This information is current as
of June 16, 2017.
IL-33 Is an Unconventional Alarmin That
Stimulates IL-2 Secretion by Dendritic Cells
To Selectively Expand IL-33R/ST2+
Regulatory T Cells
Benjamin M. Matta, Jeremy M. Lott, Lisa R. Mathews,
Quan Liu, Brian R. Rosborough, Bruce R. Blazar and Heth
R. Turnquist
Supplementary
Material
References
Subscription
Permissions
Email Alerts
http://www.jimmunol.org/content/suppl/2014/09/12/jimmunol.140048
1.DCSupplemental
This article cites 60 articles, 28 of which you can access for free at:
http://www.jimmunol.org/content/193/8/4010.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2014 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
J Immunol 2014; 193:4010-4020; Prepublished online 12
September 2014;
doi: 10.4049/jimmunol.1400481
http://www.jimmunol.org/content/193/8/4010
The Journal of Immunology
IL-33 Is an Unconventional Alarmin That Stimulates IL-2
Secretion by Dendritic Cells To Selectively Expand
IL-33R/ST2+ Regulatory T Cells
Benjamin M. Matta,* Jeremy M. Lott,* Lisa R. Mathews,*,† Quan Liu,*,‡
Brian R. Rosborough,*,x,{ Bruce R. Blazar,‖ and Heth R. Turnquist*,#
I
nterleukin-33 is a barrier tissue-expressed IL-1 family member
(1) that when released targets IL-1R–like 1, more commonly
referred to as ST2, to promote Th2 responses in T cells, mast
cells, eosinophils, basophils, and innate lymphoid cell populations
(2–6). Thus, the ST2/IL-33 axis plays a protective role in immunity
against parasitic infection (7), but may exacerbate pathologic conditions such as asthma (8), arthritis (9), eosinophilic pericarditis
(10), and dermatitis (11). Recently, pleiotropic functions of IL-33
have emerged, with several reports supporting a role for IL-33 in
promoting Th1 immune responses and antiviral immunity (12–14).
*Department of Surgery, Thomas E. Starzl Transplantation Institute, University of
Pittsburgh School of Medicine, Pittsburgh, PA 15261; †Department of Infectious
Diseases and Microbiology, University of Pittsburgh School of Public Health, Pittsburgh, PA 15261; ‡Department of Cardiovascular Surgery, Second Affiliated Hospital
of Harbin Medical University, Harbin 150086, China; xMedical Scientist Training
Program, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; {Graduate Training Program in Immunology, University of Pittsburgh School of Medicine,
Pittsburgh, PA 15261; ‖Division of Blood and Marrow Transplantation, Department
of Pediatrics, University of Minnesota, Minneapolis, MN 55455; and #Department of
Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
Received for publication February 19, 2014. Accepted for publication August 7,
2014.
This work was supported by National Institutes of Health Grants T32AI074490 (to
B.M.M.) and R00HL097155 (to H.R.T.), a Merck/United Negro College Fund Science Initiative postdoctoral fellowship (to J.M.L.), an American Heart Association
predoctoral fellowship (to B.R.R.), an American Society of Transplantation Basic
Science Faculty Development Grant (to H.R.T.), and a Roche Organ Transplantation
Research Foundation grant (to H.R.T.).
Address correspondence and reprint requests to Dr. Heth R. Turnquist, Starzl Transplantation Institute, University of Pittsburgh School of Medicine, 200 Lothrop Street,
E1554 BST, Pittsburgh, PA 15261. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: BM, bone marrow; CTV, CellTrace Violet; DC,
dendritic cell; DT, diphtheria toxin; mu, mouse; Treg, regulatory T cell; WT, wildtype.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400481
Although implicated in both Th1 and Th2 immunity, its capacity
to limit Th1 and Th17 responses in vivo (15, 16) suggests a potential
regulatory role for IL-33 and ST2 in controlling inflammation (6).
Likewise, in mouse models of cardiac transplantation, IL-33 administration prolongs fully MHC-mismatched allograft survival by
reducing Th1-mediated IFN-g production (in favor of elevated IL4, IL-5, IL-10, and IL-13) and increasing Foxp3+ regulatory T cells
(Treg) and CD11b+Gr-1int myeloid-derived suppressor cells systemically and in the graft (17–19). Our group has demonstrated that
prolongation of cardiac allograft survival by IL-33 monotherapy is
dependent on Foxp3+ Treg (18). Despite indications that IL-33
expands Treg (18), the precise characterization of IL-33–
expanded Treg and elucidation of the mechanisms contributing to
their expansion are lacking. Given the ability of IL-33 to target
CD11c+ dendritic cells (DC) (18, 20–22), and the critical role of
DC in Treg homeostasis and expansion (23, 24), we tested the
hypothesis that DC serve a critical function in orchestrating Treg
expansion mediated by IL-33.
In the current study, we identified a new functional interrelationship between IL-33 and IL-2 mediating selective Treg expansion. Specifically, although IL-33 fails to induce classical DC
maturation, it stimulates IL-2 production by CD11c+ DC that is
critical for expanding ST2-expressing Treg during interactions
with CD4+ T cells. In connection, a significant reduction in ST2
expression on DC in the absence of IL-2 may dampen DC responsiveness to IL-33. We further demonstrate that ST2 on DC is
critical for IL-33–mediated Treg expansion, as use of St22/2 DC
in vitro and depletion of CD11c+ DC in vivo inhibit this function
of IL-33. Collectively, our findings establish an important immunoregulatory function of IL-33 carried out through a novel
mechanism of CD11c+ DC-mediated Treg expansion. These data
highlight the IL-33/ST2 axis as a potential target for development
of therapeutic modalities aimed at promoting immune regulation
or expansion of Treg.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
IL-33 is a recently characterized IL-1 family member that is proposed to function as an alarmin, or endogenous signal of cellular
damage, as well as act as a pleiotropic cytokine. The ability of IL-33 to potentiate both Th1 and Th2 immunity supports its role in
pathogen clearance and disease immunopathology. Yet, IL-33 restrains experimental colitis and transplant rejection by expanding
regulatory T cells (Treg) via an undefined mechanism. We sought to determine the influence of IL-33 on hematopoietic cells that
drives Treg expansion and underlies the therapeutic benefit of IL-33 administration. In this study, we identify a feedback loop in
which conventional mouse CD11c+ dendritic cells (DC) stimulated by IL-33 secrete IL-2 to selectively expand IL-33R(ST2+)–
suppressive CD4+Foxp3+ Treg. Interestingly, this occurs in the absence of classical DC maturation, and DC-derived (innate) IL-2
increases ST2 expression on both DC and interacting Treg. ST2+ Treg represent an activated subset of Foxp3+ cells, demonstrated
to be ICOShighCD44high compared with their ST22 counterparts. Furthermore, although studies have shown that IL-33–exposed
DC promote Th2 responses, we reveal that ST2+ DC are required for IL-33–mediated in vitro and in vivo Treg expansion. Thus, we
have uncovered a relationship between IL-33 and innate IL-2 that promotes the selective expansion of ST2+ Treg over non-Treg.
These findings identify a novel regulatory pathway driven by IL-33 in immune cells that may be harnessed for therapeutic benefit or
for robust expansion of Treg in vitro and in vivo. The Journal of Immunology, 2014, 193: 4010–4020.
