SHIP Regulates the Reciprocal Development
of T Regulatory and Th17 Cells
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J Immunol 2009; 183:975-983; Prepublished online 19 June
2009;
doi: 10.4049/jimmunol.0803749
http://www.jimmunol.org/content/183/2/975
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References
Natasha R. Locke, Scott J. Patterson, Melisa J. Hamilton,
Laura M. Sly, Gerald Krystal and Megan K. Levings
The Journal of Immunology
SHIP Regulates the Reciprocal Development of T Regulatory
and Th17 Cells1
Natasha R. Locke,* Scott J. Patterson,* Melisa J. Hamilton,† Laura M. Sly,† Gerald Krystal,†
and Megan K. Levings2*
daptive immunity is controlled by subsets of CD4⫹ T
cells that are polarized by cytokines into functionally
diverse Th cell subsets. The classical paradigm that cellular immunity is mediated by Th1 cells and humoral immunity by
Th2 cells has recently been revised upon recognition of a third
lineage of Th cells that produce IL-17 (Th17 cells) and subsets of
T regulatory cells (Tregs)3 that suppress immune responses. There
is intense interest in defining how these different types of CD4⫹ T
cells develop and function, as interventions to module their relative
activities would have widespread clinical use to treat dysregulated
immune responses.
A
*Department of Surgery, University of British Columbia and Immunity and Infection
Research Centre, Vancouver Coastal Health Research Institute, Vancouver, British
Columbia, Canada; and †Terry Fox Laboratory, British Columbia Cancer Agency,
Vancouver, British Columbia, Canada
Received for publication November 11, 2008. Accepted for publication May 6, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Canadian Institutes for Health Research
(MOP57834), National Cancer Institute of Canada, Terry Fox Foundation, and StemCell Technologies. Core support was funded by the Immunity and Infection Research
Centre Michael Smith Foundation for Health Research Unit, British Columbia Cancer
Foundation, and British Columbia Cancer Agency. M.K.L. holds a Canada Research Chair in Transplantation and is a Michael Smith Foundation for Health
Research Scholar. N.R.L. and S.J.P. are recipients of Canadian Institutes for
Health Research Transplantation Training Program awards. M.J.H. holds a Michael Smith Foundation for Health Research Trainee award and a Canadian Institutes for Health Research studentship.
2
Address correspondence and reprint requests to Dr. Megan K. Levings, Department
of Surgery, University of British Columbia and Immunity and Infection Research
Centre, Vancouver Coastal Health Research Institute, 2660 Oak Street, Vancouver,
British Columbia V6H 3Z6, Canada. E-mail address: [email protected]
3
Abbreviations used in this paper: Treg, regulatory T cell; IBD, inflammatory bowel
disease; iTreg, peripherally induced regulatory T cell; MLN, mesenteric lymph node;
Teff, effector T; RA, retinoic acid; ROR, retinoic acid-related orphan receptor; WT,
wild type.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803749
PI3K is a critical regulator of tolerance. In conventional T effector (Teff) cells PI3K is activated upon TCR and CD28 costimulation, delivering signals that enhance cell cycle progression,
survival, and proliferation (1). In contrast, Foxp3⫹ Tregs have
reduced PI3K activity downstream of TCR or IL-2 receptor activation, a molecular requirement for their suppressive capacity (2)
and expression of Foxp3 (3, 4). Negative regulation of the PI3K
pathway in hematopoietic cells is primarily mediated by phosphastases, including phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and SHIP-1 and SHIP-2. These phosphatases
hydrolyze the PI3K product phosphatidylinositol 3,4,5-triphosphate, a key second messenger in the PI3K pathway, and they
negatively regulate the activity of downstream proteins such as
Akt (5). Although both SHIP-1 and SHIP-2 are expressed in hematopoietic cells (6), it is thought that SHIP-1 (hereafter SHIP) is
the major negative regulator downstream of cytokine (7), Fc (8),
and B cell (9) receptors.
In T cells, TGF-␤ plays a major role in regulating the peripheral
development of CD4⫹ T cell subsets, including peripherally induced Tregs (iTregs) (10) and Th17 cells (11), which are defined
by their ability to produce IL-17 (IL-17A), IL-17F, TNF-␣, IL-6,
and IL-22, but not IFN-␥ (11–13). Thus, depending on other cytokines in the environment, both iTreg and Th17 cells can be derived from a common precursor cell under the influence of TGF-␤
(14). Proinflammatory cytokines such as IL-6 promote Th17 differentiation via activation of STAT-3 (15) and induction of the
transcription factor retinoic acid-related orphan receptor (ROR)␥t
(16). In contrast, IL-2-mediated activation of STAT-5 prevents
Th17 cell differentiation (17) and is critical for the development of
iTregs (18). Recently it has been shown that TGF-␤-induced activation of SMAD4 is required for iTreg but not Th17 cell differentiation, suggesting that the TGF-␤-driven signaling events required for the development of these two lineages are distinct (19).
