Following the Fate of One Insulin-Reactive CD4 T cell Conversion Into Teffs and Tregs in the Periphery Controls Diabetes in NOD Mice

Type 1 Diabetes Center, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.
Diabetes (Impact Factor: 8.1). 03/2012; 61(5):1169-79. DOI: 10.2337/db11-0671
Source: PubMed
In diabetic patients and susceptible mice, insulin is a targeted autoantigen. Insulin B chain 9-23 (B:9-23) autoreactive CD4 T cells are key for initiating autoimmune diabetes in NOD mice; however, little is known regarding their origin and function. To this end, B:9-23-specific, BDC12-4.1 T-cell receptor (TCR) transgenic (Tg) mice were studied, of which, despite expressing a single TCR on the recombination activating gene-deficient background, only a fraction develops diabetes in an asynchronous manner. BDC12-4.1 CD4 T cells convert into effector (Teff) and Foxp3(+)-expressing adaptive regulatory T cells (aTregs) soon after leaving the thymus as a result of antigen recognition and homeostatic proliferation. The generation of aTreg causes the heterogeneous diabetes onset, since crossing onto the scurfy (Foxp3) mutation, BDC12-4.1 TCR Tg mice develop accelerated and fully penetrant diabetes. Similarly, adoptive transfer and bone marrow transplantation experiments showed differential diabetes kinetics based on Foxp3(+) aTreg's presence in the BDC12-4.1 donors. A single-specificity, insulin-reactive TCR escapes thymic deletion and simultaneously converts into aTreg and Teff, establishing an equilibrium that determines diabetes penetrance. These results are of particular importance for understanding disease pathogenesis. They suggest that once central tolerance is bypassed, autoreactive cells arriving in the periphery do not by default follow solely a pathogenic fate upon activation.


Available from: Georgia Fousteri
Following the Fate of One Insulin-Reactive CD4 T cell
Conversion Into Teffs and Tregs in the Periphery Controls
Diabetes in NOD Mice
Georgia Fousteri,
Jean Jasinski,
Amy Dave,
Maki Nakayama,
Philippe Pagni,
Florence Lambolez,
Therese Juntti,
Ghanashyam Sarikonda,
Yang Cheng,
Michael Croft,
Hilde Cheroutre,
George Eisenbarth,
and Matthias von Herrath
In diabetic patients and susceptible mice, insulin is a targeted
autoantigen. Insulin B chain 9-23 (B:9-23) autoreactive CD4 T
cells are key for initiating autoimmune diabetes in NOD mice;
however, little is known regarding their origin and function.
To this end, B:9-23speci c, BDC12-4.1 T-cell receptor (TCR)
transgenic (Tg) mice were studied, of which, despite expressing
a single TCR on the recombination activating genedecient back-
ground, only a f raction develops diabetes in an asynchronous
manner. BDC12-4.1 CD4 T cells convert into effector (Teff) and
-expressing adaptive regulatory T cells (aTregs) soon
after leaving the thymus as a result of antigen recognition and
homeostatic proliferation. The generation of aTreg causes the
heterogeneous diabetes onset, since crossing onto the scurfy
(Foxp3) mutation, BDC12-4.1 TCR Tg mice develop accelerated
and fully penetrant diabetes. Similarly, adoptive transfer and
bone marrow transplantation experiments showed differential di-
abetes kinetics based on Foxp3
aTregs presence in the BDC12-4.1
donors. A single-specici ty, insulin-r eactive TCR escapes thy-
mic deletion and simultaneously converts into aTreg and Teff,
establishing an equilibrium that determines diabetes penetrance.
These r esults are of particular importance for understanding
disease pathogenesis. They suggest that once central tolerance
is bypassed, autoreactive cells arriving in the periphery do not
by default follow solely a pathogenic fate upon activation.
Diabetes 61:1169 1179, 2012
ype 1 diabetes (T1D) is an autoimmune disease
where antigen-specic T cells can mediate the
destruction of insulin-producing b-cells in mice
and are thought to substantially contribute to
human diabetes pathogenesis. The NOD mouse has served
to model T1D pathogenesis for decades, and insulin-specic
T cells determine disease development in NOD mice.
Approxim ately 50% o f CD4
T-cell clones isolated from
pancreata of prediabetic NOD mice react to insulin (1).
Of these insulin-reactive clones, 90% respond specically
to the insulin B chain epitope B:9-23 (2). NOD mice with
a genetic deletion of both native insulin genes (Ins1 and
Ins2) expressing a mutated nonimmunogenic but hor-
monally active proinsulin transgene (dKO/B16:A mice) do
not develop T1D, demonstrating that immunologic rec-
ognition of insulin is crucial for the initiation of diabetes
in this model (3).
Current evidence suggests that insulin epitopes bind
weakly and with a fast dissociation rate to the NOD-specic
class II major histocompatibility complex (MHC) allele I-A
so that the limited supply of this peptide results in partial
failure during negative selection (4). However, preproinsulin-
2 expression is detected in thymic medullary epithelial cells,
is regulated by autoimmune regulator, and can result in
thymic deletion (5). Of evidence, Ins2 gene ablation signi-
cantly accelerates T1D progression in NOD mice (6,7),
whereas Ins1-decient mice are protected from diabetes (8).
Ins1 and Ins2 gene expression has been found in pan-
creatic draining lymph node s (PDLNs) and also in islets
(9). However, it is not known which insulin epitopes and
by which mechani sm they activate insulin-specic T cells
that escape negative select ion and what the functional
outcome is. Moreover, it is not well understood whether
both effector and regulatory T cells (Teffs and Tregs)
specic to insulin can be generated simultaneously in vivo.
In a recent study, two populations of B:9-23reactive cells
were described: type A that recognizes the 13-21 register
on professional antigen-presenting cells and type B that
recognizes the 12-20 register. This single amino acid shift
distinguishes both sets of B:9-23reactive cells that either
get deleted in the thymus (type A) or get activated in the
periphery by other mechanisms (type B) (10).