The Journal of Immunology
4011
BM chimeras and diphtheria toxin administration
Materials and Methods
Animals and IL-33 administration
b
d
Male C57BL/6J (B6; H2K ), BALB/c (H2K ), B6.FVB-Tg(Itgax-DTR/
EGFP)57Lan/J (CD11c-DTR), and B6.129P2-IL2tm1Hor/J (IL-22/2) mice
were purchased from The Jackson Laboratory (Bar Harbor, ME). BALB/c
St22/2 mice were obtained from A. McKenzie (Medical Research Council
Laboratory of Molecular Biology, University of Cambridge, Cambridge,
U.K.) and bred for experimental use (25). C57BL/6-Foxp3tm1Flv/J (Foxp3IRES-mRFP [FIR]) reporter mice (26) for breeding were graciously provided by F. Lakkis (Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA). All mice were housed in the specific pathogen-free
facility at the University of Pittsburgh School of Medicine. Recombinant
mouse (mu) IL-33 (BioLegend, San Diego, CA) was dissolved in PBS and
injected i.p. (0.5 mg/mu/d; for 10 d). Control animals received PBS alone.
Experiments were conducted under an Institutional Animal Care and Use
Committee–approved protocol and in accordance with National Institutes
of Health guidelines. Animals were fed a diet of Purina rodent chow
(Ralston Purina, St. Louis, MO) and received food and water ad libitum.
Propagation, purification, and treatment of CD11c+ DC
T cell isolation and MLR
T cells were purified from spleens of wild-type (WT) B6, B6 FIR, or
BALB/c mice. RBC-lysed, single-cell suspensions were incubated with
anti-CD45R/B220, anti-CD16/CD32, anti–TER–119, anti–I-A/I-E, antiCD11b, and anti-Ly6G/Ly6C obtained from BD Biosciences. For CD4+
or CD8+ T cells, anti-CD8a or anti-CD4 was included, respectively. Abbound cells were eliminated using Dynabeads (Life Technologies) following the manufacturer’s instructions. Control, LPS, or IL-33 DC were
used as stimulators in 5-d MLR of CellTrace Violet (CTV; Life Technologies, Grand Island, NY)–labeled CD4+ T cells at a 1:10 DC:T cell
ratio. Recombinant human IL-2 (50 U/ml; PeproTech, Rocky Hill, NJ), IL33 (10 ng/ml), or anti–IL-2 (10 mg/ml; JES6-1A12; BioLegend) were
added to the MLR, as indicated in the figures and figure legends.
Suppression assays
Bulk CD4+ T cells were purified from PBS- or IL-33–treated, B6 FIR mice
and stained for flow sorting (FACSAria; BD Biosciences, San Jose, CA) of
RFP+(Foxp3+) CD4+ ST2+ or ST22 Treg. Alternatively, splenic B6 FIR
CD4+ T cells were cultured with IL-33–exposed BALB/c DC, and Treg
were sorted after 5 d. Purity was consistently .92%. Sorted Treg were
tested for their ability to suppress CD3/CD28 T-activator bead (5 3 104;
Life Technologies)-induced, CTV-labeled B6 CD4+ or CD8+ T cell proliferation at a ratio of 8:1 T effector to Treg (1 3 105:1.25 3 104). Where
indicated, recombinant mu IL-12 (5 ng/ml; BioLegend) alone or in combination with IL-33 (10 ng/ml) was used to induce IFN-g in CD8+ T cell
cultures. Cultures were harvested on day 4 for flow cytometric analysis of
proliferation and IFN-g by intracellular flow analysis.
Flow cytometric analysis
Flow cytometry data were acquired using a LSRFortessa (BD Biosciences)
and analyzed using FlowJo v10 (Tree Star, Ashland, OR). Intracellular
staining was carried out using Foxp3/transcription factor–staining buffer
set (eBioscience, San Diego, CA). All fluorochrome-conjugated Abs were
purchased from BioLegend, eBioscience, or BD Biosciences, except ST2
(MD Bioproducts, St. Paul, MN).
Cytokine quantitation
Supernatants from CD11c+ cells cultured for 18 h were harvested and IL-2
quantified using IL-2 Ready-SET-Go! (eBioscience) kit, according to the
manufacturer’s instructions. Other cytokines measured in DC supernatants
were determined by Luminex (Life Technologies). IFN-g, IL-17A, and
IL-5 were quantitated in supernatants from MLR using ELISA Max kits
(BioLegend), according to the manufacturer’s instructions.
Statistics
Statistical significance was determined by Student’s t test and one- or twoway ANOVA, where appropriate, using GraphPad Prism version 5.00 for
Windows (GraphPad Software, San Diego, CA). A p value ,0.05 was
considered significant. All experiments were carried out independently for
a minimum of three times, unless indicated otherwise in the figure legends.
All bar graphs represent statistical mean 6 SD.
Results
IL-33 expands a ST2+ subset of CD4+CD25+Foxp3+ cells in
the thymus and spleen
We reported previously that IL-33 systemically expands functionally suppressive Foxp3+ Treg that prolong experimental cardiac allograft survival (17, 18). Our precise characterization of the
impact of IL-33 on Treg populations in the thymus and spleen now
reveals that administration of IL-33 profoundly modulates Treg
populations, particularly an ST2+ (IL-33R+) subset, in both primary and secondary lymphoid organs (Fig. 1). Interestingly, ST2+
Foxp3+ cells were present at a low frequency in naive, unmanipulated mice, comprising ∼10% of CD3+CD4+CD25+ cells found
in both the thymus and spleen (Fig. 1A). Administration of IL-33
expanded CD4+CD25+ cells, particularly ST2+Foxp3+ cells, and
in the spleen they approach a proportion similar to that of ST22
Foxp3+ cells (Fig. 1A). Interestingly, IL-33 decreases the total
number of cells in the thymus while increasing the total number of
splenocytes (Fig. 1B), corresponding to a significant increase in
total CD4+Foxp3+ cells, including ST2+Foxp3+ cells in the spleen
(Fig. 1C). These data substantiate that ST2+Foxp3+ cells are an existing subset of Treg and are expanded following delivery of IL-33.
Phenotypic analysis of CD4+ Foxp3+ compared with Foxp32
cells (Fig. 2A) from untreated and IL-33–treated mice revealed
expression of classical Treg markers on ST2+Foxp3+ cells, including PD-1, CTLA-4, LAG-3, OX-40, LAP (TGF-b), CD39, CD73,
CD103, and GARP (Fig. 2B). Several distinguishable characteristics of ST2+ Treg relative to their ST22 counterparts, especially
following IL-33 administration, included higher GATA-3, ICOS,
CD44, and CD69, with corresponding low expression of CD62L
(Fig. 2B). Thus, ST2+Foxp3+ cells, while sharing expression of
classical Treg markers, are distinct from their ST22Foxp3+ counterparts and display markers consistent with activated Treg (29, 30).
IL-33–expanded Treg regulate effector T cell responses
We next tested the suppressive function of Treg from PBS- and IL33–treated mice, including a comparative analysis of the ST22 and
ST2+ subsets. We found at a Treg:T effector ratio, in which conventional Treg (control ST22) failed to significantly suppress effector T cell proliferation, IL-33–expanded Treg exhibit significant
suppressive capacity against both CD4+ and CD8+ T cells (Fig.
3A). Although ST2+ Treg consistently proved more potent than
ST22 Treg, we did not observe a significant difference between the
ST22 and ST2+ subsets isolated from IL-33–treated mice.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
CD11c+ bone marrow (BM)–derived DC were propagated from total BM
cells using recombinant mu GM-CSF (1000 U/ml; R&D Systems, Minneapolis, MN) alone or in combination with recombinant mu IL-4 (1000
U/ml; Schering-Plough, Kenilworth, NJ) and harvested on day 7, as described (18, 27, 28). Briefly, culture medium supplemented with GM-CSF
and IL-4 was replenished every 2 d, and CD11c+ cells were purified using
magnetic beads (Miltenyi Biotec, Auburn, CA) on day 7. Purified CD11c+
cells were cultured overnight in complete RPMI 1640 media alone or
complete media supplemented with either 100 ng/ml LPS (Enzo Life
Sciences, Farmingdale, NY) or 20 ng/ml IL-33.