This notion is further supported by the finding that enhancement of
TGF-␤-driven iTreg and inhibition of Th17 cell development by
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Maintaining an appropriate balance between subsets of CD4ⴙ Th and T regulatory cells (Tregs) is critical to maintain immune
homeostasis and prevent autoimmunity. Through a common requirement for TGF-␤, the development of peripherally induced
Tregs is intimately linked to that of Th17 cells, with the resulting lineages depending on the presence of proinflammatory cytokines
such as IL-6. Currently very little is known about the molecular signaling pathways that control the development of Tregs vs Th17
cells. Reduced activity of the PI3K pathway is required for TGF-␤-mediated induction of Foxp3 expression and the suppressive
activity of Tregs. To investigate how negative regulators of the PI3K pathway impact Treg development, we investigated whether
SHIP, a lipid phosphatase that regulates PI3K activity, also plays a role in the development and function of Tregs. SHIP-deficient
Tregs maintained suppressive capacity in vitro and in a T cell transfer model of colitis. Surprisingly, SHIP-deficient Th cells were
significantly less able to cause colitis than were wild-type Th cells due to a profound deficiency in Th17 cell differentiation, both
in vitro and in vivo. The inability of SHIP-deficient T cells to develop into Th17 cells was accompanied by decreased IL-6stimulated phosphorylation of STAT3 and an increased capacity to differentiate into Treg cells under the influence of TGF-␤ and
retinoic acid. These data indicate that SHIP is essential for normal Th17 cell development and that this lipid phosphatase plays
a key role in the reciprocal regulation of Tregs and Th17 cells. The Journal of Immunology, 2009, 183: 975–983.
976
retinoic acid (RA) is associated with increased SMAD3 activity
(20). We have shown that in macrophages and mast cells, SHIP
expression is up-regulated by autocrine TGF-␤ (5), likely via
SMAD-dependent signaling pathways (21), suggesting that SHIP
may also be a key molecule in TGF-␤-mediated responses.
To determine whether SHIP has a role in the development
and/or function of naturally occurring cells or iTregs, we have
analyzed the phenotype and function of CD4⫹ T cells from mice
lacking a functional SHIP gene (22). Ex vivo Tregs from SHIPdeficient mice retained normal suppressive capacity in vitro and in
vivo. Deletion of SHIP, however, reduced the capacity of naive
CD4⫹ T cells to differentiate into Th17 cells and, in parallel, promoted the development of iTregs. Our results suggest that SHIP
controls the developmental balance between iTregs and Th17 cells.
Materials and Methods
Mice and cell isolation
Phospho-signaling assay
CD4⫹ T cells were incubated for 2 h in serum-free medium before stimulation. To measure phospho-Akt Ser473, cells were incubated with antiCD3 (5 ␮g/ml) and anti-CD28 (1 ␮g/ml) mAbs on ice for 15 min, followed
by crosslinking with prewarmed anti-hamster IgG F(ab⬘)2 Ab (20 ␮g/ml;
Cedarlane Laboratories) for the indicated times at 37°C with gentle shaking. Phosphorylation of STAT3 was initiated by addition of mIL-6 (100
ng/ml; R&D Systems) for the indicated times at 37°C with gentle shaking.
Activation was arrested by addition of an equal volume of ice-cold 2⫻
Foxp3-specific Fix/Perm buffer (eBioscience). Cells were permeabilized
and stained with CD4-PE-Cy7 (RM4-5), CD25-allophycocyanin (PC61),
Foxp3-PE (FJK-16s, eBioscience), phospho-Akt Ser473, and phosphoSTAT3 (both Cell Signaling Technology). A secondary goat anti-rabbit
Alexa-488 (Molecular Probes) was used to detect phospho-Abs.
RT-PCR analysis
Gene expression was measured in real time with a sequence detection
system (GeneAmp 7300; Applied Biosystems). Primer sequences were as
follows: ROR␥t, 5⬘-CCGCTGAGAGGGCTTCAC-3⬘ (forward) and 5⬘TGCAGGAGTAGGCCAC-ATTACA-3⬘ (reverse); T-bet, 5⬘-CAACCAAC
CCCTTGCCAAAG-3⬘ (forward) and 5⬘-TCC CCCAAGCAGTTGA
CAGT-3⬘ (reverse); Foxp3, 5⬘-CCCAGGAAAGACAGCAACCT T-3⬘
(forward) and 5⬘-TTCTCACAACCAGGCCACTTG-3⬘ (reverse); 18S, 5⬘ATGCCAGACACATCTGTGAGACA-3⬘ (forward) and 5⬘-CTGGCCAT
GAAGATCACACCTA-3⬘ (reverse).
The QuantiTech SYBR Green PCR kit (Qiagen) was used to quantify
mRNA levels. Data presented are normalized to 18S using the comparative
Ct method (⌬⌬Ct).
Cell culture
Medium was RPMI 1640 supplemented with 10% FBS, 10 mM HEPES, 2
mM glutamine, 1 mM sodium pyruvate, 1 mM MEM nonessential amino
acid solution, and 100 U/ml each of penicillin G and streptomycin. Cells
were stimulated with anti-CD3/CD28-coated microbeads (Invitrogen) or
plate-bound CD3 (10 ␮g/ml, 2C11) and soluble CD28 (1 ␮g/ml, 37.51).
Tregs were differentiated from CD4⫹CD25⫺CD45RBhigh T cells in the
presence of rhIL-2 (100 U/ml; Chiron) and rhTGF-␤ (1 ng/ml; R&D Systems). Retinoic acid (10 ng/ml; Sigma-Aldrich) was added where indicated. Th17 cells were differentiated from CD4⫹CD25⫺CD45RBhigh T
cells by addition of TGF-␤ (0.5 ng/ml), IL-6 (20 ng/ml, unless otherwise
indicated; R&D Systems), IL-23 (10 ng/ml; R&D Systems), anti-IL-2 (2
␮g/ml JES6-1A12; BD Biosciences), anti-IL-4 (2 ␮g/ml, BVD4-11D1; BD
FIGURE 1. Mouse Tregs exhibit attenuated Akt phosphorylation. CD4⫹ T
cells from SHIP⫺/⫺ or WT littermate control mice were stimulated with antiCD3 (5 ␮g/ml) and anti-CD28 (1 ␮g/ml) with Fc crosslinking Ab (20 ␮g/ml)
for the indicated times. Cells were stained for phospho-Akt, Foxp3, CD25, and
CD4 and analyzed by flow cytometry. A, Gating of CD4⫹CD25⫹Foxp3⫹
Tregs and CD4⫹CD25⫺Foxp3⫺ Teff cells and resulting histograms of the
mean fluorescence intensity (MFI) of phospho-Akt staining. A representative
experiment is shown. B, The average change in MFI of phospho-Akt Ser473
staining from baseline (anti-CD3 and anti-CD28 omitted control) from three
independent experiments. Bars indicate SD.
