Adaptive Islet-Specific Regulatory CD4 T Cells Control
Autoimmune Diabetes and Mediate the Disappearance of
Pathogenic Th1 Cells In Vivo1
Sarah E. Weber,2* Judith Harbertson,2* Elana Godebu,* Guthrie A. Mros,* Ryan C. Padrick,*
Bryan D. Carson,†Steven F. Ziegler,†and Linda M. Bradley3*
Adaptive regulatory T cells that develop from naive CD4 cells in response to exposure to Ag can act as immunotherapeutic agents
to control immune responses. We show that effectors generated from murine islet-specific CD4 cells by TCR stimulation with IL-2
and TGF-?1 have potent suppressive activity. They prevent spontaneous development of type 1 diabetes in NOD mice and inhibit
development of pancreatic infiltrates and disease onset orchestrated by Th1 effectors. These regulatory T cells do not require
innate CD25?regulatory cells for generation or function, nor do they share some characteristics typically associated with them,
including expression of CD25. However, the adaptive population does acquire the X-linked forkhead/winged helix transcription
factor, FoxP3, which is associated with regulatory T cell function and maintains expression in vivo. One mechanism by which they
may inhibit Th1 cells is via FasL-dependent cytotoxicity, which occurs in vitro. In vivo, they eliminate Th1 cells in lymphoid
tissues, where Fas/FasL interactions potentially play a role because Th1 cells persist when this pathway is blocked. The results
suggest that adaptive regulatory CD4 cells may control diabetes in part by impairing the survival of islet-specific Th1 cells, and
thereby inhibiting the localization and response of autoaggressive T cells in the pancreatic islets. The Journal of Immunology,
2006, 176: 4730–4739.
key components in the control of homeostasis and self-tolerance.
TR cells are now recognized to be a diverse population comprised
of distinct subsets that display a spectrum of often overlapping
phenotypes, but which may have different roles in regulating spe-
cific aspects of immunity. They can, however, be subdivided into
two major subgroups: naturally occurring or innate TR cells that
arise in the thymus early in life, and adaptive TR cells that develop
from naive CD4 cells in the periphery during the course of an
immune response (1, 2). Although the innate population has re-
ceived intense study, much less is known about adaptive TR cells
that differentiate from naive cells in the periphery in response to
the cytokine milieu that develops during their encounter with Ag to
become effector CD4 cells.
Innate TR cells represent a developmentally distinct lineage that
is characterized by a combination of surface markers that include
CD25, CD62L, CD134 (OX40), CD152 (CTLA-4), and glucocor-
ticoid-induced TNFR family-related protein, as well as the expres-
lthough the mechanisms by which they function remain
poorly understood, CD4 T cells with the capacity to reg-
ulate immune responses (TR cells)4have emerged as
sion of the X-linked forkhead/winged helix transcription factor,
FoxP3 (3). Innate TR cells can inhibit the responses of T cells by
direct cell contact in vitro, but their activity in vivo depends upon
on the cytokines, IL-10 and TGF-?1, whose precise roles in me-
diating their function remain elusive (4). In contrast, adaptive TR
populations can acquire different phenotypes depending upon the
conditions of their induction. They are thought to modulate im-
mune responses exclusively via cytokine-mediated effects and can
include Th1 and Th2 cells, as well as intermediate phenotypes (5),
in addition to IL-10- and TGF-?1-producing subsets (Tr1 and Th3,
respectively; Refs. 6 and 7), which are typically favored by mu-
cosal routes of immunization. When generated in vivo, the ana-
tomic location of their priming can also be critical in determining
their cytokine secretion phenotypes. Thus, intranasal administra-
tion of peptide Ag can promote development of IL-10-producing
TR cells (8) whereas orally administered Ag can favor TGF-?1
producing TR cells (9). Targeting of Ag to subsets of dendritic
cells, and the immunogenicity of Ag are among the factors thought
to contribute to the cytokine secretion phenotypes of adaptive TR
Innate TR cells play a fundamental role in preventing autoim-
munity through the recognition of self-Ag (4). They may also con-
tribute to control of effector T cell expansion and inflammation in
immune responses to bacterial and viral pathogens (11, 12), and to
regulation of memory T cell homeostasis (13). Adaptive TR pop-
ulations also have the potential to function in these capacities, and
such cells may arise as acute effector responses wane leading to
less immunogenic conditions. A key feature of adaptive CD4 pop-
ulations is that they can be generated ex vivo from naive CD4
cells, and used to control naive CD4 cell responses to foreign Ag
(14), as well as autoimmune responses (6). Importantly, adaptive
CD4 cells can share functional and phenotypic characteristics of
innate TR cells such as secretion of TGF-?1 and/or IL-10, and
perhaps the sustained expression of CD25 and FoxP3, suggesting
*Department of Immunology, Sidney Kimmel Cancer Center, San Diego, CA 92131;
and†Benaroya Research Institute, Virginia Mason, Seattle, WA 98101
Received for publication November 9, 2005. Accepted for publication January
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.
1This work was supported by National Institutes of Health Grant R01 DK059438.
2S.E.W. and J.H. contributed equally to this work.
3Address correspondence and reprint requests to Dr. Linda M. Bradley, Department
of Immunology, Sidney Kimmel Cancer Center; 10835 Road to the Cure, San Diego,
CA 92121. E-mail address: firstname.lastname@example.org
4Abbreviations used in this paper: TR cell, CD4 cell with regulatory function; Ltn,
lymphotactin; LN, lymph node; Bodipy 558/568, 4,4-difluoro-5-(2-thienyl)-4-bora-
3a,4a- diaza-s-indacene-3-propionic acid; TFIIB, transcription factor IIB.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00
that it will be possible to exploit such cells in the Ag-specific
regulation of responses.
