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Increased number and function of FoxP3 regulatory T cells during experimental arthritis.

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Vol. 58, No. 12, December 2008, pp 3730–3741
DOI 10.1002/art.24048
© 2008, American College of Rheumatology
Increased Number and Function of FoxP3 Regulatory T Cells
During Experimental Arthritis
Kristen Monte, Christina Wilson, and Fei F. Shih
Objective. CD4ⴙCD25ⴙFoxP3ⴙ regulatory T
(Treg) cells are critical regulators of autoimmunity. Yet
the number of Treg cells is paradoxically increased in
rheumatoid arthritis (RA) patients, and Treg cells show
variable activity in human studies. The objective of this
study was to characterize the expansion and function of
Treg cells during the initiation and progression of
experimental arthritis.
Methods. To unequivocally identify Treg cells, we
crossed FoxP3gfp mice with K/BxN mice to generate
arthritic mice in which Treg cells express green fluorescence protein. We examined the expansion and function
of Treg cells and effector T (Teff) cells during different
stages of arthritis, using flow cytometry and cell proliferation analyses.
Results. In K/BxN mice, thymic selection of KRN
T cells resulted in an enrichment of forkhead box P3
(FoxP3)–positive Treg cells. Treg cell numbers increased
during arthritis, with significant increases in spleens and
draining lymph nodes, indicating selective tropism to sites
of disease. In contrast to the in vitro unresponsiveness of
Treg cells when cultured alone, substantial proportions of
Treg cells proliferated in both nonarthritic and arthritic
mice. However, they also underwent greater apoptosis,
thereby maintaining equilibrium with Teff cells. Similarly,
enhanced Treg cell–suppressive activity during arthritis
was offset by greater resistance by their Teff cell counterparts and antigen-presenting cells.
Conclusion. In this well-established model of RA,
the interplay of Teff cells and Treg cells in K/BxN mice
recapitulated many features of the human disease. We
demonstrated an ordered expansion of Treg cells during
arthritis and dynamic changes in Treg cell and Teff cell
functions. By elucidating factors that govern Treg cell
and Teff cell development in K/BxNgfp mice, we will gain
insight into the pathophysiology of and develop novel
therapeutics for human RA.
Rheumatoid arthritis (RA) is an autoimmune
disease that results in joint inflammation and destruction. Autoreactive T cells and B cells are crucial to its
pathogenesis. Autoreactive T cells are predominantly
deleted in the thymus, but this process is not stringent.
Thus, autoreactive T cells can and do escape into the
peripheral T cell pool; subsequent activation can result
in autoimmune pathologic conditions. CD4⫹CD25⫹
FoxP3⫹ regulatory T (Treg) cells, which comprise 5–
10% of CD4⫹ T cells, are crucial for the maintenance of
peripheral tolerance (1,2).
In adult RA and juvenile idiopathic arthritis,
Treg cells were found to be enriched in the synovial fluid
of inflamed joints as compared with their levels in
peripheral blood, suggesting active homing or expansion
at sites of inflammation (3–8). These Treg cells expressed forkhead box P3 (FoxP3) and suppressed both
proliferation and cytokine production by CD4⫹CD25–
effector T (Teff) cells. Moreover, increased numbers of
Treg cells in the synovial fluid directly correlated with
limited disease (3), suggesting that Treg cells aid in
disease remission.
However, it is not known how Treg cells modulate immunity, because the number of Treg cells is
paradoxically increased during disease, and since there is
also variability in Treg cell function among different
studies. Ehrenstein et al (7), for example, demonstrated
that Treg cells from RA patients showed compromised
function as compared with those from healthy controls,
whereas other investigators (8) showed that Treg cells
obtained during active RA were equally suppressive or
were more suppressive than those from healthy controls.
Supported by grants from the NIH (K12-HD-01487-1 and
K08-AR-051980-01) and a pilot and feasibility grant from the American Cancer Society (IRG-58-010-49).
Kristen Monte, BS, Christina Wilson, BS, Fei F. Shih, MD,
PhD (current address: Pfizer Inc., Chesterfield, Missouri): Washington
University School of Medicine, St. Louis, Missouri.
Address correspondence and reprint requests to Fei F. Shih,
MD, PhD, Associate Research Fellow, Indications Discovery, Pfizer
Inc., 700 Chesterfield Parkway West, T1C, Chesterfield, MO 63017.
Submitted for publication November 14, 2007; accepted in
revised form August 15, 2008.