To determine the fate of insulin-reactive cells in more
detail, we analyzed the T-cell receptor (TCR) transgenic
(Tg) mouse line specic for B:9-23, namely BDC12-4.1.
BDC12-4.1 TCR Tg mice develop spontaneous insulitis but
no diabetes in F1 mice (FVB x NOD), whereas some di-
abetes manifests in NOD.recombination activating gene
(back-cross one generation). Disease progression
is modied by a series of genetic factors such as H-2
haplotype and the presence of additional T-/B-cell receptor
rearranged genes (RAG
versus RAG
) (11). Approxi-
mately 40% of the H-2
here BDC12-4.1.RAG
) mice develop spontaneous T1D by
40 weeks of age. This incomplete diabetes penetrance is
strikingly different from several other CD4 Tg TCR NOD
mouse models, w here diabetes development either oc-
curs in all mice or none, f or example in BDC2.5 and 2H6
TCR Tg NOD mice on the RAG- and severe combined
immunodeciencydecient backgrounds, respectively (12,13).
Currently, the function of TCR (antigen) specicity in
diabetes development is controversial. It is not clear at
From the
Type 1 Diabetes Center, La Jolla Institute for Allergy and Immunol-
ogy, La Jolla, California; and the
Barbara Davis Center for Childhood Di-
abetes, School of Medicine, University of Colorado, Denver, Colorado.
Corresponding author: George Eisenbarth,,
or Matthias von Herrath,
Received 20 May 2011 and accepted 11 January 2012.
DOI: 10.2337/db11-0671
This article contains Supplementary Data online at http://diabetes
J.J. is currently afliated with Partek, Inc., Chestereld, Missouri.
Ó 2012 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for prot,
and the work is not altered. See
-nc-nd/3.0/ for details. DIABETES, VOL. 61, MAY 2012 1169
Page 1
which level antigen can determine the fate of a T cell (14),
although it is shown that islet specicity is required for
homing in the pancreas (15). Foxp3 is the master tran-
scription factor for bona de thymic-derived, natural Tregs
(nTregs) (16). In addition to thymic nTreg differentiation,
extrathymic Foxp3
Treg development can result in mice
in particular differentiation niches that allow conversion
(adaptive [aTreg]) (17). From earlier studies, it was shown
that the thymic and peripheral Treg TCR repertoires are
similar but divers e from the T-conventional repertoire
(18,19), suggesting that minimal T conventional to aTreg
conversion occurs. In Foxp3-decient (scurfy) mice, the
pathogenic TCR repertoire is more similar to the nTreg
compartment of wild-type (wt) mice, suggesting that
nTregs bear autoreactive TCRs (20). Generating TCR Tg
mice by means of retroviral transduction showed that
nTreg-derived TCR clones cannot instruct the nTreg fate
(21). Thus, although some results highlight the instructive
nature of the TCR, TCR specicity does not completely
determine the fate of a T cell.
Most previous studies with TCR Tg mice revealed that
TCR stimulation determined by the afnity for the cog-
nate antigen and the presence or absence of endogenous
TCR rearran gement dictates whether nTregs will develop
(22,23). In the absence of TCR rearrangement (RAG
), no
nTregs develop unless their cognate is present and in suf-
cient levels ( 24). In this study, we found that BDC12-4.1
TCR Tg, B:9-23reactive, Treg cells can be efciently se-
lected in the thymus in RAG
mice but not in RAG
However, CD4
Treg cell s could be readily
detected in the periphery, spleen, pancreas, and PDLNs of
mice. De novo, Foxp3-expressing aTregs
ognition and homeostatic proliferation and were respon-
sible for the protection from T1D in 60% of the Tg mice. As
evidence, when these mice were crossed onto the C57BL/6.
scurfy background, diabetes developed faster and affected
100% of the Tg mice. We conclude that pathogenic T cells
(Teffs) and aTregs with identical B:9-23specicTCRspec-
i city can be induced in the periphery from the same
TCR-bearing precursors, but diabetes develop s because
counter-regulation by the autoreactive (insulin-specic)
aTreg is inadequate. Thus, we identi ed a TCR that can
instruct both peripheral Treg and Teff differentiation by
insulin recognition.
Mice. BDC12-4.1 TCR Tg mice were previously described (11). Heterozygous
C57BL/6 mice I-A
females (strain 4088) were purchased
from The Jackson Laboratory (Bar Harbor, ME). A minimum of two crosses
with NOD.BDC12-4.1.RAG
background was needed to generate I-A
animals. In all experiments, Tg littermates served
as genetic controls. Mice were maintained in La Jolla Ins titute for Allergy and
Immunology animal facility or at the University of Colorado Health Sciences
Center for Laboratory Animal Care in Denver under speci c pathogen-free
conditions according to each institutions animal care and use committee
guidelines for the use and care of laboratory animals.
Genotyping. Genotyping for the BDC12-4.1 Tg TCRa and TCRb, RAG,andI-A
typing were performed as described previously (11). The presence of the Foxp3
allele could be detected by PCR (forward primer, TCA GGC CTC AAT GGA CAA
AA; reverse primer, CAT CGG ATA AGG GTG GCA TA) with a band size near
1,000 base pairs (bp). Females were classied as either heterozygous Foxp3
or homozygous Foxp3
by microsatellite typing with 6-carboxyuorescein
(FAM)-conjugated forward primer, ATG AGA AGAAGG AAG ATC AG CG, and
an unlabeled reverse primer, ACC TGG GAA GGA ACT ATT GC. The
microsatellites were an alyzed with a real-time PCR ABI 3 100 Avant Analyzer
(Foster City, CA). The mutant pea ks appear at 344 bp and the wt peaks a re at
342 bp.
Glucose monitoring. Mice were followed for diabetes by weekly testing of tail
blood using either the Freestyle glucometer and test strips from Abbott Lab-
oratories (Abbott Park, IL) or the ReliOn Ultima system (Alameda, CA). Mice
whose glucose values exceeded 250 mg/dL were tested the next day and were
considered diabetic if the second reading also exceeded 250 mg/dL.