Type I diabetes is a progressive autoimmune disease, which
becomes pathogenic when innate TR cells may no longer have the
capacity to keep autoaggressive CD4 cells in check (15, 16), ulti-
mately leading to the destruction of pancreatic islet ? cells. Pro-
tection from disease can be achieved by expression of TGF-?1 in
islets (17), which may promote the recruitment, accumulation,
and/or expansion of innate TR cells that inhibit the autoimmune
response in the site (18). It is also possible that self-reactive islet-
infiltrating CD4 cells acquire characteristics of TR cells in re-
sponse to local cytokine effects such that they are not only no
longer pathogenic, but protective. Although the approach of islet-
localized cytokine expression is impractical in a clinical setting,
administration of mitogenic and nonmitogenic anti-CD3 mAb can
protect both mice and humans against diabetes onset via TGF-?-
dependent mechanism(s) in the absence of innate TR cells (19),
and remarkably can reverse pre-existing disease early after clinical
onset. These findings suggest that adaptive TR cells generated in
the periphery can be sufficient to control development of type I
diabetes even after progression to acute disease. Although poly-
clonal stimulation can be used to generate such cells, it is possible
that CD4 cells specific for the relevant autoantigens are the key
populations to be affected, because restimulation of effector cells
by Ag in the lymphoid compartment or in target organ is likely to
be crucial to elicit their effector response.
In this study, we generated adaptive TR cells from islet-specific
CD4 cells by TCR signaling together with IL-2 and TGF-?1 in
vitro. These cells displayed an effector phenotype, and responded
to restimulation in vitro and after transfer in vivo by production of
IL-10 and TGF-?1 as well as the chemokines, lymphotactin (Ltn;
Ref. 4; XCL1) and RANTES (CCL5), which are associated with
activated CD4 cells that become recruited into the pancreas (20).
This population not only prevented spontaneous development of
diabetes, but also inhibited the rapid disease onset mediated by
Th1 effectors that were also islet-specific, causing their disappear-
ance from the draining lymph nodes (LN) and spleen. These cells
did not require innate TR cells for development or function and
could be generated from islet-specific CD4 cells derived from di-
abetic mice. We show that one mechanism by which these adaptive
TR cells can inhibit the acute response of Th1 cells in vitro is via
FasL-dependent cytotoxicity, a mechanism that may also contrib-
ute to their function in vivo. Our results indicate that ex vivo-
generated, Ag-specific TR cells can be potent immunotherapeutic
agents for the control autoimmune diabetes.
Materials and Methods
NOD/LtJ, NOD/LtJ-Scid?/?, NOD/LtJ-Rag 1?/?, and NOD.NON-
Thy1.1LtJ mice were obtained from The Jackson Laboratory. BDC2.5 CD4
T cell (v?1, v?4) TCR transgenic mice (21) were from D. Mathis and C.
Benoist (Joslin Diabetes Center, Boston, MA). PCX.NOD mice that ex-
press GFP under the actin promoter were from N. Sarvetnick (The Scripps
Research Institute, La Jolla, CA). The mice were bred in the vivarium at
Sidney Kimmel Cancer Center. BDC2.5 mice were maintained as het-
erozygous for the TCR transgene and bred to NOD, NOD, Rag?/?, and
PCX.NOD mice. Female mice were used in all experiments. All experi-
ments in this study were approved by the Sidney Kimmel Cancer Center
Institutional Animal Care and Use Committee.
Anti-CD3 (2C11), -CD28 (37.51), -IFN-? (XMG1.2), -IL-4 (11B11),
-IL-10 (JES-2A5), and -TGF-?1 (1D11.16.8) were generated from the in-
dicated hybridomas and purified as described (22). Reagents from BD
Pharmingen were purified hamster IgG1, anti-CD178/FasL (MFL3), and
anti-IL10R (1B1.3a), and PE-labeled mAb specific for: CD152/CTLA-4,
CD25, ?7integrin, CD134/OX40, CD95/Fas, CD178/FasL, IFN-?, IL-10,
and anti-BrdU, and FITC and biotin-labeled anti-v?4. Allophycocyanin-
and PE-labeled streptavidin and allophycocyanin anti-CD90/Thy1.2 were
from eBioscience. PerCP-anti-CD4 was from Caltag Laboratories. Purified
anti-Ltn (XCL1) was from R&D Systems. Biotin-anti-mouse IgG and PE-
anti-rat ? from BD Pharmingen were used as second step reagents. mAb for
ELISAs included capture and detection pairs for IL-2, -4, -10, and IFN-?
from BD Pharmingen, and for the chemokines, Ltn and RANTES from
R&D Systems. Rabbit anti-FoxP3 sera was generated as described (23).
Generation of CD4 effectors
CD4 cells were enriched from the spleens and pooled LN of BDC 2.5 mice
by negative selection using magnetic sorting with a mixture from BD
Pharmingen (Imag). CD4 cells were seeded at 106/ml in 6-well plates
(Costar) in 5 ml of complete media (20) to which 10 ng/ml rIL-2 was
added. For Th1 cells, plates were coated with 10 ?g/ml anti-CD3 and the
cultures were supplemented with 5 ng/ml rIL-12 (Genetics Institute), 10
?g/ml anti-IL-4, and 10 ?g/ml anti-CD28 (20). For TR cells, plates were
coated with 50 ?g/ml anti-CD3, and cultures were supplemented 2 ng/ml
human rTGF-?1 (R&D Systems) and 10 ?g/ml anti-IFN-?. After 60–72 h,
the cells were expanded in media with rIL-2. Th1 cells were harvested at
4 days, and TR cells at 5 days.