Biosciences), and anti-IFN-␥ (4 ␮g/ml, R4-6A2; BD Biosciences). Th1
cells were differentiated with mIL-12 (200 ng/ml; R&D Systems) and antiIL-4 (2 ␮g/ml). Treg anergy was assessed by culture of Tregs (5 ⫻ 104/
well) with or without 50 U/ml rhIL-2. To measure suppression
CD4⫹CD25⫺, naive T cells (5 ⫻ 104/well) were stimulated with anti-CD3
(0.5 ␮g/ml), irradiated CD3-depleted splenocytes (1 ⫻ 106/well), and serially-diluted Tregs (5 ⫻ 104 to 0). Proliferation and suppression were
measured by [3H]thymidine incorporation in the final 16 h of a 72-h assay.
Colitis T cell transfer, histologym and scoring
Colitis was induced in 6- to 10-wk-old C57BL/6 rag⫺/⫺ mice (The Jackson
Laboratory) by i.p. injecting CD4⫹CD25⫺CD45RBlow T cells (4 ⫻ 105/
mouse) alone or together with CD4⫹CD25high Tregs (2 ⫻ 105/mouse).
Eight weeks after T cell transfer, distal and proximal colon specimens were
fixed in 10% neutral buffered formalin, embedded in paraffin blocks, and
5-␮m sections were stained with H&E and Alcian blue-periodic acid
Schiff. Tissue sections were graded semiquantitatively in a blinded fashion
from 0 to 5, representing no change to severe changes, as described (23).
For Foxp3 immunohistochemistry, 5-␮m-thick paraffin sections were dewaxed, rehydrated with alcohol, and Ags were retrieved by steaming in
sodium citrate buffer (pH 6.0, 30 min). After blocking endogenous peroxidase (3% H2O2, 3 min), biotin-labeled Foxp3 (FJK-16s; eBioscience) was
incubated overnight at 4°C. Biotin was detected with HRP-conjugated
SA-ABC kit (Dako) and staining was developed with 3,3⬘-diaminobenzidine (Sigma-Aldrich). Sections were counterstained with hematoxylin, dehydrated through alcohol, and mounted.
Intracellular staining and ELISA
Intracytoplasmic expression of cytokines was assessed after 6 h of stimulation with PMA (25 ng/ml) and ionomycin (1 ␮g/ml) in the presence of
brefeldin A (10 ␮g/ml) during the final 4 h. Cells were fixed and permeabilized using Foxp3-specific kit reagents (eBioscience) and stained with
anti-IFN-␥-PE-Cy-7 (XMG1.2), anti-TNF-␣-PE (JES5-16E3), or anti-IL17-allophycocyanin (17B7) (eBioscience). All samples were read on a BD
FACSCanto and analyzed with FCS Express V3 (De Novo Software).
ELISAs were used to quantify IFN-␥ (BD Biosciences), TNF-␣ (BD Biosciences), and IL-17 (eBioscience) present in serum samples or secreted
into culture supernatants.
Statistical analyses
Differences between colitis histology scores were analyzed using a oneway ANOVA. All other analyses were performed with two-tailed Student’s
t test at a 95% confidence interval. Paired t tests were used for analysis of
all cell cultures and unpaired t tests were used for analysis of data from
colitis T cell transfer experiments. Values of p ⬍0.05 were considered
significant and are indicated on graphs as follows: ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍
0.01; ⴱⴱⴱ, p ⬍ 0.001. Error bars represent SD.
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Female mice aged 6 –10 wk were used and maintained under specific
pathogen-free conditions in accordance with ethics protocols approved by
the University of British Columbia Animal Care Committee. C57BL/6129Sv inpp5d⫺/⫺ (SHIP-1⫺/⫺) and inpp5d⫹/⫹ (WT) littermate control
mice have been described (22). Rag1⫺/⫺ mice were obtained from The
Jackson Laboratory. CD4⫹ T cells from spleen and lymph nodes were
enriched to ⬎90% purity using EasySep CD4 negative selection kit (StemCell Technologies) supplemented with anti-CD11b-bio (5 ␮g/ml) then
sorted into CD4⫹CD25highCD45RBlow (Treg) and CD4⫹CD25⫺
CD45RBhigh (Teff) cells of ⬎98% purity on a FACSAria (BD Biosciences). CD4-PE-Cy7 (RM4-5), CD25-allophycocyanin (PC61), and
CD45RB-FITC (16A) were from BD Biosciences.
SHIP REGULATES Treg AND Th17 DEVELOPMENT
The Journal of Immunology
977
Results
Mouse Tregs have reduced activation of Akt upon TCR
stimulation
We first determined whether TCR-mediated activation of the PI3K
pathway was less robust in Tregs than in Teff cells. CD4⫹ T cells
were purified from WT mice, and phospho-Akt levels were measured in Foxp3⫹ Tregs and Teff cells (Fig. 1A). After 5 and 10 min
of TCR-mediated stimulation, Teff cells displayed significantly increased levels of Akt phosphorylation at Ser473 compared with
unactivated control cells. Consistent with results in human peripheral blood CD4⫹ T cells and Foxp3 transgenic mice (2, 24), antiCD3 and anti-CD28-induced Akt phosphorylation was almost
completely absent in Treg cells. In three independent experiments
phospho-Akt Ser473 levels were on average 3.7- ( p ⫽ 0.0038) or
6.3-fold ( p ⫽ 0.0179) lower in Treg cells than in Teff cells after 5
or 10 min of TCR stimulation, respectively (Fig. 1B).