In this case, the increased suppressive function was
offset by the responder Teff cells themselves being more
refractory to suppression and by the presence of inflammatory cytokines (9). Because these studies examined
heterogeneous patient populations, disease stages, and
therapeutic regimens, these variables likely contributed
to the differences between the studies. In addition,
investigators have differed in the criteria they use to
identify Treg cells, with some studies focusing only on
CD25bright populations while others used all CD25⫹ T
cells (8,10). Because expression of CD25 is also elevated
in activated Teff cells, the number of which is increased
during disease, varying degrees of Teff cell contamination can render interpretation difficult. It is also unclear
if arthritis resulted from a primary Treg cell dysfunction
or a secondary defect due to persistent inflammation.
To eliminate these confounding processes, we
examined Treg cell development and function in a
well-characterized murine model of RA. K/BxN mice
were generated by crossing KRN T cell receptor
(TCR)–transgenic mice with NOD mice (11). The
disease is fully penetrant in all progeny and follows a
predictable course of progressive symmetric distal
polyarthritis that resembles RA in humans. Arthritis
results from autoreactive KRN T cells recognizing
peptide 281–293 of the glycolytic enzyme glucose-6phosphate isomerase (GPI) that is bound to I-Ag7 (the
NOD-specific class II major histocompatibility complex allele) (12,13). Incomplete thymic deletion allows
autoreactive KRN CD4⫹ T cells to persist in the
periphery and to become activated by endogenously
presented GPI. KRN T cells then provide help to
GPI-specific B cells, giving rise to arthritogenic antibodies. Treg cells are enriched in arthritic K/BxN mice
(14,15), and the loss of Treg cells results in earlier and
more extended disease, suggesting that although Treg
cells do not prevent arthritis, they may nevertheless
mitigate it (15). Similar findings have been reported
for collagen-induced arthritis, in which depletion of
CD25⫹ T cells exacerbated arthritis and adoptive
transfer of CD4⫹CD25⫹ Treg cells or FoxP3transduced T cells ameliorated the disease (16–18).
To understand how antigen-specific Treg cells
develop during the course of arthritis, we crossed K/BxN
mice with FoxP3gfp reporter mice to unequivocally identify Treg cells. We then analyzed Treg cell selection in
the thymus and followed their expansion and function
during the progression of arthritis.
Mice. KRN mice have been described in detail elsewhere (11). FoxP3gfp mice were generously provided by A.
Rudensky and were bred to KRN mice to generate KRNgfp
mice in which FoxP3⫹ T cells expressed GFP (19). These were
subsequently bred to NOD/LtJ mice (The Jackson Laboratory,
Bar Harbor, ME) to generate K/BxNgfp mice. Nonarthritic
controls were KRNgfp on the C57BL/6 background. Because
FoxP3 is on the X chromosome, only male K/BxNgfp mice were
analyzed. The K/BxNgfp mice exhibited arthritis with equivalent severity and time course as the K/BxN mice (data not
shown). All mice were bred and housed under specific
pathogen–free conditions in the animal facility at the Washington University Medical Center, in accordance with the
standards of the Animal Studies Committee.
Flow cytometry. Thymi, spleens, draining lymph nodes
(LNs; popliteal, axillary, and brachial), nondraining LNs (inguinal and cervical), and mesenteric LNs were harvested from
individual mice and analyzed separately. Single-cell suspensions (3 ⫻ 106) were treated with anti-CD16 (clone 2.4G2)
prior to surface staining with specific antibodies according
to standard protocols. The following antibodies were used:
allophycocyanin (APC)–labeled anti-CD4, phycoerythrin
(PE)–labeled anti-CD4 (clone GK1.5), PE-Cy7–labeled antiCD8 (clone 53-6.7), PE-Cy7–labeled anti-CD25 (clone
PC61), APC-labeled antibromodeoxyuridine (anti-BrdU),
APC-labeled anti-CD45RB (clone C363-16A), PE-labeled
annexin V, and PE-labeled anti–Bcl-2. Antibody kits for
anti-CD4, anti-CD8, anti-CD25, and anti-CD45RB were
obtained from eBiosciences (San Diego, CA); all other
antibodies were obtained from BD Biosciences (San Diego,
CA). Streptavidin–peridinin chlorophyll A protein was obtained from BD Biosciences. TCR V␤6 was used to track the
KRN-transgenic TCR. All samples were analyzed on either
a FACSAria or a FACSCalibur flow cytometer (both from
BD Instruments) using FlowJo software (Tree Star, Ashland, OR). Lymphocytes were gated based on forwardscatter and side-scatter characteristics, and 1–2.5 ⫻ 105
gated events were collected per sample.