Proliferation, cytokine secretion, and CTL activity
BDC CD4 cells were restimulated with plate-bound anti-CD3 (10 ?g/ml)
in 200 ?l triplicate cultures in 96-well plates (Costar) as indicated in the
text. For cytokine analysis, supernatants (100 ?l) were harvested at 48 h for
naive cells and at 24 h for effectors, and tested by amplified ELISAs (24).
Latent and active TGF-?1were assayed with ELISA kits from Promega.
After collecting supernatants, [3H]thymidine (1 ?Ci/well) was added and
uptake was assessed 18 h later to measure proliferation. To test CTL ac-
tivity, Th1 cells were labeled with51Cr (Na2
added to triplicate cultures in 96-well plates with varying numbers of un-
labeled TR cells for 6 or14 h.51Cr release into supernatants was measured
using a gamma counter, and cpm from TR cell cultures were compared
with those from unlysed and lysed Th1 cells without TR cells. Alterna-
tively, Th1 cells were labeled by fluorescence with 4,4-difluoro-5-(2-thie-
nyl)-4-bora-3a,4a- diaza-s-indacene-3-propionic acid (Bodipy 558/568)
(Molecular Probes) for 20 min at 37° C (25) and 3 ? 105were mixed in
various ratios with TR cells. Viable recovery was assessed by flow cytom-
etry using 7-aminoactinomycin D to distinguish dead cells.
51CrO4) (22) and 3 ? 105were
Gene expression analysis
RNA was isolated from Th1 and TR cells after 4 and 5 days of culture,
respectively, using RNeasy Mini kits from Qiagen. Affymetrix mouse
430A 2.0 chips were used to profile gene expression levels. For each chip,
10 ?g of total RNA was labeled and scanned using Affymetrix’s standard
_manual.affx?). Data were analyzed using GeneSifter software from 4 chips
each for Th1 and Th2 cells generated from two separate experiments. A
gene was considered to be differentially expressed between two different
samples when expression was determined to be present in all samples un-
der default parameters and there was a 2-fold or greater change in net
fluorescence between the average of four samples (p ? 0.05)
BDC CD4 cells were injected i.v. into NOD, NOD Thy1.1, or NOD.Scid
mice as indicated in the text for individual experiments. In Scid recipients,
donor cells were distinguished by v?4. For transfers into NOD mice, the
donor CD4 cells were from BDC PCX.NOD mice and marked by GFP.
Thy 1.2 was used to track BDC CD4 cells in NOD Thy 1.1 mice. Blood
glucose levels were determined using an AccuChek II monitor (Boehringer
Mannheim Diagnostics) daily for 2 wk and weekly thereafter. Two con-
secutive readings ?300 mg/dl were considered indicative of diabetes. For
coinjection studies, naive BDC cells or Th1 cells were labeled with CFSE
(Molecular Probes) (25) and TR cells with Bodipy 558/568 before injection
of equal numbers (2 ? 106) into recipients. To detect donor cell division
after several days in vivo, recipients were given BrdU (Sigma-Aldrich) as
described (26). Anti-FasL blocking or isotype control mAb were injected
i.p. in a dose of 200 ?g/recipient at the time of cell transfer.
For BrdU analysis, PCX.NOD BDC cells were stained with CD4-PerCP
and biotin-v?4/allophycocyanin-strepavidin, permeabilized with Cyto-
perm/Cytofix (BD Pharmingen), and then stained with PE-anti BrdU. For
CTLA-4 staining, Th1 and TR cells were incubated with surface stains and
4731 The Journal of Immunology
permeabilized as above. For intracellular cytokine staining, the cells were
stimulated by overnight culture with 10 ?g/ml anti-CD3 at 106/ml in 24-
well plates. Brefeldin A (10 ?g/ml) was added after 2 h. After harvest, the
cells were stained for surface markers, permeabilized, and stained with
anti-cytokine Abs in concentrations that were optimized for the individual
Detection of FoxP3 protein
Analysis was performed on in vitro-generated Th1 or TR cells or in vivo-
transferred TR cells that were FACS sorted on the basis of Thy 1.2 from
the spleens and pancreatic LN 7 days after transfer into NOD Thy 1.1
recipients. The cells were washed with PBS, lysed in the presence of pro-
tease inhibitors (Roche Diagnostic Systems), and sonicated. Protein con-
centration was determined by bicinchoninic acid protein assay (Pierce).
Lysates were separated on 4–12% gradient bis-Tris gels (Invitrogen Life
Technologies), and transferred to nitrocellulose. FoxP3 was detected by
immunoblotting with anti-FoxP3 and standard chemiluminescence. As a
loading control, blots were probed for the transcription factor IIB (TFIIB)
(Santa Cruz Biotechnology). A positive control lysate was from 293T cells
transfected with mouse FoxP3 DNA.
Localization of BDC cells in tissues of NOD mice was evaluated after
labeling with51Cr as described (22) and injection of 1–2 ? 106cells with
a total of 5–6 ? 104cpm. Radioactivity present in blood, lymphoid, and
nonlymphoid organs was determined at 16 h after cell transfer using a
Pancreata were fixed in 10% buffered Formalin and embedded in paraffin.
Four-micrometer-thick sections were stained with H&E. Insulitis indices
were performed by scoring islet infiltrates (20). Between 10 and 30 islets
per pancreas were analyzed in five levels of sections that differed by 80 ?m
for a total of 120–125 individual islets for each treatment group.