Recipient B6 mice were given antibiotic-treated (Sulfatrim; Hi-Tech
Pharmacal, Amityville, NY) water for 7 d prior to irradiation. Chimeric
mice were generated by irradiating WT B6 recipient mice with two doses
(900 cGy and 400 cGy) in a span of 6 h. Two hours after the second radiation
dose, mice received 1 3 107 syngeneic WT or CD11c-DTR B6 BM. Mice
were continued on antibiotic-treated water for 7 d. For depletion of
CD11c+ cells (starting 8 wk post-BM transplant), diphtheria toxin (DT;
Sigma-Aldrich, St. Louis, MO) was administered (125 ng/mouse) every
other day for 10 d (a total of 6 doses), and mice were euthanized 2 d after
the last dose. Mice were also treated with PBS or IL-33 (0.5 mg/m/d; for
10 d) starting 1 d after the first DT treatment and ending 1 d after the last
DT treatment. Spleens were harvested 1 d after the final treatment with
IL-33, and total splenocytes were stained for flow cytometric analysis.
4012
IL-33 EXPANDS ST2+ Treg VIA DENDRITIC CELL IL-2
Although much has been elucidated on the role of IL-33 and ST2
in promoting Th2 responses, work with our collaborators revealed
that IL-12 induces ST2 expression on CD8+ T cells, thus promoting IL-33–augmented IFN-g production (13). Our present data
recapitulate these observations (Fig. 3B). However, we also reveal
that, although Treg from IL-33–treated mice did not inhibit CD8+
T cell IFN-g driven by IL-12 alone, they potently suppressed IL33–augmented CD8+ T cell IFN-g expression (Fig. 3B). ST2+
Treg reduced IFN-g production by ∼50% and displayed a trend
toward greater suppressive capacity relative to ST22 Treg from
IL-33–treated mice (Fig. 3B). In contrast, ST22 Treg from PBStreated mice did not significantly impact IFN-g expression at ratios examined (data not shown). In total, these data establish that
IL-33–expanded Treg are effective T cell suppressors and, to our
knowledge, are the first to make the important observation that IL33–expanded Treg can limit IL-33–augmented IFN-g production
by CD8+ T cells.
IL-33–exposed DC promote Th2 cytokine responses and
proliferation of ST2+Foxp3+ Treg in vitro
Administration of Flt3 ligand in mice augments naturally occurring Treg through a profound expansion of DC (23, 24). Yet, when
we directly compared administration of IL-33 to Flt3 ligand, IL-33
did not drastically augment CD11c+ cell numbers, despite its
ability to increase the incidence of CD4+Foxp3+ Treg (18). Thus,
our past findings do not support increased DC numbers as a
mechanism driving an increase in Treg following IL-33 delivery.
However, DC express ST2 (20, 21, 27), and, therefore, we sought
to determine whether IL-33 expansion of Treg is mediated instead
through a direct impact of IL-33 on DC function.
Consistent with previous observations (21), DC exposed to IL33 promoted Th2 responses in CD4+ T cells, manifested in increased secretion of IL-5, but not IFN-g (Th1) or IL-17A (Th17)
(Fig. 4A). Interestingly, we observed IL-33–exposed DC also
promoted the proliferation of ST2+Foxp3+ Treg (Fig. 4B). Although TLR4 and ST2 are both part of the TLR/IL-1R family, the
function of IL-33–exposed DC was distinct relative to DC exposed
to LPS, which augmented IFN-g and IL-17A levels, but failed to
increase IL-5 (Fig. 4A) or proliferation of ST2+ Treg (Fig. 4B).
ST2+Foxp3+ cells expanded in vitro by IL-33–exposed DC
expressed high levels of ICOS and CD44 (Fig. 4C), a phenotype
that matches splenic ST2+Foxp3+ Treg expanded by IL-33 administration in vivo (Fig. 2B). Ex vivo expanded ST2+ Treg were
functionally suppressive (Fig. 4D). Confirming that ST2 expression on DC was central to this phenomenon, St22/2 DC were poor
stimulators of ST2+ Treg proliferation (Fig. 4E) compared with
WT (St2+/+) DC. This limited proliferation of ST2+ Treg occurred
despite the addition of exogenous IL-33 in the MLR, and the
presence of St2+/+ CD4+ T cells, which could potentially respond
to IL-33 directly. Overall, these data support IL-33 targeting DC
as a critical mechanism of ST2+ Treg expansion.
IL-33 stimulates IL-2 production by CD11c+ DC
Several groups have described limited phenotypic changes associated with the Th2-promoting capacity of DC exposed to IL-33
(21, 22). However, mechanisms underlying the ability of IL33–exposed DC to influence T cell responses are unknown. We
characterized DC phenotype and cytokine production following
overnight culture with IL-33, and compared these findings with
DC exposed to LPS. In stark contrast to stimulation with LPS,
exposure of BALB/c DC to IL-33 had a subtle impact on surface
marker expression, as we observed a small, but significant increase in CD86 and programmed death ligand-1 (PD-L1), but no
significant change in MHC class II (I-Ad), CD40, CD80, or programmed death ligand-2 (PD-L2; Fig. 5A). Similar results were
observed when DC were propagated from B6 mice (data not
shown). Furthermore, unlike LPS, IL-33 did not induce significant
production of proinflammatory cytokines (Fig. 5B), indicating that
IL-33 fails to induce robust DC maturation. In fact, even when DC
were pre-exposed to IL-33, they maintained their ability to respond to subsequent stimulation with LPS (Fig. 5C).
Past investigation revealed that IL-2 signaling promotes ST2
expression on CD4+ T cells (31), and a growing body of literature
suggests that pathogen-exposed DC are an unappreciated, yet
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 1. IL-33 administration expands a ST2+ subset of CD4+CD25+Foxp3+ T cells originating in the thymus. (A) Representative flow cytometry
plots and frequency of CD4+CD25+ and CD4+CD25+Foxp3+ cells from CD3+CD4+-gated total thymocytes or splenocytes from naive or IL-33–treated (IL33 Rx) C57BL/6J (B6) mice. (B) Absolute number of total cells and (C) indicated cell populations in the thymus and spleens averaged from n = 5 mice.
*p , 0.05, **p , 0.01, ***p , 0.001.
The Journal of Immunology
4013
significant source of IL-2 for T cells (32, 33). We now make the
important observation that IL-33 stimulated a 5- to 6-fold increase
in IL-2 secretion compared with levels produced by control DC
(Fig. 5D, Supplemental Fig. 1). This finding was substantiated
using IL-22/2 DC (Fig. 5D) and DC propagation conditions with
(Fig. 5D) and without IL-4 (Supplemental Fig. 1). Interestingly,
IL-33 did not increase CD25 expression on DC above levels on
control DC, whereas LPS induced a 2.5-fold increase in its expression (Fig. 5E). Collectively, these data indicate that by maintaining low CD25 expression and significantly increasing IL-2
production, DC exposed to IL-33 provide a significant source of
IL-2 to support ST2+ Treg expansion.
CD11c+ DC production of IL-2 is necessary for
IL-33–mediated proliferation of ST2+ Treg
The role of IL-2 in driving T cell–mediated immune responses is
well established (34–36). More recently, DC delivery of IL-2 to
Treg within the synapse has been suggested to facilitate Treg
function and expansion (37, 38). Our data to this point argue that
enhanced production of IL-2 by DC in response to IL-33 may be
a critical mechanism through which IL-33 promotes ST2+ Treg
proliferation. To test this, we added a neutralizing Ab against IL-2
to the MLR and observed complete inhibition of ST2+ Treg proliferation driven by IL-33–exposed DC (Fig. 6A, 6B). Importantly,
IL-33–DC (and control DC + IL-33) demonstrated a preferential
stimulation of ST2+Foxp3+ cells (Fig. 6C) over ST22Foxp3+ cells,
and all Foxp32 T cells. To confirm that IL-2 produced by IL-33–
exposed DC was critical for ST2+ Treg expansion, we repeated the
experiment with DC propagated from IL-22/2 mice and observed
that, unlike WT DC, IL-33–exposed IL-22/2 DC did not expand
ST2+ Treg beyond that of their untreated counterparts (Fig. 6D).