Lymphocyte isolation from paws. Front and rear paws
were harvested from each mouse, and the skin was removed.
Paws from each mouse were minced in 40 ml of RPMI 1640
with 5% calf serum, 125 ␮l of type VIII collagenase (10,000
units/ml), and 200 ␮l of Dispase (50 units/ml). Minced tissues
were agitated for 2 hours at 37°C at a speed of 150 revolutions
per minute, with vortexing every 30 minutes. Digested tissues
were strained through a 70-␮m Nitex membrane. Lymphocytes
were harvested over a Ficoll gradient and analyzed by flow
In vitro T cell cultures. CD4⫹ T cells from pooled
spleens and draining LNs were enriched by positive selection
using anti-CD4 microbeads (Miltenyi Biotec, Sunnyvale, CA).
Purity was typically ⬎90%. Dendritic cells (DCs) and B cells
were prepared from spleens as described elsewhere (20). T
cells (5 ⫻ 105/well) were stimulated with 5 ⫻ 104 DCs in 1 ml
of Iscove’s medium containing 10% heat-inactivated bovine
growth serum (Hyclone, Logan, UT), 2 ␮M Glutamax (GibcoBRL, Gaithersburg, MD), 2 ⫻ 10–5M ␤-mercaptoethanol, and 50
␮g/ml of gentamicin (referred to hereinafter as ISC-10). At
various times, 1 mM BrdU was added (final concentration
10 ␮M), and cultures were analyzed 12 hours later for BrdU
incorporation and apoptosis.
T cell suppression assays. CD4⫹ T cells were enriched
by negative selection using anti-B220 and anti-CD8, followed
by negative selection using goat anti-rat Ig–coupled paramag-
Figure 1. Increased FoxP3⫹ thymocyte selection in K/BxNgfp mice. A, Thymocyte analysis by flow cytometry. Thymocytes
from 4-week-old KRNgfp and K/BxNgfp mice were labeled with allophycocyanin (APC)–conjugated CD4 and phycoerythrin
(PE)–conjugated CD8 and analyzed. Live cells were gated on CD4 and CD8. Values in each quadrant are the percentage
of positive cells in each subset. B, Histograms showing the expression of FoxP3 (top; numbers of cells) and V␤6 (bottom;
percentage of maximum) in gated CD4⫹ single-positive thymocytes, by flow cytometry. V␤6 cells were labeled with
V␤6–biotin followed by streptavidin–peridinin chlorophyll A protein (PerCP). Horizontal bars show the mean percentage
of FoxP3⫹ cells. C and D, Percentages (C) and numbers (D) of FoxP3⫹ thymocytes in CD4⫹ single-positive (SP)
thymocytes from 8 KRNgfp mice and 10 K/BxNgfp mice. Each data point represents a single mouse; horizontal lines show
the mean. P values were determined by Wilcoxon’s rank sum test.
netic beads (Chemicell, Berlin, Germany). Cells were stained
with PE-labeled anti-CD4 prior to sorting for CD4⫹FoxP3–
and CD4⫹FoxP3⫹ T cells using a FACSAria cell sorter. Cell
purity was typically ⬎98%. In some experiments, APC-labeled
anti-CD45RB was used to identify naive and memory T cells.
Proliferation assays were performed in triplicate with 1 ⫻ 105
CD4⫹ Teff cells/well, with varying ratios of Treg cells and 2 ⫻
105 irradiated splenocytes and 10 ␮M GPI peptide (Global
Peptide, Fort Collins, CO) in round-bottomed plates with
ISC-10. In other experiments, irradiated purified DCs (1 ⫻
104/well) or B cells (2 ⫻ 105/well) were used as allogeneic
stimulators for polyclonal CD4⫹ T cells with anti-CD3 (5
␮g/ml). Neutralizing anti–IL-6 (10 ␮g/ml, clone MP5-20F3;
eBiosciences) or tumor necrosis factor receptor:Fc fusion
protein (etanercept; 10 ␮g/ml) was added as indicated. Cultures were pulsed at 72 hours with 0.2 ␮Ci of 3H-thymidine/
well and harvested 18 hours later. Proliferation was measured
as the counts per minute of 3H-thymidine incorporated.