Characteristics of BDC CD4 effectors generated in the presence
of IL-2 and TGF-?1
Although CD4 cells from BDC2.5 TCR transgenic mice are ex-
posed to islet Ag, they express a predominantly naive phenotype
with high levels of the homing receptors CD62L and ?7integrin
and low levels of CD44 (27) (data not shown). As reported, few
cells express CD25 (27, 28). To generate effectors, BDC cells were
stimulated with anti-CD3 together with rIL-2. Previous studies in-
dicated that priming of naive CD4 cells in the presence of TGF-?
and rIL-2 induces TGF-?-secreting effectors but the population
includes Th1-like cells unless endogenous IFN-? is neutralized
(29). Thus, to elicit TGF-?-producing effectors with a potential to
function as TR cells, anti-IFN-? and rTGF-?1were added. We also
used a higher dose of anti-CD3 (without anti-CD28) for generation
of TR cells, which largely overcame the antiproliferative effects of
TGF-?1. Although Th1 cells developed after 4 days (20), TR cells
exhibited a 24 h lag before both populations demonstrated an
equivalent 3- to 5-fold expansion. Without IL-2, TGF-?1 failed to
support CD4 cell expansion (data not shown).
After culture, both populations were activated in appearance and
expressed high levels of CD44 and CD25 (IL-2R?) (Fig. 1A). The
cells were heterogeneous with respect to CD62L and regulation by
IL-12 may account for the higher levels found on Th1 cells than on
TR cells (30). ?7integrin was maintained on TR cells but not Th1
cells, consistent with previously reported effects of TGF-?1 on
expression of this receptor (31). Both populations expressed OX40
and high levels of intracellular CTLA-4. Recent studies show that
FoxP3 is induced in naive (CD25?) CD4 cells in response to
TGF-?1 (32) and is associated with development of a phenotype
that is indistinguishable from that of innate TR cells (14). To de-
termine whether this occurs during effector generation from BDC
CD4 cells, we analyzed expression of the FoxP3 protein by West-
ern blots. As shown in Fig. 1B, expression was detected in effec-
tors from cultures containing IL-2 and TGF-?1, but not IL-2 and
We then assessed cytokine secretion by CD4 effectors after re-
stimulation with anti-CD3 to determine the effects of TGF-?1 on
cytokine polarization. IL-2 was not detectable in the culture su-
pernatants of either Th1 or TR cells (?20 pg/ml) as is typical for
the time of harvest, the cells were analyzed for expression of the indicated markers. All were surface stains except CTLA-4, which was detected by
intracellular staining. Plots are gated on CD4?lymphocytes. Shown are v?4?cells (y-axis) vs the various markers (x-axis). B, Western blots were used
to analyze FoxP3 protein expression by 5 ? 105Th1 and TR cells effectors compared with transfected L293T cells. TFIIB was used as a loading control.
Phenotype of CD4 effector cells. A, Th1 and TR cells were induced from BDC 2.5 CD4 cells as described in Materials and Methods. At
4732 CONTROL OF DIABETES BY ADAPTIVE REGULATORY T CELLS
effectors which have active consumption. Th1, but not TR, cells
secreted IFN-? while TR, but not Th1, cells produced high levels
IL-10 (Fig. 2A, left panel). Both active and latent TGF-?1 were
measurable (Fig. 2A, middle panel), but the active form was found
only in supernatants of TR cells. The results demonstrate that
TGF-?1/IL-2 supported development of effectors that produce
both IL-10 and TGF-?1, cytokines which are associated with im-
munoregulatory functions. We also tested TR cells for secretion of
chemokines and found that compared with Th1 cells, TR cells
produced higher levels of Ltn and lower levels of RANTES (Fig.
2A, right panel). The results show that TR cells exhibit properties
associated with activated CD4 effectors, including the capacity to
produce cytokines and chemokines in response to TCR signaling.
To further evaluate functional activity, TR cells were tested for
their ability to proliferate in response to anti-CD3. Their expansion
was comparable to that of Th1 cells (Fig. 2B). The results show
that both populations can divide in the absence of exogenously
added cytokines, other T cells, or APC and that TGF-?1 does not
elicit a population that is anergic to TCR stimulation unless high
doses of IL-2 are present. However, when added to cultures of Th1
cells, TR cells completely abolished secretion of IFN-? by Th1
cells, while maintaining their production of IL-10 (Fig. 2C). The
data show that TR cells function as a regulatory population that
can down-modulate the response of Th1 cells.
Adaptive TR cells inhibit autoimmune diabetes
We showed previously that BDC Th1 cells induce diabetes when
transferred into NOD.Scid recipients (20). We used this model to
determine whether TGF-?/IL-2-induced TR cells affect the re-
sponse of Th1 cells in vivo (Fig. 3A). As expected, Th1 cells
caused rapid development of diabetes, with onset as early as 1 wk
after cell transfer, and occurring in all recipients by day 10 (Fig.