Interestingly, IL-22/2 DC also showed reduced expression of ST2
and CD25 (Supplemental Fig. 2). Overall, these data indicate that
innate IL-2 signaling in DC is necessary for steady-state expression of ST2 and, presumably, their subsequent ability to respond to
IL-33 with IL-2 production that drives selective expansion of
ST2+ Treg.
In both control DC and IL-33–exposed DC cultures, exogenous
IL-2 alone did not significantly expand ST2+ Treg, nor did it
augment IL-33–mediated ST2+ Treg expansion (Fig. 6A, 6B).
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 2. ST2+Foxp3+ cells express classical Treg markers and exhibit an activated phenotype. IL-33 was administered to B6 mice for 10 d (0.5 mg/d,
i.p.). On day 11, spleens were harvested and CD3+ cells were purified and stained for flow cytometric analysis. (A) Gating strategy on CD3+CD4+-gated
cells and (B) expression of surface and intracellular markers on CD4+Foxp32 (G1; light gray; top), CD4+Foxp3+ST22 (G2; medium gray; middle), and
CD4+Foxp3+ST2+ (G3; dark gray; bottom) cells, as indicated in (A). Bar graphs in (B) represent an average of the mean fluorescence intensity (MFI) for
each marker. Data are representative of an average of n = 5–9 individual mice from n = 3 independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001.
4014
IL-33 EXPANDS ST2+ Treg VIA DENDRITIC CELL IL-2
Instead, the addition of exogenous IL-2 did display a trend toward
increasing the incidence of ST2+ non-Treg (Fig. 6C). Addition of
IL-33 to control DC cultures, however, profoundly increased ST2+
Treg (Fig. 6), and this effect was ablated by addition of anti–IL-2
Abs (Fig. 6). Thus, treatment of DC with IL-33, either before or
during culture with CD4+ T cells, supports the selective expansion
of suppressive ST2+ Treg over ST22 Treg and non-Treg.
Following IL-33 administration (Fig. 2B) or culture with IL33–exposed DC (Fig. 4C), ICOShighCD44highST2+ Treg are the
dominant expanded Treg population. However, the addition of
anti–IL-2 reduced ICOShighST2+ Treg incidence to the same level
of that of control DC alone (Supplemental Fig. 3A), and similar
results were observed for CD44highST2+ Treg (Supplemental Fig.
3B). Correspondingly, unlike WT DC, IL-33–exposed IL-22/2
DC failed to significantly increase either CD44high or ICOShigh
ST2+Foxp3+ cells (Supplemental Fig. 3C). These in vitro data
further support a mechanism in which IL-2 production by IL-33–
exposed DC underlies ICOShighCD44highST2+ Treg proliferation
and correlates with our ex vivo analysis of ST2+ Treg following
IL-33 administration (Fig. 2B). These data lend strong support
for DC as the critical mediators of IL-33–induced expansion of
ST2+ Treg in vivo.
CD11c+ DC mediate expansion of ST2+ Treg by IL-33 in vivo
The ability of IL-33 to increase Treg in vivo is dependent on
expression of ST2 (18), and the present data reveal that IL-33–
exposed DC expand ICOShighCD44highST2+ Treg in vitro that
parallel those expanded by IL-33 in vivo. The failure of St22/2
DC to expand ST2+ Treg suggests DC may promote this phenomenon in vivo. To test this, we generated BM chimeras whereby
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 3. IL-33–expanded Treg suppress CD8+ effector T cell function. B6 Foxp3-IRES-mRFP (FIR) mice were administered IL-33 (0.5 mg/d; for
10 d). CD4+ cells were purified on day 11 from total splenocytes and stained for flow sorting of CD4+ RFP(Foxp3)+ ST22 or ST2+ Treg. Sorted Treg were
cultured with purified CTV-labeled, B6 CD4+ or CD8+ T cells at a Treg:T effector ratio of 1:8 with CD3/CD28 T-activator beads for 4 d. Cultures were
harvested and stained for flow cytometric analysis of T cell proliferation. (A) Representative flow plots and Division Index Relative to ‘No Treg’ group of T
cell proliferation based on CTV dilution profile. (B) Representative flow plots and an average of IFN-ghighCD8+ T cells unstimulated or stimulated with 5
ng/ml IL-12 alone or in combination with 10 ng/ml IL-33. Data were generated from n = 3 independent experiments. Statistical significance in (A) and (B)
was determined by one-way ANOVA. *p , 0.05, **p , 0.01. DI, division index.
The Journal of Immunology
4015
WT B6 mice were reconstituted with BM from CD11c-DTR mice,
or WT B6 as controls. Recipient mice were subsequently administered diphtheria toxin (DT) to effectively deplete CD11c+
DC concurrently during IL-33 administration (Fig. 7A). Nonirradiated controls, mice reconstituted with WT B6 BM, and mice
reconstituted with CD11c-DTR BM all show significant expansion
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 4. IL-33–exposed DC promote Th2 responses in allogeneic naive
CD4+ T cells and proliferation of ST2+
Foxp3+ cells. BALB/c BM-derived DC
were generated in 7-d culture and CD11c
purified. CD11c+ cells were incubated for
18 h in medium alone, stimulated with
LPS (100 ng/ml) or IL-33 (20 ng/ml),
washed, and then used to stimulate CTVlabeled, naive allogeneic B6 CD4+ T cells
for 5 d. On day 5, (A) concentrations of
IFN-g, IL-17A, and IL-5 in DC/T cell
coculture supernatants were quantitated
by ELISA. (B) ST2+ and Foxp3+ CD4+
cells were quantitated by flow cytometry
following surface and intracellular staining and averaged from n = 4 independent
experiments. Dashed line (ST22) and
solid line (ST2+) boxes correspond to
populations in (C). (C) Analysis of ICOS
and CD44 expression on Foxp3+-gated
cells expanded by IL-33–exposed DC
shown in (B) (ST22, dashed; ST2+, solid).
(D) BALB/c DC, generated as described
above, were used to stimulate CD4+
T cells from B6 FIR reporter mice. Flowsorted ST2+ Foxp3(RFP)+ CD4+ T cells
from IL-33–exposed DC cultures displayed potent capacity to inhibit the
anti-CD3/CD28–induced proliferation of
CD4+CD252 T cells (Teff) at a ratio of 1
Foxp3+ cell to 8 Teff. (E) WT (St22/2) or
St22/2 BALB/c CD11c+ DC were cultured with CTV-labeled B6 CD4+ T cells
in medium alone or supplemented with
10 ng/ml IL-33. After 5 d, proliferating
ST2+Foxp3+ T cells (Foxp3+ cells from
ST2+ CTVlow gate in left panel) were
quantified by flow cytometry and averaged from n = 4 independent experiments. Black boxes on flow plots indicate
populations used to generate corresponding graphs. *p , 0.05, **p , 0.01,
***p , 0.001.
of ST2+ Treg following IL-33 treatment as compared with PBStreated controls (Fig. 7B). As reported previously (23), administration of DT alone to CD11c-DTR chimeric mice reduced the
frequency of total CD4+Foxp3+ Treg (Fig. 7B). Although depletion of CD11c+ cells alone did not modulate incidence of existing
ST2+Foxp3+ cells, IL-33 administration failed to expand ST2+
4016
IL-33 EXPANDS ST2+ Treg VIA DENDRITIC CELL IL-2
Treg when CD11c+ cells were ablated concurrently (Fig. 7B).