Transwell suppression assays. DCs or B cells from
NOD mice or arthritic mice were activated for 1 hour with
plate-bound anti-CD40 (5 ␮g/ml) (clone 1C10; R&D Systems,
Minneapolis, MN) and washed extensively before being added
to the T cells. Teff cells (5 ⫻ 105), Treg cells (1.25 ⫻ 105), and
irradiated splenocytes (5 ⫻ 105) were added in 0.4-␮m Transwell inserts or were cocultured with anti-CD40–activated DCs
(5 ⫻ 104) or B cells (5 ⫻ 105) in 1 ml of ISC-10. T cells were
activated with anti-CD3 (5 ␮g/ml; clone 2C11). After 72 hours,
10 ␮l of 1 mM BrdU was added to each well, and BrdU
incorporation was assessed by flow cytometry after another 12
Statistical analysis. Wilcoxon’s rank sum test and
Student’s t-test were calculated using InStat 2.00 software
(GraphPad Software, La Jolla, CA).
Thymic selection of FoxP3ⴙ Treg cells in
K/BxN mice. We used K/BxNgfp mice to examine the
development and function of FoxP3⫹ Treg cells during
arthritis. In these mice, Treg cell–suppressive activity
resided specifically in the FoxP3⫹ (green fluorescence
protein [GFP]–positive) population, regardless of CD25
expression (19). Therefore, we defined Treg cells as
CD4⫹FoxP3⫹ T cells and Teff cells as CD4⫹FoxP3– T
FoxP3 expression in thymocytes was analyzed in
4-week-old KRNgfp and K/BxNgfp mice to minimize the
effects of chronic inflammation on thymocyte development. In KRNgfp mice, CD4⫹ single-positive thymocytes
comprised 1–3% of total thymocytes (Figure 1A). Negative selection by endogenous GPI resulted in an ⬃3fold decrease in thymic cellularity (mean ⫾ SD 21.5 ⫾
2.37 ⫻ 107 in KRNgfp mice and 8.3 ⫾ 1.75 ⫻ 107 in
K/BxNgfp mice), resulting primarily from losses in the
double-positive subset (Figure 1A). Consistent with our
previous observations, Treg cells were enriched 2–3-fold
among CD4⫹ single-positive thymocytes in K/BxNgfp
mice as compared with KRNgfp mice (Figures 1B and C)
To examine the effect of KRN TCR expression
on FoxP3 selection, we analyzed the expression of the
transgene-encoded TCR V␤6, because there is no clonotypical antibody to the KRN TCR. Consistent with
the results obtained in other transgenic TCR systems,
recognition of its cognate peptide appears to be a
prerequisite for KRN Treg cell selection (21,22). In
KRNgfp mice, which lacked GPI–I-Ag7 complexes,
V␤6bright KRN T cells were seen only among the Teff
cells, suggesting that Treg cells required endogenous
TCR expression (resulting in lower V␤6 levels) for
selection (Figure 1B). Conversely, in K/BxNgfp mice,
negative selection by endogenous GPI resulted in preferential deletion of V␤6bright Teff cells. The remaining
Teff cells expressed equivalently lower levels of V␤6, as
in the Treg cells. Statistically higher numbers of CD4⫹
single-positive FoxP3⫹ thymocytes were found in
K/BxNgfp mice (Figure 1D). The enrichment of Treg
cells in K/BxNgfp mice resulted from both selective
deletion of CD4⫹FoxP3– thymocytes, especially
V␤6bright T cells, and induction of FoxP3⫹ thymocytes.
Development of KRN Treg cells during arthritis
in K/BxNgfp mice. The frequencies of Treg cells were
increased similarly in the spleen and peripheral LNs.
While GFP expression correlated largely with CD25
expression in KRNgfp mice, 10–15% of CD25⫹ cells
were FoxP3– and were activated Teff cells (19). This
proportion of CD25⫹FoxP3– Teff cells was doubled in
K/BxNgfp mice, with significant overlap with FoxP3⫹
Treg cells, highlighting the power of the FoxP3-GFP
reporter in this autoimmune model (Figure 2A). To
show that FoxP3⫹ T cells function as Treg cells, sorted
CD4⫹FoxP3⫹ T cells were tested in a suppression assay
using CD4⫹FoxP3– T cells as responder Teff cells. Treg
cells did not proliferate in response to NOD dendritic
cells (DCs) presenting either endogenous GPI or KRN
superagonist G7m (13). However, they nevertheless
suppressed the proliferation of Teff cells when cocultured in a 1:1 ratio (Figure 2B).