SHIP-deficient Tregs are anergic and suppressive in vitro
FIGURE 3. SHIP-deficient Tregs
suppress the adoptive transfer of colitis.
Rag⫺/⫺ mice were injected with CD4⫹
CD25⫺CD45RBhigh T cells from
SHIP⫺/⫺ or WT mice alone or with
2 ⫻ 105 CD4⫹CD25⫹CD45RBlow
Tregs from SHIP⫺/⫺ or WT mice. After
8 wk, proximal and distal colon tissue
were analyzed. A, A representative
proximal colon section for each group
stained with H&E (⫻40 magnification;
bar indicates 500 ␮m) or Alcian blueperiodic acid Schiff (AB-PAS, ⫻200
magnification; bar indicates 200 ␮m).
B, Proximal and distal colon sections
were scored blindly by two investigators according to Powrie and colleagues
(23), and results are the mean colitis
score from six to eight mice per group.
ⴱⴱⴱ, p ⬍ 0.0001. C, MLN cells were
stimulated with PMA and ionomycin
and analyzed for intracellular TNF-␣
and IFN-␥. Plots shown are from an individual mouse, gated on CD4⫹ T cells,
and are representative of three to four
mice per group.
Reduced activity of the PI3K/Akt pathway is known to be necessary for Treg function (2– 4), but the upstream molecular events
that prevent normal activation of Akt in Tregs are unknown. We
investigated whether reduced activity of Akt in Tregs was related
to expression of SHIP, a lipid phosphatase known to limit PI3K
activity (5). Pooled spleen and lymph node cells from 6- to 8-wkold SHIP⫺/⫺ and WT littermate controls were analyzed by FACS
for expression of CD4, CD25, and Foxp3 (Fig. 2A). Six- to 10wk-old SHIP⫺/⫺ mice had a consistent reduction in the proportion
of CD4⫹CD25⫺ Teff cells, and an increased proportion of CD4⫹
T cells expressed CD25 and Foxp3 compared with WT littermates
(SHIP⫺/⫺ 24.98 ⫾ 5.99% vs WT 9.24 ⫾ 1.31%, n ⫽ 7, p ⫽
0.0002) (25, 26). The increase in the proportion of Tregs also
resulted in an increase in their absolute numbers (data not shown),
consistent with previous reports (25, 26). We next determined
whether the absence of SHIP altered the proliferative and/or suppressive capacity of Tregs. SHIP-deficient Teff cells proliferated to
the same extent as WT Teff cells (Fig. 2B). In the absence of IL-2,
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FIGURE 2. SHIP deficiency does not affect Treg anergy or suppressive
capacity in vitro. A, Spleen and lymph node cells were isolated from SHIP⫺/⫺
or WT littermate control mice and analyzed by flow cytometry. Representative
dot plots showing the percentage of total spleen and lymph node cells that
express CD4 and CD25 and the percentage of CD4⫹ cells that express Foxp3
and CD25 are shown. B and C, CD4⫹CD25⫺CD45RBhigh Teff cells (1 ⫻ 105)
purified from SHIP⫺/⫺ or WT mice by FACS sorting. Proliferation of Teff
cells or Tregs (1 ⫻ 105/well) was assessed by stimulation with anti-CD3 (1
␮g/ml) and T-depleted irradiated splenocytes (5 ⫻ 105) in the presence or
absence of IL-2 (50 U/ml). To test suppression, cells were stimulated with
anti-CD3 (1 ␮g/ml) and T-depleted irradiated splenocytes (5 ⫻ 105) for 72 h
in the presence of the indicated ratios of CD4⫹CD25⫹CD45RBlow Tregs purified from either SHIP⫺/⫺ or WT mice. [3H]thymidine was added to the
cultures for the final 16 h before harvesting. For C, data are expressed as
percentage suppression, calculated by the formula: 1 ⫺ (proliferation in the
presence of Tregs/proliferation of Teff cells alone) ⫻ 100. Data are representative of three independent experiments. Data shown represent means ⫾ SD of
triplicate samples.
978
SHIP REGULATES Treg AND Th17 DEVELOPMENT
Tregs from both genotypes failed to proliferate, indicating that
SHIP is not required for Treg anergy. Additionally, Teff cell proliferation was suppressed by WT and SHIP⫺/⫺ Tregs equally (Fig.
2C). These results are in agreement with previous findings that
SHIP-deficient Tregs retain their suppressive capacity (25, 26).
SHIP-deficient Tregs suppress the adoptive transfer of colitis
SHIP regulates the colitogenic potential of Teff cells
Surprisingly, during the course of these experiments we consistently observed that the transfer of SHIP⫺/⫺ Teff cells appeared to
induce less severe changes in colon morphology including more
moderate mucosal inflammatory cell infiltration and epithelial hyperplasia and reduced mucin depletion from goblet cells (Fig. 3A).
This difference was statistically significant when colitis scores in
the proximal colon were compared (SHIP⫺/⫺ Teff cells, 1.88 ⫾
0.40 vs WT Teff cells, 3.13 ⫾ 0.71, n ⫽ 8; p ⬍ 0.001, Fig. 3B).
Furthermore, analysis of inflammatory cytokines present in serum
showed that mice that received SHIP⫺/⫺ Teff cells had significantly lower levels of systemic IFN-␥ (SHIP⫺/⫺ Teff cells,
32.30 ⫾ 6.42 pg/ml (n ⫽ 5) vs WT Teff cells, 65.98 ⫾ 11.91 pg/ml
(n ⫽ 4); p ⫽ 0.0009) and TNF-␣ (SHIP⫺/⫺, 132.6 ⫾ 76.91 pg/ml
(n ⫽ 5) vs WT, 530.3 ⫾ 243.4 pg/ml (n ⫽ 4); p ⫽ 0.010) (Fig.