These critical data are in accordance with our findings in vitro and
again reveal that, although T cells, including Treg, may express
ST2, IL-33 does not facilitate expansion of ST2+ Treg in the absence of CD11c+ DC that can respond to IL-33 (Fig. 8).
Discussion
IL-33 is expressed by epithelial and endothelial cells in most organs (1, 39, 40), and proinflammatory cytokines or TLR ligands
increase its expression in tissues and myeloid cells (14, 41–44).
Any means supporting IL-33 secretion are uncertain, yet unprocessed IL-33 is functional when released during necrosis (6, 45).
Thus, IL-33 is described as an alarmin, or endogenous factor activating immune responses when released from damaged tissue
(14, 46–48). DC, expressing TLR/IL1R family members such as
TLR4 and ST2 (20, 21, 27), are well equipped to recognize
alarmins, such as IL-33 or high-mobility group box 1, which, like
LPS, targets TLR4 (49). Both LPS and high-mobility group box 1
mature DC into potent immunostimulatory cells that provide the
ample MHC, costimulatory molecules, and IL-1b, -6, -23, and IL12p70 needed for Th1 and Th17 polarization (50). Likewise, it has
been recently established that IL-12p70 potently inhibits Treg
proliferation (51). Thus, IL-33 is an unconventional alarmin that
fails to induce classical DC maturation or support Th1 immunity.
Although we substantiated a capacity of IL-33–exposed DC to
generate Th2 responses (21), we establish that IL-33–exposed DC
support expansion of ST2+Foxp3+ Treg. In total, these data suggest IL-33 is an unconventional alarmin that preserves DC in
a relatively immature state, but induces functional changes me-
diating their ability to polarize naive T cells into Th2 effectors and
expand Treg.
DC have the ability to produce IL-2 following TLR stimulation (32,
33), and our present analysis revealed significantly elevated IL-2
production by DC following overnight stimulation with IL-33.
When DC were generated from BM in GM-CSF and IL-4, IL-33
stimulated more IL-2 production by DC compared with LPS,
whereas LPS induced increased IL-2 compared with IL-33 when DC
were propagated using GM-CSF alone. Nonetheless, IL-33 induced
a significant increase over control DC in both culture conditions, and
the overall fold increase was similar in DC from cultures with GMCSF with or without IL-4. Also, whereas we observed lower IL-2
produced by DC generated in GM-CSF alone in response to LPS
compared with amounts previously reported (52), our studies used
100-fold less LPS (100 ng/ml versus 10 mg/ml). In total, these
data demonstrate that IL-33 stimulates DC IL-2 secretion in vitro
and suggest that DC signaling induced by IL-4, or closely related
IL-13, may shape ST2 signaling to promote IL-2 secretion.
IL-33 initiates ST2 signaling that leads to activation of NFAT and
AP-1 (53, 54), two transcription factors that are critical for IL-2
production (36). Interestingly, NFAT family members have also
recently been implicated in the regulation of monocyte and macrophage TLR-mediated responses (55). As ST2 is a member of the
TLR/IL-1R superfamily, our observation that IL-33, like LPS,
induces IL-2 is consistent with a common signaling pathway
shared between TLR4 and ST2 (7). However, IL-33 induces IL-2
production, yet fails to initiate the profound DC maturation triggered by LPS exposure. Given these different functional outcomes,
it will be important to complete future studies identifying overlap
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 5. IL-33 induces IL-2 production by CD11c+ DC in the absence of classical phenotypic maturation. BALB/c, B6 wild-type, or IL-22/2 BMderived CD11c+ DC were generated in 7-d culture and cultured for 18 h in medium alone or supplemented with IL-33 (20 ng/ml) or LPS (100 ng/ml) (A–E),
or pre-exposed to IL-33 for 18 h or 2 h prior to stimulation with LPS (C). Cells were harvested and stained for flow cytometric analysis of surface marker
expression (A, C), and supernatants were harvested for cytokine quantitation by (B) Luminex or (D) ELISA. Histograms (A) are representative of n = 3
independent experiments. Data in (B) and (D) are representative of n = 4 (LPS) or n = 5 (Control; IL-33) independent experiments. (E) CD25 expression on
BALB/c CD11c+ DC. For (C): *, versus control; #, versus IL-33. *p , 0.05, **p , 0.01, ***p , 0.001. ND, not detectable.
The Journal of Immunology
4017
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 6. CD11c+ DC-derived IL-2 promotes selective expansion of ST2+ Treg following IL-33 exposure. CD11c+ (A–C) BALB/c, (D) WT B6, or IL22/2 DC were cultured overnight in media alone or supplemented with IL-33 (20 ng/ml). DC were cultured in MLR with CTV-labeled (A–C) B6 FIR or (D)
BALB/c CD4+ T cells for 5 d. Some wells were supplemented with IL-33 (10 ng/ml), neutralizing IL-2 Ab (10 mg/ml), IL-2 (50 U/ml), or a combination, as
indicated. After 5 d, cells were harvested and stained for flow cytometric analysis. Representative flow plots and an average of (A) Foxp3 and (B) Foxp3 and
ST2 expression on CD4+-gated cells or (C) ST2 expression versus CTV on CD4+ Foxp3+ (top panels) and CD4+Foxp32 (bottom panels) cells. Results in
(A–C) were averaged from n = 4 independent experiments. (D) Representative flow plots of ST2 expression versus CTV on CD4+Foxp3+ cells from cultures
with WT B6 or IL-22/2 DC and averaged from n = 3 independent experiments. The graph represents the fold change in (Figure legend continues)
4018
IL-33 EXPANDS ST2+ Treg VIA DENDRITIC CELL IL-2
and divergence in NFAT, AP-1, and other pathways in DC involved in TLR–IL-1R family signaling following IL-33 stimulation to that generated by TLR ligands, such as LPS.
IL-2 is known to induce ST2 expression by CD4+ T cells, especially in conjunction with IL-33 (31, 56). Moreover, IL-2 is both
important for Th2 differentiation (57) and critical for Treg development (35, 37, 58). We demonstrate that neutralization of IL-2
in the presence of IL-33–exposed DC completely blocks ST2+
Treg expansion. Because activated T cells are a major source of
IL-2 (34), the importance of DC-derived IL-2 in ST2+ Treg expansion was established using DC generated from IL-22/2 mice.
Significantly, IL-33–exposed IL-22/2 DC failed to induce proliferation of ST2+ Treg over that of untreated DC, which is in
contrast to their WT counterparts. The importance of IL-2 derived
from IL-33–exposed DC in selective ST2+ Treg expansion is
further supported by our observation that addition of exogenous
IL-2 alone to control or IL-33–exposed DC–T cell cocultures
failed to significantly augment ST2+ Treg proliferation. In total,
our data support DC delivery of IL-2 to CD25high Treg, potentially
into the DC:Treg synapse (38), as a driving factor underlying
the ability of IL-33–exposed DC to selectively support proliferation of ST2+ Treg over other T cell subsets during interactions
with the bulk CD4+ T cell population. However, our studies do not
rule out the possibility that non-DC sources of IL-2, in the presence of IL-33 and TCR stimulation provided by DC, could contribute to ST2+ Treg proliferation. Further mechanistic studies
into the relationships between TCR stimulation and DC/T cell
IL-2 and IL-33 signaling are clearly warranted.
ST2+ cells, including Treg, are present in non–IL-33–treated
mice, and the failure of IL-33 to expand ST2+ Treg in vivo when
CD11c+ cells are depleted supports a dominant role for DC
driving ST2+ Treg expansion. Because IL-33 only slightly modulates CD11c+ DC incidence (18), it is unlikely that IL-33 expands Treg through amplification of DC numbers, as does Flt3
ligand (24). That IL-33 fails to potently expand ST2+ Treg in
interacting CD4+ T cells when DC are St22/2 also supports IL33–induced changes in DC underlying their ability to expand
ST2+ Treg. We do not find evidence that IL-33 increases DC IL-33
message nor induces IL-33 secretion (data not shown), suggesting
IL-33–exposed DC do not secrete IL-33 to directly expand ST2+
Treg. However, additional experimentation is necessary to precisely define whether ST2-mediated cross-presentation of IL-33
by DC plays any role in the expansion of ST2+ Treg or how the
function and homeostasis of ST2+ Treg are impacted by IL-33
once innate IL-2 has initiated their expansion.