Flow cytometry. Cells were washed in uorescence- activated cell sorter
(FACS) staining buffer (0.1% sodium azide, 1 mmol/L EDTA, and 2% FCS in
PBS) and incubated with 10 ml/mL Fc block (2.4G2) (BD-Pharmingen, San
Diego, CA) for 10 min. After the blocking step, cells were stained for CD4,
CD8a, CD44, CD62L, CD69, CD25 and CD127, killer cell lectin-like receptor
subfamily G (KLRG)-1, CD103, V b 2(Vb2) (BDC12-4.1), or V b 4(Vb4) (2H6)
(eBioscience or BD-Pharmingen, San Diego, CA). For intracellular Foxp3,
Helios, or Ki-67 detection, cells were xed with Foxp3 Fix/Perm buffer and
stained with the corresponding monoclonal antibodies (eBioscience). For
cytokine detection, splenocytes were activated in vitro for 4 h with anti-CD3/
28 (10/5 mg/mL, respectively) or phorbol myristic acid/ionomycin (50/1000 ng/ml,
respectively) in the presence of brefeldin A (BfA, 10 mg/mL) (Sigma-Aldrich,
St. Louis, MO). Cells were costained for Foxp3, interferon-g (IFN-g), and
interleukin-10 (IL-10) after xation with the Foxp3 Fix/Perm kit. All antibody
incubations were performed at 4°C for 30 min (isotype controls were included).
Cells were immediately acquired on a FACSCalibur or LSRII ow cytometer
(BD-Pharmingen) and analyzed using FlowJo software (Treestar).
Immunouorescence histological staining. Pancreas sections (6-mm) were
cut by cryostat and dried overnight. For positive control, spleen sections were
included. Upon rehydr ation with TBS-PI (Tris-buffered saline containing pro-
tease inhibitor, mini EDTA-free tablets; Roche) and xation (0.4% para-
formaldehyde, 15 min at room temperature [RT]), sections were blocked in 5%
BSA and 2% goat serum TBS-PI (30 min RT). T o block endogenous biotin
activity, the avidin/biotin blocking kit from Vector was used according to the
manufacturers instructions. As primary antibodies, antiCD4-FITC (clone
RM4-5, 1/50 dilution; BD-Pharmingen), guinea pig anti-swine insulin (1/100
dilution; Dako), and biotinylated anti-Foxp3 (clone FJK-16S, 1/50 dilution;
eBioscience) were used. Primary antibody incubations were carried for 1.52h
at RT in a damp chamber. For detecting CD4, the Alexa Fluor 488 signal am-
plication kit for uorescein dyes (Invitrogen) was used according to the
manufacturers instructions. For detecting Foxp3 and insulin, Alexa Fluor
647coupled streptavidin (1/1,000 dilution; Invitrogen) and Alexa Fluor 594
labeled polyclonal goat antiguinea pig IgG antibody (1/1,000 dilution; Mo-
lecular Probes) were added overnight at 4°C or for 1.5 h at RT, respectively.
Finally, nuclei were stained with Hoechst 33258 dye (1/200 dilution, 20 min;
Invitrogen). After washing, sections were mounted with SlowFade Gold
mounting reagent (Invitrogen), and coverslips were sealed with nail polish.
Images were recorded using FluoView FV10i confocal microscope (Olympus),
and analysis was performed with Fluoview ASW2.1 (Olympus) and Image Pro
Analyzer 7.0 (Media Cybernetics) software packages.
Enzyme-linked immunosorbent assay. Puri
ed, FACS-sorted CD4SP T cells
from the thymi of BDC12-4.1.RAG
mice were cultured in 96-well plates
at 0.25 3 10
cell/well in the presence of 0.5 3 10
/well of T celldepleted
splenocytes (TDS) as antigen-presenting cells and insulin B:9-23 peptide se-
quence 1 (PHLVEALYLVCGERG) or 2 (SHLVEALYLVCGERG) at 50 mg/mL.
TDS consisted of spleens magnetically depleted of CD90.2
T cells with the
use of aCD90.2 antibody-coated microbead s (Miltenyi Biotech Inc., Auburn,
CA) from NOD.Ins2
or Ins1
donor mice. S upernatants were collected
3 days after culture for IL-4, transforming growth factor-b1 (TGF-b1), IFN-g,
and IL-10 detection. Cytokines present in the supernatants were qu antied
by a sandwich enzyme-linked immunosorbent assay ( ELISA) using standard
commercially available kits (Pharmingen OptEIA mouse; BD-Pharmingen).
Splenocyte transfer. Spleens were extracted under aseptic conditions and
homogenized by grinding between two glass microscope slides or passing
through cell strainers. Erythrocytes (red blood cells) were lysed using red blood