To determine Treg cell expansion during arthritis, we quantified the frequency and numbers of Treg
cells in the spleen, joint draining LNs, nonjoint draining
LNs, and mesenteric LNs during different phases of
arthritis: nonarthritic KRNgfp control, acutely arthritic
(4–6 weeks) K/BxNgfp mice, and chronically arthritic
(8–10 weeks) K/BxNgfp mice. In all organs surveyed, the
frequencies of Treg cells were increased in a hierarchical
manner, with higher increases in the spleens and joint
draining LNs relative to the nondraining LNs and mesenteric LNs in K/BxNgfp mice compared with KRNgfp
mice (Figure 2C). (For spleens, P ⬍ 0.0001, nonarthritic
versus acutely arthritic mice, and P ⫽ 0.0003, nonarthritic versus chronically arthritic mice, by Wilcoxon’s
rank sum test. For draining LNs, P ⫽ 0.02, nonarthritic
versus acutely arthritic mice, P ⫽ 0.0037, nonarthritic
versus chronically arthritic mice, and P nonsignificant,
acutely arthritic versus chronically arthritic mice. In both
acutely arthritic and chronically arthritic mice, P ⬍ 0.005
for comparisons between each of the different organs.)
There was no statistically significant difference in the
distribution of Treg cells in the various organs in nonarthritic mice.
When we quantified the total numbers of Treg
cells in arthritic mice, Treg cell numbers were significantly increased in the spleens of acutely arthritic mice
and in the spleens and draining LNs of chronically
arthritic mice as compared with nonarthritic mice (P ⬍
0.05 for spleen cells and draining LNs between nonarthritic mice and their counterparts in both acutely and
chronically arthritic mice). Within the nonarthritic
group, there was no statistically significant difference in
the Treg cell numbers in various organs. Within the
acutely arthritic group, differences between spleens and
the various LNs were significant (P ⬍ 0.05). Within the
chronically arthritic group, Treg cell numbers in the
spleen and draining LNs were significantly different
from those in nondraining LNs and mesenteric LNs.
Therefore, Treg cells first expanded in the spleens and
then spread to the draining LNs during disease progression (Figure 2D). Comparison of Teff cell and Treg cell
numbers in the spleens and LNs showed that Treg cell
expansion was met by similar expansion by Teff cells,
such that the ratios of Teff cells to Treg cells were
maintained during arthritis (Figure 2E).
We also examined Teff cells and Treg cells in the
paws. In nonarthritic mice, ⬍1% were CD4⫹ T cells.
Of these, 5.9 ⫾ 3.2% (mean ⫾ SD) were Treg cells
Figure 2. Site-specific expansion of FoxP3⫹ Treg cells during arthritis. A, Splenocyte analysis by flow cytometry.
Splenocytes from 4-week-old KRNgfp and K/BxNgfp mice were labeled with allophycocyanin (APC)–conjugated CD4 and
phycoerythrin (PE)–conjugated CD25 and analyzed. Shown are percentages in live cells gated on CD4 and CD25 (top) and
expression of CD25 and FoxP3–green fluorescence protein (GFP) in gated CD4⫹ splenocytes (bottom). Values in each
quadrant are the percentage of positive cells in each subset. B, T cell suppression assay. Treg cells (TR; CD4⫹FoxP3⫹) and
Teff cells (TE; CD4⫹FoxP3–) were sorted and stimulated with irradiated NOD splenocytes, with or without 1 ␮M G7m
peptide, either separately or in coculture (1:1 ratio). C, Percentages of CD4⫹FoxP3⫹ Treg cells in spleens and lymph nodes
(LNs; draining [dLNs], nondraining [ndLNs], and mesenteric [mLNs]) from 8 nonarthritic, 10 acutely arthritic, and 6
chronically arthritic mice. D, Total numbers of CD4⫹FoxP3⫹ Treg cells in spleens and LNs from 8 nonarthritic, 10 acutely
arthritic, and 6 chronically arthritic mice. E, Total numbers of CD4⫹FoxP3– Teff cells in spleen and LNs from 8
nonarthritic, 10 acutely arthritic, and 6 chronically arthritic mice. Values in B–E are the mean ⫾ SD. F, Analysis of
lymphocytes from inflamed paws. Lymphocytes were isolated from inflamed paws and analyzed for PE-labeled CD4 and
GFP-labeled FoxP3 by flow cytometry. Live lymphocytes were identified by forward-scatter and side-scatter characteristics,
as well as propidium iodide exclusion. Values in each quadrant are the percentage of positive cells in each subset; numbers
in parentheses are the percentage of CD4⫹ T cells. Results are representative of 5 nonarthritic, 6 acutely arthritic, and 6
chronically arthritic mice. Med ⫽ medium.