4A). This reduction in systemic IFN-␥ and TNF-␣ is indicative of
reduced Th1 cell development and inflammation, and it correlates
with the reduced severity of colitis induced by transfer of SHIPdeficient Teff cells.
Negative regulation of iTreg development by SHIP is T cell
intrinsic
More detailed analysis of recipients of SHIP⫺/⫺ Teff cells revealed
that MLN from these mice contained significantly higher numbers
of Foxp3⫹ cells than did MLN from recipients of WT Teff cells
(SHIP⫺/⫺, 10.69 ⫾ 2.20% vs WT, 1.82 ⫾ 0.32%, n ⫽ 6 – 8; p ⬍
0.0001) (Fig. 4B). Notably, the percentage of MLN cells expressing Foxp3 in recipients of SHIP⫺/⫺ Teff cells was not significantly
different from that in mice that received WT Teff and Treg cotransfer (10.12 ⫾ 1.32%, n ⫽ 9; p ⫽ 0.53). Although the WT Teff
FIGURE 4. SHIP negatively regulates Treg development in vivo. Mice
were sacrificed 8 weeks after T cell transfer. A, Serum was analyzed by
ELISA for amounts of IFN-␥ and TNF-␣. Results are the average of three
to five individual mice per group. B, MLN cells were analyzed for expression of Foxp3. Representative FACS plots and the percentage Foxp3⫹ in
gated CD4⫹ cells in five to eight individual mice per group are shown. Bars
indicate means. C, Immunohistochemical staining of proximal colon sections for Foxp3 (brown) and hematoxylin counterstain (blue). A representative section for each group is shown at ⫻200 magnification. Bar indicates
100 ␮m. D, To determine purity of transferred cells for colitis experiments,
an aliquot of each injected population was analyzed for intracellular Foxp3
and surface CD25 expression.
plus WT Treg population contained ⬃25% Foxp3⫹ cells at the
time of transfer, after 8 wk in vivo this was reduced to ⬃10%.
This reduction in Foxp3⫹ cells reflects the normal homeostatic
level of Tregs and is consistently seen in the T cell transfer
model of colitis (29).
Immunohistochemical staining of colon tissue samples (Fig. 4C)
revealed that Foxp3⫹ cells were also located within the colon tissue of mice that received SHIP⫺/⫺ Teff cells: Foxp3⫹ cells were
found scattered throughout inflammatory infiltrates and also immediately below the epithelial basement membrane of intact epithelium. In contrast, very few Foxp3⫹ cells were present in mucosal inflammatory infiltrates of mice that received WT Teff cells
and no inflammation or Foxp3⫹ cells were found in PBS
control mice.
We speculated that the increase in iTregs in mice receiving
SHIP⫺/⫺ Teff cells may be due to insufficient purification and
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Since in vitro suppression assays do not necessarily predict in vivo
function (27), we investigated whether the absence of SHIP alters
the capacity of Tregs to prevent the adoptive transfer of inflammatory bowel disease (IBD). Whereas transfer of highly purified
CD4⫹CD25⫺CD45RBhigh Teff cells into rag1⫺/⫺ mice induces
colitis, cotransfer of Tregs prevents disease (28). Mice that received Teff cells from WT mice developed severe colitis (Fig. 3, A
and B). In contrast, mice that received WT Teff cells in the presence of a 2:1 ratio (Teff cells-Tregs) of Tregs, from either WT or
SHIP⫺/⫺ mice, showed no apparent clinical signs of disease, had
normal colon morphology, and significantly reduced proximal colitis scores (WT Teff cells and WT Tregs, 0.58 ⫾ 0.34; WT Teff
cells and SHIP⫺/⫺ Tregs, 0.67 ⫾ 0.56, n ⫽ 6, p ⬍ 0.001) compared with transfer of WT Teff cells alone (3.13 ⫾ 0.72, n ⫽ 8).
The ability of mesenteric lymph node (MLN) cells to produce
IFN-␥ and TNF-␣ was also measured as an indication of the severity of inflammation (Fig. 3C). Cotransfer of WT Tregs suppressed the development of MLN cells capable of producing
IFN-␥ compared with transfer of Teff cells alone (% CD4 cells
IFN-␥⫹: WT Teff cells, 29.53 ⫾ 3.61% vs WT Teff cells and WT
Tregs, 12.05 ⫾ 4.52%, n ⫽ 4; p ⫽ 0.0009), as did the cotransfer
of SHIP⫺/⫺ Treg cells (14.87 ⫾ 4.78%, n ⫽ 3; p ⫽ 0.0055). In
contrast, neither WT (% CD4 cells TNF-␣⫹: 32.98 ⫾ 6.18%, n ⫽
4; p ⫽ 0.168 (NS)) nor SHIP⫺/⫺ Tregs (41.66 ⫾ 15.09%, n ⫽ 3;
p ⫽ 0.868 (NS)) significantly altered TNF-␣ production from WT
Teff cells (43.43 ⫾ 11.82%, n ⫽ 4).
The Journal of Immunology
979
possible carryover of alternatively activated M2 macrophages,
which are expanded in SHIP⫺/⫺ mice (30). However, analysis of
the cells pretransfer revealed that CD4⫹CD25⫺CD45RBhigh Teff
cells from WT and SHIP⫺/⫺ mice did not differ in the percentages
of contaminating Foxp3⫹ cells (Fig. 4D) or CD4⫺ cells (⬍1%).