cell lysis buffer (R7757) (Sigma-Aldrich) or ACK buffer. Splenocytes were
washed in sterile PBS three times. Cells were resuspended in the appropriate
volume of sterile PBS before transfer. Varying amounts of primary, unsorted
splenocytes (1.5 3 10
to 6 3 10
cells) were injected intraperitoneally in 200 mL
into 68-week-old I-A
Bone marrow transplantation. Bone marrow (BM) was harvested under
aseptic conditions from the tibias and femurs of 45-week-old donors in MACS
buffer (PBS, 0.5% BSA, and 2 mmol/L EDTA) and washed twice. Mature B and
T cells were depleted by negative selection with CD4, CD8, and CD45RB/B220
magnetic microbeads and the autoMACS Magnetic Cell Sorter (Miltenyi Bio-
tech Inc.) according to the manufacturers instructions. Ten million BM cells
(in 200 m L) were injected retro-orbitally into sublethally X rayir radiated
(200 rads), 68-week-old I-A
Statistical analysis. The statistical signicance of the difference between
means was determined using the two-tailed Student t test. All statistica l
analyses, including Kaplan-Meier survival curve analyses, were performed
using GraphPad Prism v ersion 4.01 for Windows (GraphPad Software,
1170 DIABETES, VOL. 61, MAY 2012
Page 2
San Diego, CA ). P values were as follows: *P , 0.05, **P , 0.01, and
***P , 0 .001.
mice have both B:9-23specicTeffs
and Foxp3
Tregs in the periphery, but accumulate
Teffs as they age. Earlier studies had shown that BDC12-
4.1 mice only develop T1D on the RAG
background with
;40% penetrance by 40 weeks of age, whereas RAG
develop insulitis but no overt diabetes (11). On the RAG
background, no other TCRa (TCRa) chains were expressed,
and only CD4 single-positive (SP) cells were able to ma-
ture in the thymus (Supplementary Fig. 1). To investigate
whether Treg activity would contribute to diabetes re-
sistance in BDC12-4.1.RAG
mice, we performed detailed
phenotypic analysis. A signicant proportion (6070% in the
spleen and 20% in the PDLNs) of the remaining CD4
cells displayed marked activation, exhibiting a typical Teff
phenotype (CD44
)by710 weeks of age (Fig. 1A
and B), whereas CD69 levels were more pronounced in the
FIG. 1. BDC12-4.1.RAG
mice have both B:9-23specic Teffs and Foxp3
Tregs in the periphery, but accumulate Teffs as they become older. A and
B: Splenocytes and PDLN-derived cells from 710-week-old BDC12-4.1.RAG
mice were analyzed by ow cytometry after gating on the CD4
population (B:9-23sp ecic T cells) for the presence of CD44
Teffs. Mice showed downregulation of TCRb expression in the spleen (;30% of
cells were Vb2
) (data not shown). Representative ow cytometric plots displaying the CD44/CD62 L prole in the spleen and PDLNs after
gating on the CD4
population (A), and cumulative data for more than ten 710-week-old mice are displayed (B), with each symbol representing
individual mice. C: The frequency of Teffs was monitored over time (with age) in the spleen and PDLNs of BDC12-4.1.RAG
mice. DF: Tissues from the
same mice were analyzed by ow cytometry after gating on the CD4
population for the presence of CD25
(Treg). It is interesting
to note that Tregs and Teffs both appear to undergo cyclical changes in the pancreatic lymph node but not the spleen, possibly indicative of recurring
changes in the Teff/Treg equilibrium. G: Pancreatic sections from 10-week-old BDC12-4.1.RAG
mice were prepared and stained for insulin (red), CD4
(green), and Foxp3 (blue). Triple immunouorescence labeling was performed as described in
capture was performed for the selected region, which is indicated with a white box. H: Spleen sections were stained similarly to serve as a positive
control. White arrows indicate the presence of Foxp3
T cells. (A high-quality digital representation of this gure is available in the online issue.)
Page 3
PDLNs (data not shown). Interestingly, when we searched
for the presence of Tregs in BDC12-4.1.RAG
mice, on
average, ;3% of CD4
cells in the spleen and
;5% in the PDLNs were CD25
(Fig. 1DF). Foxp3
Tregs were also detected with immunouorescence staining
directly in the pancreatic inltrate of BDC12-4.1.RAG
mice (Fig. 1G and H). Thus, BDC12-4.1 T cells can develop
into Teffs as well as aTregs likely after exiting the thymus in
a naïve state (see next section).
Next, we monitored the Teff and aTreg frequencies over
time and found that fewe r CD44
cells accumu-
lated in the PDLNs compared with the spleen, correlating
with the slightly increased aTreg frequency in that site
(Fig. 1C and F). In the spleen, the number and activation
status of Teffs consistently increased with age, whereas
aTreg frequency remained relatively stable. In contrast, in
the PDLNs, Teffs and aTregs both underwent cyclical in-
creases and decreases.
Insulin-specic Foxp3
Tregs do not originate in the
thymus but are induced in the periphery. Foxp3
cells can be generated in the thymus as a result of agonist
recognition during thymic selection. To dene the precise
origin of Foxp3
cells in BDC12-4.1.RAG
mice, we ana-
lyzed thymocytes by ow cytometry. The percentage of
viable CD4/CD8 double-positive (DP) thymocytes was
high, but they did not exhibit the typical characteristics of
cognate antigen recognition; no CD4/CD8 coreceptor
downregulation and marginal CD69 downregulation were
observed (Supplementary Fig. 2). However, TCRb levels
were somewhat reduced, and a 90% reduction in mature
CD4 SP cells was seen in RAG
compared with RAG
mice. This suggests that some degree of negative selection
took place, most probably at the transition from the DP to
SP stage (Supplementary Fig. 2). Had insulin-specic
negative selection been complete, no CD4SP cells should
be detected (25).
FIG. 2. Insulin-specic Foxp3
Tregs do not originate in the thymus. Representative thymi from BDC12-4.1 TCR Tg mice on the RAG
and RAG
genetic backgrounds at 710 weeks of age were analyzed. A: The DP, CD4 SP, CD8SP, double-negative (DN), and CD4
SP gating are shown.
B: A representative dot blot is shown. After gating on the respective popul ations, analysis for the presence of nTregs (CD25
) was per-
formed. On the RAG
background, no nTregs could be identied in either DP, CD4SP, or CD4
SP gated thymocyte populations (C ). On the RAG
background, nTreg development was readily detected as a result of TCR rearrangement (pairing of the Tg Vb chain with non-T g, endogenous
Va chains). (A high-quality color representation of this gure is available in the online issue.)
1172 DIABETES, VOL. 61, MAY 2012
Page 4
Interestingly, no Foxp3
nTregs could be det ected
within the CD4SP population on the RAG
(Fig. 2), arguing strongly that the Foxp3
Tregs detected
in the periphery were in fact induced BDC12-4.1 aTregs.