3A). In contrast, 95% of the recipients of TR cells alone remained
disease-free throughout the experiment. When TR cells were coin-
jected with Th1 cells in a ratio of 2 ? 106:1 ? 106, respectively,
there was marked protection, with 80% of the recipients exhibiting
normal blood glucose levels. Experiments conducted for ?90 days
confirmed that protection was long-lasting (data not shown). After
titrating TR cells, we found that the 2:1 ratio was the optimum for
disease prevention; with lower doses of TR cells, the numbers of
protected recipients dropped in a dose-dependent manner (data not
To assess in vivo changes that accompanied injection of TR
cells, we examined the pancreata of NOD.Scid recipients for the
presence of islet infiltrates (Fig. 3B). At the time of diabetes onset,
the recipients of Th1 cells showed extensive mononuclear cell in-
filtrates and islets were no longer visible (left panel). In contrast,
recipients of TR cells had very limited islet infiltration (middle
panel). In mice given both Th1 and TR cells, pancreatic infiltrates
were also very limited (right panel), suggesting that Th1 cells
failed to accumulate in this site when TR cells were also present.
To quantitate the levels of islet infiltration that occurred when TR
cells were injected alone or together with Th1 cells, we analyzed
the insulitis indices for the two groups of recipients. As shown in
Fig. 3C, at 30 days after transfer of TR cells, the majority of islets
were either surrounded by, or invaded by infiltrating cells (peri-
insulitis, and insulitis, respectively). Although destruction of islets
had been contained in recipients of both TR and Th1 cells, pro-
gression to a greater level of insulitis had occurred. The data in-
dicate that TR cells, like Th1 cells become established in the pan-
creas, but that the pathogenicity of Th1 effectors was kept in check
by TR cells.
To determine whether TR cells have the potential to alter the
spontaneous diabetes onset, TR cells were injected into NOD mice
at 7 days of age during the preinsulitis phase which extends to 3
wk of age. For these young recipients, TR cells were administered
plate-bound anti-CD3. Supernatants were tested for the indicated cytokines and chemokines by ELISA. B, Th1 and TR cells were titrated as shown and
cultured as for A. Proliferation was measured by [3H]thymidine uptake. C, Th1 cells (1 ? 105) were cultured alone or together with the indicated ratios
of TR cells. Cytokine secretion was measured as for A.
Responses of CD4 effectors cells. A, 1 ? 105Th1 and TR cells that were induced as for Fig. 1 were restimulated in separate cultures with
4733 The Journal of Immunology
i.p. in a dose 20-fold lower than that used for adult animals. As
shown in Fig. 3C, TR cells protected female NOD mice from be-
coming diabetic. These data indicate that TGF-?1/IL-2-induced
TR cells controlled development of disease in addition to blocking
the pathogenic response of islet-specific Th1 cells. When an equiv-
alent number of Th1 cells were injected into young NOD mice, all
recipients died by 1 wk after cell transfer (data not shown).
Adaptive TR cells arise from CD25?CD4 cells
Previous studies showed that BDC mice contain innate TR cells
that express CD25 (28), and CD25?TR cells have been reported
to enhance TGF-?1-dependent conversion of CD25?CD4 cells
into CD25?cells that exhibit regulatory functions (33). To inves-
tigate whether the innate TR cells contribute to development of TR
cells induced with TGF-?1/IL-2, we generated BDC Rag?/?mice,
which lack this population. As reported (34), these mice develop
diabetes by 3–4 wk of age. In addition, as shown in Fig. 4A, BDC
Rag?/?CD4 cells isolated from 3-wk-old donors induce diabetes
after transfer into NOD.Scid recipients, whereas an equal number
of wild-type BDC CD4 cells have limited diabetogenicity unless
they are previously activated (35). To determine whether TR cells
could develop from BDC CD4 cells in the absence of innate TR
cells, TGF-?1/IL-2 effectors were induced from BDC Rag?/?
CD4 cells and cotransferred with Th1 cells into NOD.Scid recip-
ients in a 2:1 ratio. The data in Fig. 4B demonstrate that despite
their autoaggressive potential, BDC Rag?/?CD4 cells had the
capacity to develop into TR cells that control diabetes. The results
show that innate TR cells are not required for the generation or
function of TR cells in response to TGF-?1/IL-2.
In vivo localization, response, and phenotype of TGF-?1/IL-2
Because Th1 and TR cells express different levels of the homing
receptors CD62L, and ?4?7(Fig. 1A), we analyzed their capacity
to localize in lymphoid as well as nonlymphoid tissues compared
with naive BDC CD4 cells using51Cr labeling of the donor pop-
ulations to track their distribution in NOD recipients. As shown in
Fig. 5A, the effectors were found in the blood and homed with
similar efficiency to the spleen and pancreatic LN, but poorly to
other peripheral LN compared with naive cells. As reported (36),
the effectors showed a greater capacity than naive cells to enter the
lungs and liver, but neither Th1 nor TR cells were detected in the
pancreas. The data suggest the pancreatic LN and spleen are the
sites in the lymphoid compartment where TR and Th1 cells have
the potential to interact.
To determine the phenotype of TR cells in vivo, we assessed
FoxP3 expression after adoptive transfer. For these experiments,
NOD Thy 1.1 mice were used as recipients of TR cells generated
from BDC CD4 cells to enable distinction of donor cells by Thy
1.2. After 7 days, TR cells were isolated by FACS sorting from the
pancreatic LN and spleen, and tested for FoxP3. As shown in Fig.
5B, TR cells from both sites retained expression of FoxP3. We also
examined surface marker expression in pancreatic LN at 1 wk after
transfer of effectors generated from BDC PCX.NOD CD4 cells
NOD.Scid female mice (n ? 20/group) who were monitored for onset of diabetes by blood glucose levels. B, Representative pancreatic histology for Th1
recipients (day 7), TR recipients, and Th1 ? TR recipients (day 30) by H&E staining. C, Insulitis indices for NOD.Scid recipients of TR cells or Th1 ?