A related and intriguing finding was that LPS induced a prominent increase in CD25 expression on DC, whereas IL-33 did not.
Presumably, this permits maximum availability of innate IL-2 to
target local cells. Furthermore, IL-22/2 DC themselves exhibit
reduced ST2 expression, supporting a mechanism in which innate
IL-2 facilitates both autocrine and local cell expression of ST2.
Based on these data, we propose a regulatory loop in which
proliferating ST2+ cells with IL-33 DC versus control DC for WT and IL-2 KO. Black boxes on flow plots indicate populations used to generate corresponding graphs. *p , 0.05, **p , 0.01, ***p , 0.001. AU, arbitrary units for fold change reported in graphs in (C) and (D).
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 7. Ablation of CD11c+ cells during IL-33 administration prevents IL-33–mediated expansion of ST2+Foxp3+ Treg in vivo. (A) Representative
flow plots and frequency of CD11c+ splenocytes in CD11c-DTR BM chimeras receiving PBS, IL-33, DT, or DT + IL-33. (B) Representative flow plots and
frequency of CD4+-gated, ST2+Foxp3+ Treg in PBS or IL-33–treated control mice (No BMT) and irradiated mice reconstituted with WT B6 or CD11cDTR BM and treated with DT alone, or DT + IL-33. Flow plots are representative, and graphs are an average of n = 3–9 mice per group from n = 3
independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001.
The Journal of Immunology
4019
innate IL-2 regulates ST2 expression on local leukocytes to promote Th2 responses and preferentially expand ST2+ Treg (Fig. 8).
We previously demonstrated that administration of IL-33 requires recipient ST2 expression and Treg to promote heart transplant survival (18). The present examinations reveal that ST2+
Treg are present in the primary and secondary lymphoid organs of
naive mice and profoundly expand after IL-33 administration with
the assistance of DC. A population of ST2-expressing Treg was
shown recently to infiltrate damaged muscles and facilitate their
repair (59). Thus, ST2+ Treg may possess unique biologic functions in addition to their demonstrated suppressive capacity. Other
groups have described CD44high and ICOShigh Treg subsets that
exhibit enhanced suppressor function compared with their corresponding counterparts (27, 28). We found that ST2+ Treg
exhibited the greatest suppressor activity against CD8+ effector
T cell function, including an ability to dampen IL-33–augmented
CD8+ T cell IFN-g production. Therefore, in circumstances in
which IL-33, released as an alarmin, may act on other ST2+ leukocytes and promote disease immunopathology (8–11), consumption of IL-33 by ST2+ Treg may be an important regulator of
IL-33–associated inflammatory responses. Given the ability of IL33–exposed DC to selectively expand ST2+ Treg over non-Treg, it
is easy to envision the development of IL-33–exposed DC as a
means to facilitate Treg expansion to support efforts for their use
as regulatory cell therapy to prevent graft-versus-host disease
following hematopoietic stem cell transplant or support induction
of transplantation tolerance (60, 61).
This study makes several new observations regarding IL-33,
DC, and Treg immunobiology. We identify an unappreciated mechanism of IL-33–mediated immune regulation in which ST2+ Treg
are expanded through IL-33–driven production of IL-2 by CD11c+
DC. The result of this newly established IL-33/IL-2 axis in
CD11c+ DC is the ability to preferentially expand an activated
subset of suppressive Foxp3+ Treg expressing ST2. Finally, our
demonstration, both in vitro and in vivo, that IL-33–exposed DC
potently and selectively expand ST2+ Treg sets the stage to develop and potentially harness this new knowledge in ongoing
experimental and clinical attempts to apply the regulatory capacity
of both DC and Treg.
Acknowledgments
We thank Dr. Hongmei Shen and the Starzl Transplant Institute Flow
Cytometry Core Facility for their assistance.
Disclosures
The authors have no financial conflicts of interest.
References
1. Pichery, M., E. Mirey, P. Mercier, E. Lefrancais, A. Dujardin, N. Ortega, and
J. P. Girard. 2012. Endogenous IL-33 is highly expressed in mouse epithelial
barrier tissues, lymphoid organs, brain, embryos, and inflamed tissues: in situ
analysis using a novel Il-33-LacZ gene trap reporter strain. J. Immunol. 188:
3488–3495.
2. Schmitz, J., A. Owyang, E. Oldham, Y. Song, E. Murphy, T. K. McClanahan,
G. Zurawski, M. Moshrefi, J. Qin, X. Li, et al. 2005. IL-33, an interleukin-1-like
cytokine that signals via the IL-1 receptor-related protein ST2 and induces T
helper type 2-associated cytokines. Immunity 23: 479–490.
3. Licona-Limón, P., L. K. Kim, N. W. Palm, and R. A. Flavell. 2013. TH2, allergy
and group 2 innate lymphoid cells. Nat. Immunol. 14: 536–542.
4. Mirchandani, A. S., R. J. Salmond, and F. Y. Liew. 2012. Interleukin-33 and the
function of innate lymphoid cells. Trends Immunol. 33: 389–396.
5. Koyasu, S., and K. Moro. 2011. Innate Th2-type immune responses and the
natural helper cell, a newly identified lymphocyte population. Curr. Opin. Allergy Clin. Immunol. 11: 109–114.
6. Liu, Q., and H. R. Turnquist. 2013. Implications for interleukin-33 in solid organ
transplantation. Cytokine 62: 183–194.
7. Liew, F. Y., N. I. Pitman, and I. B. McInnes. 2010. Disease-associated functions
of IL-33: the new kid in the IL-1 family. Nat. Rev. Immunol. 10: 103–110.
8. Kurowska-Stolarska, M., P. Kewin, G. Murphy, R. C. Russo, B. Stolarski,
C. C. Garcia, M. Komai-Koma, N. Pitman, Y. Li, W. Niedbala, et al. 2008. IL-33
induces antigen-specific IL-5+ T cells and promotes allergic-induced airway
inflammation independent of IL-4. J. Immunol. 181: 4780–4790.
9. Xu, D., H. R. Jiang, Y. Li, P. N. Pushparaj, M. Kurowska-Stolarska, B. P. Leung,
R. Mu, H. K. Tay, A. N. McKenzie, I. B. McInnes, et al. 2010. IL-33 exacerbates
autoantibody-induced arthritis. J. Immunol. 184: 2620–2626.
10. Abston, E. D., J. G. Barin, D. Cihakova, A. Bucek, M. J. Coronado, J. E. Brandt,
D. Bedja, J. B. Kim, D. Georgakopoulos, K. L. Gabrielson, et al. 2012. IL-33
independently induces eosinophilic pericarditis and cardiac dilation: ST2
improves cardiac function. Circ. Heart Fail. 5: 366–375.
11. Imai, Y., K. Yasuda, Y. Sakaguchi, T. Haneda, H. Mizutani, T. Yoshimoto,
K. Nakanishi, and K. Yamanishi. 2013. Skin-specific expression of IL-33 activates group 2 innate lymphoid cells and elicits atopic dermatitis-like inflammation in mice. Proc. Natl. Acad. Sci. USA 110: 13921–13926.
12. Bourgeois, E., L. P. Van, M. Samson, S. Diem, A. Barra, S. Roga, J. M. Gombert,
E. Schneider, M. Dy, P. Gourdy, et al. 2009. The pro-Th2 cytokine IL-33 directly
interacts with invariant NKT and NK cells to induce IFN-gamma production.