Our ndings also suggest that the CD25
detected in the thymus in RAG
mice were the result of
TCR rearrangement (pairing between the Tg Vb2chain
with non-Tg V a chains). These observations highlight
the si gnicance o f de novo aTreg induction in enforc ing
tolerance. Autoreactive T cells convert into aTregs to
FIG. 3. B DC12-4.1 CD4SP cells convert int o Tef fs and Tregs upon antigen recognition in vitro. CD4
(CD4SP) thymocytes from BDC12-
mice were FACS-sorted and cultured in the presence of TDS from Ins2
NOD mice. The stimulation lasted for 48 h in the absence
(without [w/o]) or presence of 50 mg/mL B:9-23 peptide (Ins1 or Ins2 sequence), B:9-23(1) and B:9-23(2), respectively. For control, plate-bound
anti-CD3 (1 mg/mL) plus anti-CD28 (0.5 mg/mL) were used to stimulate the cells. Cells were consequently stained and analyzed by ow cytometry
after gating on CD4
for the expression of CD44 and CD62 L (A) or CD25 and Foxp3 (B). Cell supernatant was quantied with ELISA for the
presence of four cytokines: IL-10, IL-4, IFN-g, and TGF-b1 4 days after stimulation (C). (A high-quality color representation of this gure is
available in the online issue.)
Page 5
keep Teffs of the same or other specicities under their
Insulin recognition in vitro can induce Foxp3
upregulation in BDC12-4.1 TCR Tg cells. In contrast
to humans, mice express two insulin genes, Ins1 and Ins2,
and the B:9-23 peptides, to which BDC12-4.1 Tg cells react,
differ in their amino acid sequence at position 9 (S for INS2
and P for INS1). ELIspot analysi s showed that BDC12-4.1
Tg cells produce IFN-g upon stimulation with both B:9-23
sequences, whereas they are not specic for B24-C36 or
B16:A (Suppl ementary Research Design and Methods and
Fig. 3 ). We hypothesized that one of the mechani sms in-
volved in acquiring Foxp3 expression in the periphery
would include insulin recognition. Therefore, we sorted
CD4SP cells from the thymus of BDC12-4.1.RAG
and placed them in culture with TDS from Ins2
loaded with either B:9-23 peptide (INS1 or 2). After 48 h
stimulation with either B:9-23 INS1 (1) or INS2 (2), upre-
gulation of CD44 in conjunction with C D62 L down-
regulation occurred (Fig. 3A). Most importantly, B:9-23
stimulation also induced generation of CD25
aTreg (Fig. 3B), suggesting that insulin recognition can
induce BDC12-4.1 T cells to acquire both effector and
regulatory properties. It is striking that a T cell wit h a xed
TCR can convert into a Treg upon antigen encounter in
vitro in the absence of exogenously added TGF-b1. ELISA
analysis of the cell culture supernatants revealed genera-
tion of IL-10, IFN-g, and IL-4 and low amounts of TGF-b1
(Fig. 3C). This TGF-b1 expression could explain the Foxp3
induction and aTreg formation in the in vitro cultures.
Altogether, activ ation of naïve, thymically derived CD4
BDC12-4.1 TCR Tg cells (RAG
background) in vitro
resulted in the generation of effector and Foxp3-expressing
T cells and the production of cytokines with proin-
ammatory and immunoregulatory properties.
Peripheral Foxp3
aTregs coproduce IL-10 and IFN-g.
In order to address how the Foxp3-expressing CD4
T cells
in BDC12-4.1.RAG
TCR Tg mice might exert their Treg
function, we determined their cytokine prole. As shown
in Fig. 4A, polyclonal activation of ex vivo splenocytes
showed that non-Treg cells (CD25
) produced
predominantly IFN-g, whereas Treg cells (CD25
coproduced IFN-g and IL-10. Interestingly, the IFN-g/IL-10
coproduction signature was specicfortheTregcells
FIG. 4. aTregs from BDC12-4.1.RAG
mice coexpress IFN-g and IL-10, whereas Teffs produce solely IFN-g. Total splenocytes from BDC12-
(A and B) and BDC12-4.1.RAG
(C and D) were incubated in vitro for 4 h with BfA (10 mg/mL) either alone (control, no activation) or
in the presence of BfA and anti-CD3/28 (10/5 mg/mL, respectively) or phorbol myristic acid/ionomycin (50/1000 ng/ml, respectively). Cells were
costained for Foxp3, IFN-g, and IL-10 after xation with the Foxp3 Fix/Perm kit. The cytokine pro le is shown after gating on the Treg
) or non-Treg (CD4
) population. In the control (no activation), gating was performed on total CD4
cells. One representative
mouse (for each genotype) of at least three that were tested in two independent experiments is shown. (A high-quality color representation of this
gure is available in the online issue.)
1174 DIABETES, VOL. 61, MAY 2012
Page 6
derived from BDC12-4.1.RAG
and not BDC12-4.1.RAG
mice (Fig. 4B). Thus, BDC12-4.1 mice generate Foxp3
aTregs that coproduce IFN-g and IL-10 in the periphery.
Since BDC12-4.1.RAG
mice exhibit a strong degree of
lymphopenia (11), we wondered whether a proportion of
cells were the result of homeostatic expansion.