The absence of M2 macrophages in recipients of SHIP⫺/⫺ Teff
cells was confirmed by flow cytometric analysis for CD11b⫹ macrophages and investigation of arginase I activity in spleen populations (data not shown). We also considered the possibility that
the small fraction (ⱕ1%) of SHIP⫺/⫺ Foxp3⫹ Tregs that were
transferred had a significant growth advantage compared with WT
Tregs, but since we did not see a difference in the in vitro proliferative capacity of SHIP⫺/⫺ Tregs (Fig. 2B), preferential proliferation of these cells is unlikely to be the major factor leading to
their increase in vivo. Thus, somewhat counterintuitively, in the
absence of this negative regulator of the PI3K pathway, Teff cells
appear to have an intrinsic capacity to differentiate more frequently
into iTregs.
SHIP inhibits in vitro Treg differentiation in the presence of
strong TCR stimulation
To directly test the possibility that there is a T cell-intrinsic role
for SHIP in iTreg development, we tested the capacity of
CD4⫹CD25⫺CD45RBhigh T cells from WT and SHIP⫺/⫺ mice to
develop into Foxp3⫹ cells in vitro in the presence of TGF-␤. These
experiments were performed with different modes of TCR activation since iTreg development is influenced by the strength of TCR
stimulation (31). We also tested the effect of TGF-␤ in the presence of RA, the active metabolite of vitamin A, since it enhances
TGF-␤-induced Foxp3 expression (31, 32) and enables Treg differentiation even in the presence of high levels of costimulation
(31). Upon weak TCR stimulation with immobilized anti-CD3 and
soluble CD28, similar percentages of Foxp3⫹ Treg cells were elicited by TGF-␤ from WT (75.53 ⫾ 8.02%, n ⫽ 4) and SHIP⫺/⫺
(75.22 ⫾ 12.98%, n ⫽ 4) T cells (Fig. 5A). The addition of RA to
this condition did not reveal a difference in iTreg differentiation (%
Foxp3⫹ cells: WT, 76.98 ⫾ 16.01% vs SHIP⫺/⫺, 68.81 ⫾ 21.18,
n ⫽ 3; p ⫽ NS). Similarly, upon mild TCR stimulation with a low
(1:5) ratio of anti-CD3/CD28-coated beads, TGF-␤ induced similar Foxp3 expression in WT and SHIP⫺/⫺ T cells (% Foxp3⫹
cells: WT, 45.14 ⫾ 9.68% vs SHIP⫺/⫺, 33.27 ⫾ 15.62, n ⫽ 3; p ⫽
0.575 (NS)) and the addition of RA enhanced this conversion in
both genotypes (% Foxp3⫹ cells: WT, 61.39 ⫾ 7.50% vs
SHIP⫺/⫺, 58.99 ⫾ 29.94, n ⫽ 3; p ⫽ 0.191 (NS)).
In contrast, under conditions of strong TCR stimulation, with
one anti-CD3/CD28-coated bead for each T cell, iTreg differentiation was significantly increased in SHIP-deficient cells in the
presence of TGF-␤ alone (% Foxp3⫹ cells: WT, 0.21 ⫾ 0.06% vs
SHIP⫺/⫺, 4.48 ⫾ 1.54%, n ⫽ 4; p ⫽ 0.018) or TGF-␤ and RA (%
Foxp3⫹ cells: WT, 48.50 ⫾ 13.95% vs SHIP⫺/⫺, 75.03 ⫾ 9.37%,
n ⫽ 4; p ⫽ 0.037). Culture of SHIP⫺/⫺ Teff cells with CD3/CD28coated beads and RA in the absence of TGF-␤ did not result in any
detectable Foxp3 expression (data not shown), indicating that elevated autocrine TGF-␤ production in the absence of SHIP is unlikely to be responsible for the increased iTreg differentiation.
We also tested whether SHIP⫺/⫺ iTregs induced by TGF-␤, in
the absence or presence of RA, were functional. These experiments were performed with cells differentiated with weak TCR
activation so that the proportion of induced Foxp3⫹ cells was similar between WT and SHIP⫺/⫺ T cells. We found that the SHIP⫺/⫺
iTreg cells were functional and did not significantly differ from
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FIGURE 5. TGF-␤- and retinoic
acid-mediated Treg differentiation is
increased in the absence of SHIP under strong TCR stimulatory conditions. A, CD4⫹CD25⫺CD45RBhigh T
cells from WT or SHIP⫺/⫺ mice were
cultured for 4 days under neutral (100
U/ml IL-2), TGF-␤ (IL-2 and 1 ng/ml
TGF-␤), or RA and TGF-␤ (IL-2,
TGF-␤, and 10 ng/ml RA) conditions
with either 10 ␮g/ml plate-bound antiCD3 and 1 ␮g/ml soluble anti-CD28or anti-CD3/CD28-coated beads at a
ratio of one bead per cell or one bead
to five cells. Data are representative
of three independent experiments. B,
To test for suppressive capacity, WT
CD4⫹ T cells were stimulated with
anti-CD3 (1 ␮g/ml) and T-depleted
irradiated splenocytes (5 ⫻ 105) in
the absence or presence of iTregs
differentiated with 10 ␮g/ml platebound anti-CD3 and 1 ␮g/ml soluble
anti-CD28 with TGF-␤ or TGF-␤ and
RA. After 72 h, [3H]thymidine was
added to the cultures for the final 16 h
before harvesting. Data are expressed
as percentage suppression and represent the average ⫾ SD of two independent experiments.