TR cells were determined by scoring 125 individual islets for each treatment group for the presence of infiltrates. D, One-week-old NOD mice were injected
i.p. with 1 ? 105TR cells and compared with age-matched NOD mice that were not injected (n ? 18 females/group) for diabetes incidence.
Adaptive TR cells inhibit autoimmune diabetes. A, Th1 cells (1 ? 106) and TR cells (2 ? 106) were injected i.v. alone or together into
4734 CONTROL OF DIABETES BY ADAPTIVE REGULATORY T CELLS
into separate groups of NOD recipients (Fig. 5C). Both subsets lost
CD25 and remained heterogeneous for CD62L, indicating that un-
like for innate TR cells, these markers do not distinguish adaptive
TR cells in vivo.
We showed previously that Th1 cells expand in vivo after adop-
tive transfer and maintain their cytokine polarization (20). We now
assessed in vivo expansion and cytokine secretion by TR cells.
Like Th1 cells, TR cells showed higher levels of BrdU uptake in
pancreatic LN compared with spleen, suggesting that the autoan-
tigen recognized by BDC CD4 cells is present in sufficient levels
to induce a response primarily in this site (Fig. 5D). To assess the
stability of the cytokine phenotype of TR cells in vivo, we used
intracellular staining. TR cells from the pancreatic LN but not the
spleen retained the capacity to produce IL-10, TGF-?1, and Ltn
(Fig. 5E) suggesting that their response to Ag in this site plays a
role in maintaining their effector phenotype. IL-2, IFN-?, and
TNF-? were not detected (data not shown), indicating that their
cytokine secretion patterns are stable during the time of the
Selective effects of TR cells on Th1 cells
To study the effects of TR cells on naive or Th1 cells in vivo, they
were cotransferred in a 1:1 ratio into NOD recipients after marking
the populations with the fluorescent dyes, Bodipy 558/568 or
CFSE, respectively. After 4 days, naive BDC cells underwent sev-
eral divisions in the pancreatic LN, and neither their numbers nor
capacity to proliferate were affected by TR cells (Fig. 6A). In con-
trast, when Th1 cells were injected with TR cells, there was a
marked disappearance of Th1 cells from the pancreatic LN (Fig.
6B), but not from nondraining LN (Fig. 6C, day 2 after cell trans-
fer). Comparable results were obtained when the dyes used to label
Th1 and TR cells were reversed. The response was not altered by
treatment of recipients of Th1 and TR cells with mAb to TGF-?1
or to IL10 and IL-10R compared with control Ab (data not shown),
suggesting that the potential production of these cytokines by TR
cells in vivo does not lead to impaired survival of Th1 cells.
Expression of FasL by Th1, but not Th2, cells
To identify possible molecular mechanism(s) that could participate
in TR-mediated disappearance of Th1 cells in vivo, we performed
gene profiling of Th1 and TR cells using RNA isolated from the
activated effector populations from primary cultures. We detected
677 gene differences as measured by a ?2-fold change in expres-
sion. Although perforin (37) and granzyme-mediated (38) cyto-
toxic activity has been associated with innate TR cells, we did not
find these molecules to be expressed in greater levels than in Th1
cells (Fig. 7A). In contrast, Th1 cells expressed higher levels of the
TNF superfamily member, FasL (4.2-fold), and of the Bcl-2 family
proapoptotic protein member, Bim (3.2-fold), suggesting greater
susceptibility to apoptosis. Although we did not detect differences
between Th1 and TR cells in Bim expression by intracellular stain-
ing (data not shown), we confirmed that while both populations
expressed similar levels of Fas, only Th1 cells expressed FasL
(Fig. 7B). On the basis of this data, we determined whether TGF-
?1/IL-2-induced TR cells might be cytotoxic.
Cytotoxic activity of adaptive TR cells
Because our previous studies showed that effectors retain high vi-
ability for 48 h after withdrawal from stimulation (39), we used
51Cr labeling of Th1 cells to assess cytotoxicity by release of ra-
dioactivity after mixing with various numbers of TR cells. As
shown in Fig. 8A, there was marked lysis after 14 h but not 6 h of
coculture. To validate these results, we used florescent labeling of
Th1 cells. Thus, Bodipy 558/568-labeled Th1 cells were mixed
with unlabeled TR cells and recovery of viable Th1 cells was as-
sayed after overnight culture. As shown in Fig. 8B, although both
populations retained high viability and were recovered in similar
numbers when cultured separately, when combined, there was a
dose-dependent loss of Th1 cells. To determine whether the Fas/
FasL pathway participated in this effect, we included a blocking
mAb to this FasL in the cultures (Fig. 8B). The data demonstrate
that cytotoxicity by TR cells was prevented by inhibition of Fas/
FasL interactions. In contrast, in the same cultures, recovery of TR
cells was unaffected by the presence of FasL-blocking mAb (Fig.
8C). The results show that TR cells can mediate direct killing of
Th1 cells but do inflict fratricide on themselves. The data support
the hypothesis that a cytotoxic mechanism might contribute to con-
trol of Th1 cells by TR cells in vivo.
To begin to test this prediction, anti-FasL-blocking or isotype
control mAb were administered to NOD mice at the time of in-
jection of CFSE-labeled Th1 cells and Bodipy-labeled TR cells. As
shown in Fig. 8D, the disappearance of Th1 cells that occurs by
day 2 in the presence of TR cells was reversed, suggesting that
Fas/FasL interactions may contribute either directly or indirectly to
the mechanism(s) underlying TR-mediated loss of Th1 cells in
vivo. Although the in vitro assays of TR cytotoxicity and the in
vivo induction of Th1 cell disappearance by TR cells suggest that
A, CD4 cells were isolated from wild-type BDC mice, or from BDC mice
that were crossed to NOD Rag?/?mice, and were injected into separate
groups of NOD.Scid recipients (2 ? 106/mouse). Diabetes incidence is
shown for recipients of wild-type (n ? 13) or Rag?/?(n ? 7) BDC cells.