Since homeostatically expanded Foxp3
cells express the
signature (26), we p erformed ow
cytometry staining analysis for both markers after gating
on the CD25
(Treg) population. Indeed, as shown
in Fig. 5A, .20% of the Tregs coexpressed CD103/KLRG-1
and therefore were likely derived from homeostatic ex-
pansion. We also found that Teff and Treg populations
stained marginally positive for Ki-67 (Fig. 5B). In control
animals, Treg renewal was more pro-
nounced. To conrm the nonthymically derived lineage
of Foxp3
cells in BDC12-4.1.RAG
mice, we performed
additional staining for the nTreg-specicmarker,Helios
(27). Indeed, ,5% of the Treg population in BDC12-4.1.RAG
mice expressed Helios (Fig. 5B and C). In summary, Foxp3
aTregs develop in the periphery of BCDC12-4.1.RAG
mice as a result of insulin recognition and homeostatic
Lack of Foxp3 in vivo results in 100% diabetes
penetrance in BDC12-4.1 TCR Tg mice in an MHC-
dependent manner. We found a strong correlation be-
tween the age and glucose intolerance in prediabetic
mice (Supplementary Fig. 4). This sug-
gested that despite the induction of aTregs, Teffs accumu-
lated and eliminated more and more b-cells. However, only
a proportion of mice eventually turn diabetic. In order to
address whether Foxp3
cells in the periphery were re-
sponsibl e fo r thi s T1D prot ection in 60 % o f BDC 12-4.1.
mice, we bac k-crosse d Foxp3 mutant ( scurfy)
C57BL/6 mice to Tg BDC12-4.1.RAG
NOD mice for two
generations (see gure legend for details). As depicted in
Fig. 6A and B, spontaneous autoimmune diabetes was
signicantly accelerated with 100% penetrance when the
scurfy Foxp3 mutation was introduced into BDC12-4.1.
mice. At least one copy of I-A
was needed
for diabetes development, and one copy of the I-A
somewhat delayed, but did not prevent, diabetes (Fig. 6C).
FIG. 5. aTregs in BDC12-4.1.RAG
mice do not express Helios, show marginal proliferation, and result from homeostatic expansion. A:Splenocyte-
derived Treg from 7 1 0-week- old BDC12-4.1.RAG
or RAG
signature by ow cytometry. More than 20% of CD4
Foxp3-expressing cells in the RAG
background were likely the result of homeostatic
expansion. B: Majority of Tregs in BDC12-4.1.RAG
mice are not proliferating and do not express Helios compared with Tregs from BDC12-4.1.RAG
Ki-67 and Helios histogram overlay was performed after gating on Treg (CD25
For control, thymi from the same mice were analyzed. Histogram overlay depicts the expression of Ki-67 and Helios after gating on DP (red line),
CD4SP (blue line), and Treg (CD4
) populations (orange line).
Page 7
However, despite the presence of a s ingle I-A
allele, all
Foxp3-mutant (mut) BDC12-4.1.RAG
mice still progressed
to diabetes (Fig. 6C).
The above results conclusively conrmed our initial
hypothesis that in BDC12-4.1.NOD.RAG
mice, pathogenic
as well as regulatory cells coemerge, and the generation of
Tregs was crucial to curb diabetes development. A set
of adoptive transfer experiments was performed to con-
rm pathogenic and regulatory roles of BDC12-4.1 CD4
cells. We reasoned that if the scurfy mutation truly elimi-
nated any regulation by BDC12-4.1 T cells, then adoptive
transfer of total splenocytes or BM transplantation (BMT)
from BDC12-4.1.RAG
mice into NOD.RAG
) recipients would
cause diabetes much faster than transfer of cells from
donors. As shown in Fig. 7A, BMT from
donors into both NOD and g7/
recipients led to more diabetes development
when the donors had the Foxp3 scurfy mutation (Foxp3
than when the donors did not (Foxp3
). Similar obser-
vations were made when splenocytes were transferred
(Fig. 7 B). Interestingly, in this set of experiments, only
when NO D.RAG
, and not I-A
hosts did diabetes occur, underlining the importance of
other insulin-dependent diabetes susceptibility genes for
inuencing disease progression. Taken together, these
results provide strong evidence that the aTreg population
observed in BDC12-4.1.RAG
TCR Tg mice provide ac-
tive peripheral tolerance and control pathogenic cells of
Here we show that the heterogeneous and much-reduced
T1D development in mice uniformly expressing a TCR
specic for a critical insulin B chain epitope (BDC12-4.1.
TCR Tg mice) was due to de novo generation of
insulin-specic Tregs in the periphery. A signicant pro-
portion of insulin B:9-23specic T cells escaped negative
selection and then recognized insulin in the periphery,
which led to their differentiation into Teffs and aTregs. This
is the rst time where a T cell with a xed (autoreactive)
TCR can follow both Teff and aTreg fates when exposed to
similar differentiation niches in vivo.
In accordance, in vitro CD4SPsorted thymocytes that
responded to B:9-23 stimulation produced IL-10 and TGF-b1
and a fraction became Foxp3
. With increasing age, the
number of Teffs increased in the spleen, whereas the
FIG. 6. Lack of Foxp3 (scurfy mutation) signicantly accelerates diabetes onset and increases penetrance in BDC12-4.1.RAG
mice. I-A
(g7/g7 wt) TCR Tg male mice were crossed with I-A
(C57BL/6 background) females (b/b wt/mut). The
resulting back-cross generation 1 (BC1) I-A
female and I-A
male mice were
selected and intercrossed to generate all combinations. Of those, I-A
(g7/g7 mut) and I-A
(g7/g7 wt) male (A)orI-A
(g7/g7 wt/mut) and I-A
mut/mut) female (B) offspring were monitored for diabetes development. In the absence of functional Foxp3, both male and female I-A
mice diabetes develops faster, and disease is 100% penetrant (P < 0.0001). C: Diabetes development in BDC12-4.1.RAG
depends on
both MHC I-A
(g7) and intact Foxp3. T1D development curve shown for the resulting BDC12-4.1.RAG
TCR Tg male mice from the afore-
mentioned intercross. Whereas diabetes incidence in Foxp3 wt mice depends on the presence of the protective MHC allele I-A
(0% diabetes in
g7/b wt vs. 20% in g7/g7 wt), mice bearing the scurfy mutation (mut) develop fast and fully invasive diabetes irrespective their MHC genotype
(100% diabetes in both g7/b mut and g7/g7 mut); females show similar results (data not shown).
