest that it is critical to use Foxp3 rather
than CD25 whenever possible as the marker for Treg isolation
when studying Treg, particularly when examining their proliferative responses and death. A small contamination of Tcon in the
CD4+CD25+ population could well outnumber the rare proliferative ICOS+ Treg and thus significantly impact data interpretation.
ICOS+ and ICOS2 Treg have been noted for their difference in
several aspects. Human ICOS+ Treg use IL-10 to suppress dendritic cells and TGF-b to suppress T cells, whereas ICOS2 Treg
only use TGF-b for suppression (30). Human ICOS+ Treg have
also been found to contain stronger suppressive activity than that
of ICOS2 Treg (29, 31, 33). Our data showing that murine ICOS+
Treg are much more suppressive than ICOS2 Treg are consistent
with the data of human Treg. Of note, human ICOS+ Treg were
found to be more apoptotic than ICOS2 Treg (30). At present, the
reason for the difference in survival between human and murine
ICOS+ Treg is unclear. Murine ICOS2 Treg become apoptotic
within a few hours after in vitro TCR stimulation (data not
shown). Because the human Treg were purified based on CD25
expression, it would be of interest to determine whether these
CD25+ and ICOS+ Treg contain activated conventional T cells,
which may have a high death rate. ICOS appears to be unimportant for Treg generation in the thymus but is involved in Treg
homeostasis. Deficiency of either ICOS or its ligand results in
decreases of Treg numbers in the peripheral lymphoid organs (31,
34). In addition, expression of ICOS ligand by tumor cells promotes Treg expansion, which may contribute to tumor immune
suppression (35). Together, these observations and our data suggest that ICOS may directly promote Treg survival. The importance of ICOS to maintain Treg pool size and survival may help to
explain the recent finding that inhibition of ICOS signal in mice
may reduce Treg number, promote autoimmunity, and exacerbate
airway hyperresponsiveness (36–39).
Human CD4+ Treg can be divided into resting and activating/
memory subsets. It has been reported that the human CD4+
CD45RO+Foxp3+CD25hi Treg are highly proliferative but susceptible to apoptosis (40). In another report, human CD4+ Treg
were divided into CD45RA+Foxp3low resting, CD45RA2Foxp3hi
activated, and CD45RA2Foxp3low cytokine-secreting subpopulations. Both CD45RA+Foxp3low and CD45RA2 Foxp3hi Treg
1704
ICOS DEFINES PROLIFERATIVE VERSUS DEATH-PRONE Treg
FIGURE 6. Effects of IL-2 on Treg
proliferation and survival. Experiments were performed and analyzed
similarly to those of Fig. 1 except that
IL-2 was added at a concentration of
1000 U/ml in the test group. A, CFSE
and Live/Dead staining on gated total
Tcon and Treg. B, Death rates of Tcon
and Treg. C, Overlay of CFSE staining
in live Tcon and Treg. Data shown are
representative of three experiments.
are suppressive, whereas CD45RA2Foxp3low Treg are nonsuppressive. In addition, CD45RA+Foxp3low Treg appear to be the
precursors of CD45RA2Foxp3hi Treg, which are terminally differentiated and death prone (41). The CD45RA+Foxp3low Treg are
more proliferative and survive better than the CD45RA2Foxp3hi
Treg after TCR stimulation, which mimics the murine ICOS+ and
ICOS2 Treg to certain extents. However, the differences between
these human Treg subsets defined by Foxp3 levels and CD45RA
expression are not as drastic as those between the murine Treg
subsets defined by ICOS expression.
Our study has revealed common and different signaling features
between freshly isolated Treg in FBS-containing medium and in
PBS with or without TCR stimulation. Treg in FBS-containing
medium (Treg-FBS) display increased phosphorylation of ERK1/2
and Rsk1, substrate for ERK1/2, but decreased phosphorylation
of PLCg1 compared with those of Tcon. However, in Treg rested
in PBS (Treg-PBS), ERK1/2 and Rsk1 phosphorylation is drastically decreased and is much lower than that in Tcon in PBS. The
same is true for Treg-PBS stimulated with anti-CD3. These observations suggest that Treg may be hyperresponsive to growth
factors or cytokines in the serum-containing medium such as recently reported with leptin (42), and such signaling is independent
of PLCg1 activation. Additionally, distal signaling events that lead
to ERK1/2 activation should not be defective in Treg as ERK1/2
phosphorylation occurs at a high level in Treg-FBS. Although
Treg are hyperresponsive to yet to be identified stimuli in the
serum-containing medium, they are defective in TCR signaling.