Eur. J. Immunol. 39: 1046–1055.
13. Yang, Q., G. Li, Y. Zhu, L. Liu, E. Chen, H. Turnquist, X. Zhang, O. J. Finn,
X. Chen, and B. Lu. 2011. IL-33 synergizes with TCR and IL-12 signaling
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 8. IL-33 stimulates IL-2 secretion by
CD11c+ DC that drives ST2 expression and ST2+ Treg
expansion. LPS signaling through TLR4 induces classical DC maturation characterized by upregulation of
costimulatory molecules and production of proinflammatory cytokines such as IL-12 that drive Th1
immune responses in naive CD4+ T cells. DC exposed
to LPS secrete little IL-2, but upregulate CD25 to deplete available IL-2. A baseline level of IL-2 secretion
is required for expression of ST2 on DC in the steady
state, which may control their ability to respond to IL33. Exposure of DC to IL-33 enhances their production
of IL-2, but does not alter expression of costimulatory
molecules or proinflammatory cytokine production.
CD25 levels remain constant, thus allowing for maximum bioavailability of IL-2 for T cells. DC-derived IL-2
promotes ST2 expression and expansion of ICOShigh
CD44highFoxp3+ Treg, which constitutively express
high levels of CD25. IL-2 may also support ST2 expression on naive CD4+ T cells and promote the development of Th2 responses by IL-33–exposed DC.
4020
14.
15.
16.
17.
18.
19.
20.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37. Cheng, G., A. Yu, and T. R. Malek. 2011. T-cell tolerance and the multifunctional role of IL-2R signaling in T-regulatory cells. Immunol. Rev. 241:
63–76.
38. Kulhankova, K., T. Rouse, M. E. Nasr, and E. H. Field. 2012. Dendritic cells
control CD4+CD25+ Treg cell suppressor function in vitro through juxtacrine
delivery of IL-2. PLoS One 7: e43609.
39. Moussion, C., N. Ortega, and J. P. Girard. 2008. The IL-1-like cytokine IL-33 is
constitutively expressed in the nucleus of endothelial cells and epithelial cells
in vivo: a novel ‘alarmin’? PLoS One 3: e3331.
40. K€uchler, A. M., J. Pollheimer, J. Balogh, J. Sponheim, L. Manley,
D. R. Sorensen, P. M. De Angelis, H. Scott, and G. Haraldsen. 2008. Nuclear
interleukin-33 is generally expressed in resting endothelium but rapidly lost
upon angiogenic or proinflammatory activation. Am. J. Pathol. 173: 1229–1242.
41. Talabot-Ayer, D., N. Calo, S. Vigne, C. Lamacchia, C. Gabay, and G. Palmer.
2012. The mouse interleukin (Il)33 gene is expressed in a cell type- and
stimulus-dependent manner from two alternative promoters. J. Leukoc. Biol. 91:
119–125.
42. Zhao, W. H., and Z. Q. Hu. 2012. Up-regulation of IL-33 expression in various
types of murine cells by IL-3 and IL-4. Cytokine 58: 267–273.
43. Hazlett, L. D., S. A. McClellan, R. P. Barrett, X. Huang, Y. Zhang, M. Wu,
N. van Rooijen, and E. Szliter. 2010. IL-33 shifts macrophage polarization,
promoting resistance against Pseudomonas aeruginosa keratitis. Invest. Ophthalmol. Vis. Sci. 51: 1524–1532.
44. Polumuri, S. K., G. G. Jayakar, K. A. Shirey, Z. J. Roberts, D. J. Perkins,
P. M. Pitha, and S. N. Vogel. 2012. Transcriptional regulation of murine IL-33 by
TLR and non-TLR agonists. J. Immunol. 189: 50–60.
45. Cayrol, C., and J. P. Girard. 2009. The IL-1-like cytokine IL-33 is inactivated
after maturation by caspase-1. Proc. Natl. Acad. Sci. USA 106: 9021–9026.
46. Haraldsen, G., J. Balogh, J. Pollheimer, J. Sponheim, and A. M. K€uchler. 2009.
Interleukin-33 - cytokine of dual function or novel alarmin? Trends Immunol. 30:
227–233.
47. Oboki, K., T. Ohno, N. Kajiwara, H. Saito, and S. Nakae. 2010. IL-33 and IL-33
receptors in host defense and diseases. Allergol. Int. 59: 143–160.
48. Chan, J. K., J. Roth, J. J. Oppenheim, K. J. Tracey, T. Vogl, M. Feldmann,
N. Horwood, and J. Nanchahal. 2012. Alarmins: awaiting a clinical response. J.
Clin. Invest. 122: 2711–2719.
49. Li, G., X. Liang, and M. T. Lotze. 2013. HMGB1: the central cytokine for all
lymphoid cells. Front. Immunol. 4: 68.
50. Yang, D., Y. V. Postnikov, Y. Li, P. Tewary, G. de la Rosa, F. Wei, D. Klinman,
T. Gioannini, J. P. Weiss, T. Furusawa, et al. 2012. High-mobility group
nucleosome-binding protein 1 acts as an alarmin and is critical for
lipopolysaccharide-induced immune responses. J. Exp. Med. 209: 157–171.
51. Zhong, H., and K. Yazdanbakhsh. 2013. Differential control of Helios(+/-) Treg
development by monocyte subsets through disparate inflammatory cytokines.
Blood 121: 2494–2502.
52. Granucci, F., S. Feau, V. Angeli, F. Trottein, and P. Ricciardi-Castagnoli. 2003.
Early IL-2 production by mouse dendritic cells is the result of microbial-induced
priming. J. Immunol. 170: 5075–5081.
53. Andrade, M. V., S. Iwaki, C. Ropert, R. T. Gazzinelli, J. R. Cunha-Melo, and
M. A. Beaven. 2011. Amplification of cytokine production through synergistic
activation of NFAT and AP-1 following stimulation of mast cells with antigen
and IL-33. Eur. J. Immunol. 41: 760–772.
54. Brint, E. K., K. A. Fitzgerald, P. Smith, A. J. Coyle, J. C. Gutierrez-Ramos,
P. G. Fallon, and L. A. O’Neill. 2002. Characterization of signaling pathways
activated by the interleukin 1 (IL-1) receptor homologue T1/ST2: a role for Jun
N-terminal kinase in IL-4 induction. J. Biol. Chem. 277: 49205–49211.
55. Minematsu, H., M. J. Shin, A. B. Celil Aydemir, K. O. Kim, S. A. Nizami,
G. J. Chung, and F. Y. Lee. 2011. Nuclear presence of nuclear factor of activated
T cells (NFAT) c3 and c4 is required for Toll-like receptor-activated innate inflammatory response of monocytes/macrophages. Cell. Signal. 23: 1785–1793.
56. Guo, L., I. S. Junttila, and W. E. Paul. 2012. Cytokine-induced cytokine production by conventional and innate lymphoid cells. Trends Immunol. 33: 598–
606.
57. Cote-Sierra, J., G. Foucras, L. Guo, L. Chiodetti, H. A. Young, J. Hu-Li, J. Zhu,
and W. E. Paul. 2004. Interleukin 2 plays a central role in Th2 differentiation.
Proc. Natl. Acad. Sci. USA 101: 3880–3885.
58. Burchill, M. A., J. Yang, C. Vogtenhuber, B. R. Blazar, and M. A. Farrar. 2007.
IL-2 receptor beta-dependent STAT5 activation is required for the development
of Foxp3+ regulatory T cells. J. Immunol. 178: 280–290.
59. Burzyn, D., W. Kuswanto, D. Kolodin, J. L. Shadrach, M. Cerletti, Y. Jang,
E. Sefik, T. G. Tan, A. J. Wagers, C. Benoist, and D. Mathis. 2013. A special
population of regulatory T cells potentiates muscle repair. Cell 155: 1282–1295.