1176 DIABETES, VOL. 61, MAY 2012
Page 8
number of Tregs remained relatively constant, w hich
explains the development of T1D in a fraction of the
mice. Accordingly, mice became more
intolerant to glucose as they aged. Insulin-specicTeffs
and Tregs underwent cyclical changes in the PDLNs,
which could be a sign of a more active, iterative immu-
nological process in that location; indeed, earlier studies
had described similar cyclical alterations in islet-reactive
CD8 responses in NOD mice. Alternatively, insulin-specic
Teffs could have migrated to the pancreas, causing the
decrease in cell counts in the PDLNs. In agreement, his-
tological examination showed the presence of few Foxp3
but far more Foxp3
cells in the pancreas, especially
in islets that were massively inltrated.
In the mouse, Foxp3 is the most specic marker for
Treg cells (28), and accumulating evidence suggests
that Foxp3
Treg can be generated from peripheral naïve
or already differentiated T cells in vivo under specic
conditions (29). Such aTregs have been described to
develop at lower levels of antigen availability under tol-
erogenic priming conditions (for example at mucosal
surfaces or in t he p resence of TGF-b1) and are favored
by homeostatic expansion (23). Usually, most Foxp3
Treg originate in the thymus as a result of the regular
T-cell selection process (25). Thymic differentiation of
naturally occurring Tregs is regulated by TCR afnity,
CD28 costimulation, common g-c cytokine (IL-2) recog-
nition, and TGF-b1 production (30,31). Previous studies
with TCR Tg mouse models, where agonist peptides were
expressed in the thymus, showed that self-peptide rec-
ognition not only induces negative selection but also
nTreg differentiation, which suggests that the TCR reper-
toire of Treg cells overlaps somewhat with that of patho-
genic T cells (23,32).
Since insulin is expressed in the thymus, we hypothe-
sized that BDC12-4.1 Foxp3
nTregs may also develop.
However, only on the RAG
background could thymic
nTregs be observed, whereas on the RAG
they were
completely absent. We have evidence to suggest that in the
background, the presence of a dditional
TCR a chains enabled thymic Treg commitment. However,
since nTregs did not develop in the thymus of RAG
either insulin B:9-23 expression is not adequate to drive their
differentiation, medullary epithelial cells lack some im-
portant costimulatory signals, or the afnity/avidity of
the trimolecular TCR/peptide/I-A
complex is too low
in NOD mice. In the future, it would be of interest to an-
alyze thymi from BDC12-4.1 mice on diabetes-resistant ge-
netic backgrounds, i.e., on BALB/c.
In the periphery, where the environment is largely
lymphopenic, T cells start to proliferate. Perhaps these en-
vironmental cues together with stronger insulin recognition
and presentation in the PDLNs permit autoreactive T-cell
activation and differentiation into Teffs and aTregs. De-
spite the immediate effects of the immune system on the
function and viability of insulin-producing b-cells, other
factors, such as androgens, could have contributed to
their decline (33). This is seen by the persistent gender
discrepancy in several genetic scenarios (i.e., RAG
homozygosity, etc.). However, in order to address
how the insulin-specic aTreg modulated disease pro-
gression, we examined their cytokine production prole.
aTregs in BDC12-4.1.RAG
mice coproduce IFN-g and
I L-1 0, whereas Teffs produced solely IFN-g. It is not clear
whether some IFN-g can be important for Treg function as
we and others had shown previously (34,35). To corroborate
this notion, T-bet expression (the master regulator of IFN-g
expression) in Treg cells is essential for their suppressive
FIG. 7. BMT and splenocyte adoptive transfers into RAG
recipients from BDC12-4.1.RAG
but not Foxp3
donors resulted in di-
abetes development. A: BMT from BDC12-4.1.RAG
donors into recipients that are I-A
either on the NOD or C57BL/6 background leads
to signicantly faster diabetes when the donors have the Foxp3 scurfy mutation (Foxp3
) as compared with Foxp3
(P = 0.0016 for NOD.RAG
and P = 0.003 for I-A
recipients). B: The same acceleration of diabetes is observed after splenocyte transfer into NOD.RAG
In this experimental setting, homeostatic expansion is thought to have contributed to the pathogenicity of the cells. However, I-A
recipients do not support the effector function of Foxp3
B:9-23specic (BDC12-4.1 TCR Tg) CD4
T cells, suggesting that other insulin-
dependent diabetes loci are important to support their function in this scenario (P = 0.003 for NOD.RAG
and P = 0.25 for I-A
recipients). In all cases, cells were derived from nondiabetic donors.
Page 9
activity in the scurfy (Foxp3-decient) model (36). The
CD103/KLRG-1 coexpression signature (26) together with
t he absence of high amounts of folate receptor-4 and
Helios, both distinctive markers for nTregs (37) (data not
shown), strongly su pports the adaptive origin.
The BDC12-4.1 TCR Tg mouse is the rst single-Tg,
RAG-decient animal that exhibits spontaneous in vivo
development of aTregs and Teffs with identical TCR
sequence. Perhaps insulin is a unique autoantigen, and
presentation in the context of I-A
provides a permissive
signal to instruct the acquisi tion of aTreg and Teff phe-
notype in cells bearing identical TCRs. It is important to
note that the heterogeneous disease observed in BDC12-
animals, where the TCR repertoire is restricted
to one insulin-specic Tg TCR, is usually not observed in
other Tg or retrogenic CD4
TCR models for T1D. For
example, BDC2.5 mice recognizing the chromogranin auto-
antigen on immunodecient backgrounds develop fulminant
diabetes in 100% of the animals, suggesting that no aTreg
development takes place (38,39). Of evidence, when BDC2.5.
mice are placed onto the scurfy background, no
alteration in disease kinetics is seen.
The present ndings support an intriguing TCR Tg model,
where autoreactive cells escaping negative selection convert
in the periphery into Teffs and aTregs, where their balance
in the periphery determines the degree of islet destruction
and consequently diabetes development. We conclude that it
might be useful in further studies to assess therapies that
might tip the balance toward antigen-specic Tregs rather
than Teffs, and monitoring their frequency and the ratio
could be an important biomarker to predict disease pro-
gression after certain types of therapeutic intervention.