The defects of TCR signaling in Treg are likely proximal to
PLCg1, as its phosphorylation is significantly decreased. Diminished IkBa and NF-kB phosphorylation as well as ERK1/2
phosphorylation is likely caused by diminished PLCg1-derived
diacylglycerol, which is the upstream activator for both the NF-kB
pathway and the Ras–ERK1/2 pathway (43).
Multiple reports have demonstrated that PI3K and mTOR signaling inhibits Treg differentiation and/or function and that Treg
display decreased mTOR and Akt activities (42, 44–49). Our
The Journal of Immunology
1705
FIGURE 7. Differential expression of ICOS defines proliferative and death-prone Treg subsets. A, Assessment of Treg death and survival of freshly
isolated Treg based on ICOS expression. Top panels, CD4 and Foxp3 staining and ICOS expression on CD4+Foxp3+ Treg. Bottom panels, Live/Dead
staining of ICOS+ and ICOS2 Treg. B, Ki67 staining of ICOS+ and ICOS2 Treg and Tcon. C, CFSE-labeled splenocytes were stimulated with an antiCD3 Ab (1 mg/ml; 2C11) for 24 or 48 h. Treg proliferation and survival were examined as for Fig. 1 except that Treg were gated for ICOS+ and ICOS2
subsets. Histogram shows overlay of CFSE staining of ICOS+ and IOCS2 Treg after 48 h TCR stimulation. D, Increased Bad expression in ICOS2 Treg.
Bad and Bax mRNA levels were determined by real-time quantitative PCR with cDNA synthesized from total RNA isolated from sorted ICOS+CD4+
Foxp3GFP+ and ICOS2CD4+Foxp3GFP+ Treg as templates. E, Assessment of suppressive function of ICOS+ and ICOS2 Treg using the contact inhibition assay. CFSE-labeled Thy1.1+Thy1.2+ Tcon were mixed with Thy1.2+ ICOS+ or ICOS2 Treg at the indicated ratios and mitomycin C-treated
TCRb2/2d2/2 splenocytes. Cells were left unstimulated or stimulated with an anti-CD3 Ab for 72 h and then stained for Thy1.1, Thy1.2, and CD4. Top
panels show CFSE intensity in gated Thy1.2+ Tcon from unstimulated or anti-CD3–stimulated cultures. Bottom panels are overlaid histograms of CFSE
intensity in gated Thy1.1+Thy1.2+ Tcon from anti-CD3–stimulated cultures in the presence of either ICOS2 Treg or ICOS+ Treg. At the 1:1 Tcon to Treg
ratio, only ICOS2 Treg were tested.
studies are consistent with the notion that mTOR signaling is
differentially regulated in Treg and Tcon. mTORC2 activation is
decreased in both Treg-FBS and Treg-PBS with or without TCR
stimulation, indicating that Treg are defective in mTORC2–Akt
signaling. At present, it is unclear how much the decreased Akt
activity in Treg may contribute to Treg death. In both Treg-FBS
and Treg-PBS with or without TCR stimulation, S6K1 phosphorylation is decreased but 4E-BP1 phosphorylation is increased
compared with those in Tcon. The dissociation of S6K1 and
4E-BP1 phosphorylation is quite striking and intriguing, as phosphorylation of these two proteins is usually mediated by mTORC1
and occurs concordantly in other cell types (23). Such dissociation
could be caused by altered composition of mTORC1 leading to
change of substrate preference and differential expression of
phosphatases for S6K1 and 4E-BP1 in Treg. Of note, S6K1
phosphorylation has been found to be increased in human CD4+
CD25+ T cells in one study. In that study, 4E-BP1 phosphorylation
was not examined. In another study, S6K1 and 4E-BP1 phosphorylation were both decreased in human CD4+CD25+ T cells
compared with those in CD4+CD252 cells (46, 48, 49). The
reason for the discrepancies between those studies and ours is
unclear at present.
1706
In summary, we have demonstrated that Treg display both
hyperproliferative and death-prone properties and that ICOS can
define Treg into two subsets based on these properties. The ICOS+
Treg are rare but are superior in survival, are hyperproliferative in
response to TCR stimulation, and contain strong suppressive activity. In contrast, the ICOS2 Treg are the predominant subset, are
death prone, exhibit defective proliferation capabilities, and contain weak suppressive activity. Our data suggest modulating Treg
survival as an important therapeutic strategy for autoimmune
diseases and cancer.
Acknowledgments
We thank Dr. Michael Kulis and Tommy O’Brien for critically reviewing
the manuscript, Li Xu for technical assistance, and Nancy Martin and Mike
Cook at Duke Cancer Center Flow Cytometry Core Facility for sorting cells.
Disclosures
The authors have no financial conflicts of interest.
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