980
SHIP REGULATES Treg AND Th17 DEVELOPMENT
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FIGURE 6. SHIP-deficient T cells are deficient in Th17 differentiation in vitro. A, CD4⫹CD25⫺CD45RBhigh T cells from WT or SHIP⫺/⫺ mice were
stimulated for 6 days with 10 ␮g/ml plate-bound anti-CD3 and 1 ␮g/ml soluble anti-CD28 under neutral, Th1, or Th17 polarizing conditions. B,
CD4⫹CD25⫺CD45RBhigh T cells were stimulated for 6 days with the indicated ratio of anti-CD3/CD28-coated beads to cells under Th17 polarizing
conditions. C, The amount of IL-17 in triplicate culture supernatants from B was measured by ELISA. D, Naive cells were stimulated with plate-bound
anti-CD3 (10 ␮g/ml) and soluble anti-CD28 (1 ␮g/ml) under Th17 differentiation conditions with increasing amount of IL-6 for 4 days. In all cases cells
were restimulated with PMA and ionomycin and the percentage of cells producing IL-17, IFN-␥, or expressing Foxp3 was assessed by flow cytometry. E,
CD4⫹ T cells from SHIP⫺/⫺ or WT controls were stimulated with mIL-6 (100 ng/ml) for the indicated times. Cells were stained for phospho-STAT3 and
CD4 and analyzed by flow cytometry. F, CD4⫹CD25⫺ T cells were purified and analyzed for expression of ROR␥t, Foxp3, and T-bet mRNA by
quantitative RT-PCR. Data in A–E are representative of a minimum of three individual experiments. For E and F, data are the average ⫾ SD from three
independent experiments.
WT iTregs in terms of their capacity to proliferate (data not
shown) or to suppress the proliferation of WT Teff cells (Fig. 5B).
SHIP is required for optimal Th17 cell differentiation in vitro
It is now known that TGF-␤ is involved in the development of both
iTregs and Th17 cells, with the latter developing in the presence of
TGF-␤ and proinflammatory cytokines such as IL-6 (33). We
therefore hypothesized that SHIP may be involved in the reciprocal development of these two cell types. To test this, Teff cells
from WT or SHIP⫺/⫺ mice were cultured under Th17, Th1, or
neutral differentiation conditions. In agreement with Tarasenko
et al. (34), the absence of SHIP resulted in enhanced Th1 cell
differentiation, as evidenced by an increase in the percentage of
cells capable of producing IFN-␥ (Fig. 6A). In contrast, the absence of SHIP significantly impaired Th17 cell differentiation, resulting in fewer cells expressing IL-17 by intracellular staining
analyses (% IL-17⫹ cells: SHIP⫺/⫺, 15.16 ⫾ 0.23% vs WT,
30.70 ⫾ 0.97%, n ⫽ 3; p ⫽ 0.0017).
The Journal of Immunology
981
SHIP is required for in vivo development of Th17 cells in a
colitis model
Since a differential requirement for SHIP depending on the
strength of TCR activation was found in iTreg differentiation,
Th17 cell differentiation was compared under strong (one bead to one
cell) and mild (one bead to five cells) TCR activation (Fig. 6B). Th17
cell development was most efficient under strong stimulatory conditions, and in the absence of SHIP there was a 2-fold reduction in
IL-17-expressing cells and an 8-fold reduction in IL-17 secretion
(SHIP⫺/⫺, 1.48 ⫾ 1.65 ng/ml vs WT, 11.8 ⫾ 2.8 ng/ml, n ⫽ 3; p ⫽
0.0074) (Fig. 6C). With mild TCR activation, SHIP⫺/⫺ Teff cells
failed to differentiate into Th17 cells, and levels of secreted IL-17
were below the limits of detection (Fig. 6, B and C).
To determine whether reduced Th17 cell development was due
to altered responsiveness to IL-6 in SHIP⫺/⫺ Teff cells, IL-6
was titrated into differentiation assays. These experiments revealed
that Th17 cell differentiation was reduced in SHIP⫺/⫺ Teff cells
regardless of the concentration of IL-6 present (Fig. 6D). Under
these conditions, in the absence of IL-2, IL-6 inhibited expression
of Foxp3 equally in WT and SHIP⫺/⫺ Teff cells, indicating that
SHIP is not required for IL-6-mediated suppression of Foxp3
expression.
The development of iTregs vs Th17 cells is known to require
cytokine-induced activation of STAT5 and STAT3, respectively
(35). Although IL-2-induced activation of STAT5 did not differ
significantly between WT and SHIP⫺/⫺ CD4⫹ T cells (data not
shown), SHIP⫺/⫺ CD4⫹ T cells had a significant defect in IL-6stimulated phosphorylation of STAT3 (Fig. 6E). Additionally, ex
vivo CD4⫹CD25⫺ T cells had reduced basal expression of ROR␥t
and enhanced expression of T-bet and Foxp3 mRNA (Fig. 6F).
Combined with evidence that SHIP⫺/⫺ T cells have enhanced
IFN-␥-induced phosphorylation of STAT1 (34), these data suggest
that SHIP may have an underappreciated role in cytokine-mediated
regulation of STAT activation and control over the expression of
Th cell lineage-defining transcription factors.
Discussion
We have shown here that SHIP has a key role in regulating CD4⫹
T cell differentiation via its capacity to promote Th17 and limit
iTreg development. In vitro and in vivo analyses revealed that in
the absence of SHIP, T cells have an enhanced capacity to develop
into iTregs and a parallel decrease in Th17 cell development.
These data suggest that modulation of the PI3K pathway is central
to T cell-mediated immune regulation and suggest that pharmacological manipulation of SHIP activity may represent a new strategy
for manipulating the balance between Tregs and Th17 cells.
We previously reported that human peripheral blood Foxp3⫹
Tregs show reduced activation of the PI3K pathway downstream
of the TCR (2). We show herein that murine Tregs share the same
alteration in PI3K pathway activation, as demonstrated by phosphorylation of Akt at Ser473. Mounting evidence indicates that
limitation of Akt activity in Tregs is essential for the development
of the normal Treg transcriptional signature (4), induction, but not
maintenance, of Foxp3 expression (3, 4) and suppressive capacity
(2). On the other hand, at least some level of PI3K activity appears
to be critical for Treg suppression, since Tregs with a kinase dead
mutant of the main PI3K isoform activated downstream of TCR
stimulation, p110␦, are unable to prevent the spontaneous development of colitis (36), and IL-2-mediated preservation of Treg
function is dependent on PI3K (37).