60. Brunstein, C. G., J. S. Miller, Q. Cao, D. H. McKenna, K. L. Hippen,
J. Curtsinger, T. Defor, B. L. Levine, C. H. June, P. Rubinstein, et al. 2011.
Infusion of ex vivo expanded T regulatory cells in adults transplanted with
umbilical cord blood: safety profile and detection kinetics. Blood 117: 1061–
1070.
61. Wood, K. J., A. Bushell, and J. Hester. 2012. Regulatory immune cells in
transplantation. Nat. Rev. Immunol. 12: 417–430.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
21.
to promote the effector function of CD8+ T cells. Eur. J. Immunol. 41:
3351–3360.
Bonilla, W. V., A. Fröhlich, K. Senn, S. Kallert, M. Fernandez, S. Johnson,
M. Kreutzfeldt, A. N. Hegazy, C. Schrick, P. G. Fallon, et al. 2012. The alarmin
interleukin-33 drives protective antiviral CD8⁺ T cell responses. Science 335:
984–989.
Jiang, H. R., M. Milovanović, D. Allan, W. Niedbala, A. G. Besnard,
S. Y. Fukada, J. C. Alves-Filho, D. Togbe, C. S. Goodyear, C. Linington, et al.
2012. IL-33 attenuates EAE by suppressing IL-17 and IFN-g production and
inducing alternatively activated macrophages. Eur. J. Immunol. 42: 1804–1814.
Duan, L., J. Chen, H. Zhang, H. Yang, P. Zhu, A. Xiong, Q. Xia, F. Zheng,
Z. Tan, F. Gong, and M. Fang. 2012. Interleukin-33 ameliorates experimental
colitis through promoting Th2/Foxp3⁺ regulatory T-cell responses in mice. Mol.
Med. 18: 753–761.
Brunner, S. M., G. Schiechl, W. Falk, H. J. Schlitt, E. K. Geissler, and
S. Fichtner-Feigl. 2011. Interleukin-33 prolongs allograft survival during chronic
cardiac rejection. Transpl. Int. 24: 1027–1039.
Turnquist, H. R., Z. Zhao, B. R. Rosborough, Q. Liu, A. Castellaneta, K. Isse,
Z. Wang, M. Lang, D. B. Stolz, X. X. Zheng, et al. 2011. IL-33 expands suppressive CD11b+ Gr-1(int) and regulatory T cells, including ST2L+ Foxp3+
cells, and mediates regulatory T cell-dependent promotion of cardiac allograft
survival. J. Immunol. 187: 4598–4610.
Yin, H., X. Y. Li, X. B. Jin, B. B. Zhang, Q. Gong, H. Yang, F. Zheng, F. L. Gong,
and J. Y. Zhu. 2010. IL-33 prolongs murine cardiac allograft survival through induction of TH2-type immune deviation. Transplantation 89: 1189–1197.
Turnquist, H. R., J. Cardinal, C. Macedo, B. R. Rosborough, T. L. Sumpter,
D. A. Geller, D. Metes, and A. W. Thomson. 2010. mTOR and GSK-3 shape the
CD4+ T-cell stimulatory and differentiation capacity of myeloid DCs after exposure to LPS. Blood 115: 4758–4769.
Rank, M. A., T. Kobayashi, H. Kozaki, K. R. Bartemes, D. L. Squillace, and
H. Kita. 2009. IL-33-activated dendritic cells induce an atypical TH2-type response. J. Allergy Clin. Immunol. 123: 1047–1054.
Besnard, A. G., D. Togbe, N. Guillou, F. Erard, V. Quesniaux, and B. Ryffel.
2011. IL-33-activated dendritic cells are critical for allergic airway inflammation. Eur. J. Immunol. 41: 1675–1686.
Darrasse-Jèze, G., S. Deroubaix, H. Mouquet, G. D. Victora, T. Eisenreich,
K. H. Yao, R. F. Masilamani, M. L. Dustin, A. Rudensky, K. Liu, and
M. C. Nussenzweig. 2009. Feedback control of regulatory T cell homeostasis by
dendritic cells in vivo. J. Exp. Med. 206: 1853–1862.
Swee, L. K., N. Bosco, B. Malissen, R. Ceredig, and A. Rolink. 2009. Expansion
of peripheral naturally occurring T regulatory cells by Fms-like tyrosine kinase 3
ligand treatment. Blood 113: 6277–6287.
Townsend, M. J., P. G. Fallon, D. J. Matthews, H. E. Jolin, and A. N. McKenzie.
2000. T1/ST2-deficient mice demonstrate the importance of T1/ST2 in developing primary T helper cell type 2 responses. J. Exp. Med. 191: 1069–1076.
Wan, Y. Y., and R. A. Flavell. 2005. Identifying Foxp3-expressing suppressor
T cells with a bicistronic reporter. Proc. Natl. Acad. Sci. USA 102: 5126–5131.
Turnquist, H. R., T. L. Sumpter, A. Tsung, A. F. Zahorchak, A. Nakao, G. J. Nau,
F. Y. Liew, D. A. Geller, and A. W. Thomson. 2008. IL-1beta-driven ST2L
expression promotes maturation resistance in rapamycin-conditioned dendritic
cells. J. Immunol. 181: 62–72.
Rosborough, B. R., D. Raı̈ch-Regué, B. M. Matta, K. Lee, B. Gan,
R. A. DePinho, H. Hackstein, M. Boothby, H. R. Turnquist, and A. W. Thomson.
2013. Murine dendritic cell rapamycin-resistant and rictor-independent mTOR
controls IL-10, B7-H1, and regulatory T-cell induction. Blood 121: 3619–3630.
Firan, M., S. Dhillon, P. Estess, and M. H. Siegelman. 2006. Suppressor activity
and potency among regulatory T cells is discriminated by functionally active
CD44. Blood 107: 619–627.
Vocanson, M., A. Rozieres, A. Hennino, G. Poyet, V. Gaillard, S. Renaudineau,
A. Achachi, J. Benetiere, D. Kaiserlian, B. Dubois, and J. F. Nicolas. 2010.
Inducible costimulator (ICOS) is a marker for highly suppressive antigenspecific T cells sharing features of TH17/TH1 and regulatory T cells. J. Allergy Clin. Immunol. 126: 280-289, 289.e1-7.
Guo, L., G. Wei, J. Zhu, W. Liao, W. J. Leonard, K. Zhao, and W. Paul. 2009.
IL-1 family members and STAT activators induce cytokine production by
Th2, Th17, and Th1 cells. Proc. Natl. Acad. Sci. USA 106: 13463–13468.
Granucci, F., C. Vizzardelli, N. Pavelka, S. Feau, M. Persico, E. Virzi,
M. Rescigno, G. Moro, and P. Ricciardi-Castagnoli. 2001. Inducible IL-2 production by dendritic cells revealed by global gene expression analysis. Nat.
Immunol. 2: 882–888.
Zelante, T., J. Fric, A. Y. Wong, and P. Ricciardi-Castagnoli. 2012. Interleukin-2
production by dendritic cells and its immuno-regulatory functions. Front.
Immunol. 3: 161.
Boyman, O., and J. Sprent. 2012. The role of interleukin-2 during homeostasis
and activation of the immune system. Nat. Rev. Immunol. 12: 180–190.
Thornton, A. M., E. E. Donovan, C. A. Piccirillo, and E. M. Shevach. 2004.
Cutting edge: IL-2 is critically required for the in vitro activation of CD4+
CD25+ T cell suppressor function. J. Immunol. 172: 6519–6523.
Malek, T. R. 2008. The biology of interleukin-2. Annu. Rev. Immunol. 26: 453–479.
IL-33 EXPANDS ST2+ Treg VIA DENDRITIC CELL IL-2