B, TR cells were generated from BDC Rag?/?CD4 cells and Th1 cells
were induced from wild-type BDC CD4 cells as for Fig. 1. One ? 106Th1
cells were injected into individual NOD.Scid recipients either alone (n ?
10) or together with 2 ? 106TR cells (n ? 11) and diabetes incidence
Adaptive TR cells develop in the absence of CD25?cells.
4735 The Journal of Immunology
TR cells induce apoptosis of Th1 cells, we were unable to directly
demonstrate Th1 cells in the early stages of apoptotic death in
TR-treated recipients by annexin V staining (data not shown). Not
surprisingly, there were similar levels of the antiapoptotic protein
Bcl-2 in Th1 cells recovered from recipients that did or did not
receive TR cells (data not shown).
In this study, we show that Ag-specific TR cells develop from
autoreactive CD4 cells in response to TCR stimulation in the pres-
ence of IL-2 and TGF-?1 and can be used as a cell-based therapy
to inhibit the development of spontaneous and Th1 cell-mediated
diabetes. After in vitro induction, these adaptive TR cells exhibit
characteristics of an activated effector population which prolifer-
ates and produces the cytokines, IL-10 and TGF-?1, and the che-
mokines, Ltn and RANTES. They home to, and respond to Ag in
the draining pancreatic LN and they appear to target autoaggres-
sive Th1 cells by eliminating them from this site and from the
spleen. As a consequence, the autoaggressive response is con-
trolled, and although islets become infiltrated, the massive inflam-
mation and islet-destruction that is orchestrated by Th1 cells is
completely curtailed. These findings demonstrate that Ag-specific
TR cells generated ex vivo can acquire properties that enable them
to block an autoimmune response in the lymphoid tissues, and
thereby may prevent accumulation of pathogenic cells in the target
organ and development of disease. In autoimmune diabetes where
autoantigen-specific CD4 cells are present in prediabetic individ-
uals, this approach may be an effective alternative or adjunct ther-
apy to in vitro expanded innate TR cells (28) for which it may
prove more difficult to obtain Ag-specific populations.
The adaptive TR cells described in this study have several fea-
tures that distinguish them. It has been reported that TGF-?1 pro-
motes conversion of naive CD25?CD4 cells into a population that
is phenotypically and functionally indistinguishable from innate
CD25?TR cells (14, 32, 40). However, although CD4 cells re-
sponding to TCR stimulation in the presence of IL-2 and TGF-?1
in our model up-regulate FoxP3, a characteristic marker of innate
TR cells (41), they do not display an anergic phenotype in vitro. In
contrast to a previous report (14), TR cells generated ex vivo with
TGF-?1 did not inhibit the proliferation of naive Ag-specific cells
in vivo. Moreover, they failed to retain CD25 expression after in
vivo transfer, underscoring a recent report that FoxP3 marks TR
cells independently of CD25 (42). In addition, by gene array anal-
ysis, we find that adaptive TR cells, unlike innate TR cells, retain
the capacity for synthesis of IL-2 (L. M. Bradley, unpublished
observations). The development and function of islet-specific TR
cells did not depend upon innate CD25?cells which are present in
low but detectable frequencies in BDC 2.5 mice (28). Indeed, TR
cells could be generated in the absence of CD25?cells from islet-
specific populations that contained diabetogenic TR cells. This
suggests that induction of adaptive TR cells ex vivo may remain
possible after the autoaggressive response is underway, and that
this strategy has the potential to be a feasible therapeutic approach
after onset of clinical manifestations of disease when the activity
of innate TR cells is no longer sufficient to maintain control.
Our results indicate that TGF-?1 regulates the development of
effector cells that secrete both TGF-? and IL-10. This phenotype is
intermediate to that of Tr1 cells which predominantly produce
IL-10 (6), and Th3 cells which primarily secrete TGF-?1 (9). The
results support the concept that adaptive TR cells can display a
spectrum of cytokine phenotypes (5), and reveal the potential for
using different cytokine milieus to promote the development of TR
cells that produce specific cytokines that will be most effective in
down-regulating autoimmune or allergic responses in particular
sites. Because islet-specific TR cells that develop in response to
TGF-?1 retained their cytokine polarization patterns in vivo in
phenotype, and responses of adaptive
TR cells. A, Naive BDC CD4 cells, Th1
cells, or TR cells were51Cr-labeled and
injected into separate NOD recipients
(n ? 3/group). After 16 h, the indicated
tissues were harvested and the radioac-
tivity counted to detect localization of
the transferred cells. B, FoxP3 was an-
alyzed as for Fig. 1 from 5 ? 104TR
cells sorted from the spleens or pancre-
atic LN of NOD Thy 1.1 recipients at 7
days after transfer of 2 ? 106NOD
BDC TR cells. C, Surface marker ex-
pression by Th1 and TR cells induced
from BDC 2.5 ? PCX.NOD mice in
pancreatic LN at 7–10 days after trans-
fer into NOD mice. D, NOD mice in-
jected with 2 ? 106Th1 cells or TR
cells from BDC ? PCX.NOD mice
(n ? 4/group) were given BrdU on day
the spleen and pancreatic LN was as-
sessed on day 7 for Th1 cells and day
10 for TR cells. E, TR cells from D
were tested for synthesis of the indicated
cytokines by intracellular staining.