We, like others (25, 26), observed a dramatic increase in the
ratio of Teff cells to Tregs as well as an increase in the absolute
number of Tregs in the spleen and lymph nodes of SHIP⫺/⫺ mice.
Collazo et al. (26) recently reported that SHIP⫺/⫺ mice also have
an increase in the proportion, but not the absolute number, of thymic Foxp3⫹ Tregs, suggesting that increased thymic output likely
does not account for the expansion of peripheral Foxp3⫹ Treg
cells. Indeed, since T cell-specific deletion of SHIP does not result
in an increase in the number of splenic Foxp3⫹ cells, there is
clearly a T cell extrinsic role for SHIP in the regulation of Treg
development (34). Nevertheless, our data support the conclusion
that there is also a T cell-intrinsic role for SHIP in the peripheral
development of iTregs since in vivo adoptive transfer experiments
revealed that a significantly higher proportion of SHIP⫺/⫺ Teff
cells became Foxp3⫹ in comparison to WT Teff cells.
Further investigation into how SHIP may be involved in Treg
development revealed a critical role for this lipid phosphatase in
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FIGURE 7. SHIP deficiency impairs Th17 cell differentiation in vivo.
MLN and spleen cells isolated 8 wk after transfer of the indicated T cell
populations were stimulated with PMA and ionomycin. A, Intracellular
IFN-␥ and IL-17 production by MLN cells in gated CD4⫹ cells. Data are
representative of three mice per group. B, Amounts of IL-17 in culture
supernatants from cells stimulated with PMA and ionomycin for 24 h were
determined by ELISA. Each dot represents the average of triplicate wells
from one mouse; bars represent the average from three mice per group.
Given the compelling in vitro evidence that SHIP⫺/⫺ Teff cells fail
to differentiate into Th17 cells in vitro, we investigated whether
there was a similar block in the in vivo development of Th17 cells
in the colitis model. A lower proportion of MLN cells from recipients of SHIP⫺/⫺ Teff cells exhibited a Th17 cytokine profile compared with mice that received WT Teff cells (% IL-17⫹IFN-␥⫺
cells: SHIP⫺/⫺, 3.61 ⫾ 0.70% vs WT, 9.26 ⫾ 1.97%, n ⫽ 4; p ⫽
0.0028) (Fig. 7A). Similar results were obtained from spleens (%
IL-17⫹IFN-␥⫺ cells: SHIP⫺/⫺ Teff cells, 2.12 ⫾ 1.02% vs WT
Teff cells, 8.66 ⫾ 2.07%, n ⫽ 3; p ⫽ 0.0162, data not shown).
Levels of secreted IL-17 were also significantly reduced in both
MLN (SHIP⫺/⫺ Teff cells, 8.88 ⫾ 2.94 ng/ml vs WT Teff cells,
15.08 ⫾ 1.24 ng/ml, n ⫽ 3; p ⫽ 0.0282) and spleen (SHIP⫺/⫺
Teff cells, 1.79 ⫾ 1.23 vs WT Teff cells, 5.56 ⫾ 0.24 ng/ml,
n ⫽ 3; p ⫽ 0.0064) cell cultures from recipients of SHIP⫺/⫺
compared with WT Teff cells (Fig. 7B). These data correlate
with reduced Th17 differentiation from SHIP-deficient naive T
cells in vitro and further show that the absence of SHIP hampers
Th17 cell development in vivo.
982
than their WT counterparts. This deficiency in IL-17 production
was independent of the concentration of IL-6 present, and it was
not due to increased inhibitory effects of IFN-␥ on Th17 cell development, since the amounts of IFN-␥ under Th17 polarizing conditions were equal in WT and SHIP⫺/⫺ T cells. Nevertheless, since
T-bet is known to negatively regulate Th17 differentiation (11, 41,
42) and SHIP⫺/⫺ CD4⫹ T cells have high basal levels of T-bet and
low ROR␥t mRNA, this imbalance in expression of lineage-defining transcription factors likely contributes to the molecular basis
for the altered T cell differentiation in the absence of SHIP.
An outstanding question is which signaling pathways require
SHIP for normal Th17 cell development. Evidence that SHIP⫺/⫺
Teff cells do not have altered TCR-mediated activation of Erk,
Akt, Zap-70, or phospholipase C-␥1 (34) suggests that the relevant
pathways may be cytokine driven. Indeed, we found that IL-6stimulated phosphorylation of STAT3 was significantly impaired
in SHIP⫺/⫺ CD4⫹ T cells. Moreover, TGF-␤ is known to induce
SHIP expression and lipid phosphatase activity (21) and changes
in this signaling pathway could contribute to the development of
both naturally occurring Tregs and iTregs (43). Further investigation will be required to define how SHIP controls cytokine-mediated regulation of the STAT and PI3K pathways and thereby acts
as a critical determinant in Th17 vs iTreg development.
Acknowledgments
We thank K. Assi for expert assistance in blinded scoring of tissue sections,
Paul Orban and Ciriaco Piccirillo for critical review of the manuscript, and
Kevin Maloy for helpful discussions.
Disclosures
The authors have no financial conflicts of interest.
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Increasing evidence, however, indicates that SHIP may not simply
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T cell-specific deletion of SHIP has been demonstrated to enhance Th1 differentiation due to increased sensitivity to cytokinemediated T-bet induction (34). Indeed, the enhanced potential to
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