This work was supported by a U01 National Institute of
Allergy and Infectious Diseases prevention center grant to
M.v.H., and the National Institutes of Health (DK-32083),
Autoimmunity Prevention Center (AI050864), Diabetes
Endocrine Research Center (P30-DK-57516), Juvenile Di-
abetes Research Foundation (4-2007-1056 and 11-2005-15),
Childrens Diabetes Foundation, and Brehm Coalition sup-
port to G.E.
No potential conicts of interest relevant to this article
were reported.
The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the
G.F. and J.J. designed and conducted experiments,
analyzed the data, and wrote the manuscript. A.D., T.J., and
Y.C. performed experiments. M.N. performed experiments,
provided important guidance, contributed to the discussion
of the results, and critically improved the manuscript. P.P.
performed immunouorescence staining. F.L., G.S., M.C.,
and H.C. provided important guidance, contributed to the
discussion of the results, and critically improved the manu-
script. G.E. and M.v.H. supervised the study and wrote the
manuscript. G.E. and M.v.H. are the guarantors of this work
and, as such, had full access to all the data in the study and
take responsibility for the integrity of the data and the ac-
curacy of the data analysis.
TheauthorsthankMalinaMcClure for excellent mouse
husbandry, Natalie Amirian for assisting with histological
preparation and staining, and Priscilla Colby for administra-
tive assistance (all from La Jolla Insitute for Allergy and
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  • Source
    • "Intracellular Foxp3 staining was performed using Foxp3 staining kit (eBioscience) and manufacturer's protocol. Intracellular cytokine staining was performed as described [13]. Cells were immediately acquired on a LSRII flow cytometer (BD Biosciences) and analyzed using the FlowJo software (Treestar). "
    [Show abstract] [Hide abstract] ABSTRACT: The infusion of ex vivo-expanded autologous T regulatory (Treg) cells is potentially an effective immunotherapeutic strategy against graft-versus-host disease (GvHD) and several autoimmune diseases, such as type 1 diabetes (T1D). However, in vitro differentiation of antigen-specific T cells into functional and stable Treg (iTreg) cells has proved challenging. As insulin is the major autoantigen leading to T1D, we tested the capacity of insulin-specific T-cell receptor (TCR) transgenic CD4+ T cells of the BDC12-4.1 clone to convert into Foxp3+ iTreg cells. We found that in vitro polarization toward Foxp3+ iTreg was effective with a majority (>70%) of expanded cells expressing Foxp3. However, adoptive transfer of Foxp3+ BDC12-4.1 cells did not prevent diabetes onset in immunocompetent NOD mice. Thus, in vitro polarization of insulin-specific BDC12-4.1 TCR transgenic CD4+ T cells toward Foxp3+ cells did not provide dominant tolerance in recipient mice. These results highlight the disconnect between an in vitro acquired Foxp3+ cell phenotype and its associated in vivo regulatory potential.
    Full-text · Article · Nov 2014 · PLoS ONE
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    [Show abstract] [Hide abstract] ABSTRACT: Foxp3(+) CD4(+) T helper cells called regulatory T (T reg) cells play a key role in controlling reactivity to self-antigens and onset of autoimmunity. T reg cells either arise in thymus and are called natural T reg (nT reg) cells or are generated in the periphery through induction of Foxp3 and are called inducible T reg (iT reg) cells. The relative contributions of iT reg cells and nT reg cells in peripheral tolerance remain unclear as a result of an inability to separate these two subsets of T reg cells. Using a combination of novel TCR transgenic mice with a defined self-antigen specificity and conventional mouse models, we demonstrate that a cell surface molecule, neuropilin-1 (Nrp-1), is expressed at high levels on nT reg cells and can be used to separate nT reg versus iT reg cells in certain physiological settings. In addition, iT reg cells generated through antigen delivery or converted under homeostatic conditions lack Nrp-1 expression. Nrp-1(lo) iT reg cells show similar suppressive activity to nT reg cells in controlling ongoing autoimmune responses under homeostatic conditions. In contrast, their activity might be compromised in certain lymphopenic settings. Collectively, our data show that Nrp-1 provides an excellent marker to distinguish distinct T reg subsets and will be useful in studying the role of nT reg versus iT reg cells in different disease settings.
    Full-text · Article · Sep 2012 · Journal of Experimental Medicine
  • [Show abstract] [Hide abstract] ABSTRACT: It is widely accepted that Type 1 diabetes is a complex disease. Genetic predisposition and environmental factors favour the triggering of autoimmune responses against pancreatic β-cells, eventually leading to β-cell destruction. Over 40 susceptibility loci have been identified, many now mapped to known genes, largely supporting a dominant role for an immune-mediated pathogenesis. This role is also supported by the identification of several islet autoantigens and antigen-specific responses in patients with recent onset diabetes and subjects with pre-diabetes. Increasing evidence suggests certain viruses as a common environmental factor, together with diet and the gut microbiome. Inflammation and insulin resistance are emerging as additional cofactors, which might be interrelated with environmental factors. The heterogeneity of disease progression and clinical manifestations is likely a reflection of this multifactorial pathogenesis. So far, clinical trials have been mostly ineffective in delaying progression to overt diabetes in relatives at increased risk, or in reducing further loss of insulin secretion in patients with new-onset diabetes. This limited success may reflect, in part, our incomplete understanding of key pathogenic mechanisms, the lack of truly robust biomarkers of both disease activity and β-cell destruction, and the inability to assess the relative contributions of various pathogenic mechanisms at various time points during the course of the natural history of Type 1 diabetes. Emerging data and a re-evaluation of histopathological, immunological and metabolic findings suggest the hypothesis that unknown mechanisms of β-cell dysfunction may be present at diagnosis, and may contribute to the development of hyperglycaemia and clinical symptoms. © 2012 The Authors. Diabetic Medicine © 2012 Diabetes UK.
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