4736 CONTROL OF DIABETES BY ADAPTIVE REGULATORY T CELLS
draining LN and contribute to prevention of diabetes in NOD mice,
both IL-10 and TGF-?1 have the potential to participate in the
long-term control of the autoreactive response.
Thus, TGF-?1 produced by adaptive TR cells could block the
development of autoaggressive Th1 cells from naive cells by in-
hibiting the induction of T-bet (29, 43), or cause deviation of islet-
specific cells to a nonpathogenic phenotype, which may occur
when TGF-?1 is expressed in islets (17). TGF-?1 can reduce cy-
tokine secretion by activated CD4 cells (44) but it does not cause
their apoptosis or limit their capacity to expand (45). Moreover,
TGF-?1 can induce Th1 cells to produce IL-10 (46). IL-10 can
inhibit the differentiation and responses of Th1 by down-regulating
IL-12 production by APC (47, 48). IL-10 can also directly inhibit
cytokine production by Th1 cells, and enhance responses of acti-
vated T cells to TGF-?1 through regulation of its receptors (45).
Despite the potential for TGF-?1/IL-10 cross-talk, neither cyto-
kine exhibits cytotoxicity for T cells, and in blocking experiments
in vivo, neither cytokine was required for the adaptive TR popu-
lation to acutely control Th1 cells by impacting their survival (data
not shown). Nevertheless, these cytokines, and TGF-?1 in partic-
ular, could contribute to the maintenance of TR cells over more
We observed the loss of Th1 cells from pancreatic LN and the
spleen, but not from pooled PLN when cotransfer with TR cells
was performed, and have shown that TR cells can be directly cy-
totoxic for Th1 cells in the absence of either Ag or APC, support-
ing the concept that TR cells have the potential to regulate Th1
responses by this mechanism. The killing effect of TR cells in vitro
was mediated by the Fas/FasL pathway, which also contributes to
the in vivo disappearance of Th1 cells. Because TR cells do not kill
themselves in vitro, other surface receptors, which could include
and adhesion/costimulatory molecules, might also participate in
this response by facilitating the interactions of Th1 and TR cells
and thereby enable Fas/FasL engagement. However, it is excep-
tionally difficult to detect apoptotic cells in vivo due to their rapid
clearance. In addition, in vivo, Ag may facilitate Th1 and TR in-
teractions, possibly at the level of APC, and result in the engage-
ment of other mechanism(s) that regulate survival of Th1 cells.
Thus, although TR cells have the potential to directly impact the
survival of Th1 cells through non-Ag-specific processes such as
cytotoxicity, Ag may nonetheless confer specificity to the regula-
tion by bringing TR and Th1 cells in close proximity at the level
of APC. Although further study will be required to resolve the
means by which TR cells inhibit development of diabetes, the re-
sults underscore that both adaptive and innate TR cells may use
multiple processes to achieve immune regulation, and that by the
use of ex vivo generation and/or expansion of TR cells, conditions
that lead to specific functional readouts to target particular aspects
of immune processes can be designed.
A potentially important aspect of adaptive TR cells is that they
may have the capacity to give rise to memory cells that could
cells (A) or Th1 cells (B and C) were labeled with CFSE and 2 ? 106were
injected alone or together with an equal number of Bodipy 558/568-labeled
TR cells into NOD recipients. For A and B, the indicated tissues were
assessed on day 4 after transfer for the presence of donor cells by flow
cytometry. For C, analysis was performed on day 2.
Selective effects of TR cells on Th1 cells. Naive BDC CD4
BDC Th1 and TR cells that were induced as for Fig. 1, and used for gene
expression analysis by Affymetrix microarrays. Expression of selected
genes involved in apoptosis (p ? 0.05). Gene differences ?2-fold were
considered significant. B, Fas and FasL expression by Th1 and TR BDC ?
PCX. NOD cells in the pancreatic LN at 7 days after their transfer into
separate groups of NOD mice.
Expression of FasL by Th1 cells. A, RNA was isolated from
4737 The Journal of Immunology
mediate long-term protection because FoxP3 was retained by TR
cells. Their capacity to produce TGF-?1 in sites where Ag is
present may support their self-renewal through autocrine usage.
Although our data suggest that adaptive TR cells can be sufficient
to control diabetes, it is likely that adaptive and innate TR cells can
work in concert. TGB-?1 and IL-10 production by innate TR cells
could augment the expansion and responses adaptive TR cells. In
view of a recent report that innate TR cells can use exogenous
TGF-?1 to mediate suppression (49), adaptive TR cells may also
serve to augment their function. Because innate TR cells in NOD
mice are inadequate to prevent diabetes onset, the use of adaptive
TR cells that are specific for disease-associated Ag could serve to
control the acute response, and thereby enable the innate TR pop-
ulation to regain long-term control.
Because adaptive TR cells share characteristics of effectors,
these cells could arise in the context of immune responses to cer-
tain pathogens where the location of infection or the characteristics
of the microorganism, itself, promote the development of cytokine
polarized effectors which down-regulate responses. The presence
of such cells may account for the regulatory activities found to be
associated with CD25?cells immediately ex vivo (50), which
have been identified previously in disease models (4). Harnessing
the adaptive immune response using Ag-specific CD4 cells with
specific functional and phenotypic characteristics to regulate not
only autoimmune diseases, but also allergy, and graft rejection,
will add to the arsenal of strategies to combat these conditions.
The authors have no financial conflict of interest.
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4739The Journal of Immunology