Figure 3. Higher rates of Treg cell proliferation than Teff cell proliferation in vivo. A, Fraction of bromodeoxyuridine
(BrdU)–positive Treg cells (TR) and Teff cells (TE) in spleens, draining lymph nodes (dLNs), and nondraining LNs
(ndLNs) from KRNgfp mice and acutely arthritic K/BxNgfp mice. Values are the mean and SD of 4 mice per group.
Results are representative of 4 separate experiments. B, Plot of the numbers of BrdU⫹ Treg cells versus Teff cells in
draining LNs from 20 arthritic mice. The bisecting diagonal line indicates 1:1 correspondence. With a few exceptions,
more Treg cells than Teff cells incorporated BrdU. C, Bcl-2 expression in splenic Treg cells and Teff cells from arthritic
mice. Cells were gated on FoxP3–green fluorescent protein (GFP) and CD4–allophycocyanin (right) and analyzed for
intracellular Bcl-2 expression (left). Values in each quadrant are the percentage of each subset in splenocytes. Dark line
shows phycoerythrin (PE)–labeled Bcl-2; shaded histogram shows the isotype control. Horizontal bars show the
percentage of Bcl-2 cells. D, Frequency of Bcl-2⫹ cells and mean fluorescence intensity (MFI) of Bcl-2 expression in
Teff cells and Treg cells from draining LNs obtained from 4 arthritic mice. Horizontal bars show the mean. Results are
representative of 2 independent experiments. P values were determined by Fisher’s t-test. E, Percentage of annexin V⫹
(Ann V⫹) Teff cells and Treg cells in draining LNs from 5 arthritic mice. Horizontal bars show the mean. Results are
representative of 2 independent experiments. P value was determined by Fisher’s t-test.
(Figure 2F). During acute arthritis, CD4⫹ T cells
comprised 2.1 ⫾ 0.7%, with 58.5 ⫾ 10.9% being Treg
cells. As the inflammation waned in the paws, Treg
cells became less frequent, comprising 39.9 ⫾ 10.1%
of CD4⫹ T cells. Our data were consistent with
findings in human RA showing an accumulation of
Treg cells during active inflammation.
Greater in vivo turnover of Treg cells than Teff
cells in K/BxNgfp mice. Because increased numbers of
Treg cells in the spleen and LNs during arthritis can
arise from increased proliferation or migration, we
directly examined Teff cell and Treg cell proliferation by
in vivo BrdU incorporation. Nonarthritic KRNgfp mice
and acutely arthritic K/BxNgfp mice were injected intra-
peritoneally with 1 mg of BrdU 12 hours prior to killing.
The proportions of BrdU⫹ V␤6⫹CD4⫹FoxP3– (KRN
Teff cells) and V␤6⫹CD4⫹FoxP3⫹ (KRN Treg cells)
were quantified. In nonarthritic KRNgfp mice, higher
proportions of Treg cells (⬃10%) than Teff cells (1–3%)
incorporated BrdU (Figure 3A). This tonic proliferation
by Treg cells suggests active surveillance at steady-state,
even in nonlymphopenic mice.
In arthritic K/BxNgfp mice, recognition of GPI
increased the proportion of cycling Teff cells and Treg
cells by 3-fold, resulting in 5–10% of Teff cells and
30–35% of Treg cells incorporating BrdU. Consistently
higher proportions of cycling Treg cells were observed in
the LNs (Figure 3A) and accounted for the greater
increase in T cell numbers seen in the LNs as the disease
progressed from an acute to a chronic phase (Figure
To directly compare the numbers of cycling Teff
cells and Treg cells, we plotted the numbers of BrdU⫹
Teff cells versus the numbers of BrdU⫹ Treg cells in
draining LNs from arthritic mice (Figure 3B). With few
exceptions, more Treg cells than Teff cells incorporated
BrdU. In some mice, the number of BrdU⫹ Treg cells
was 3-fold higher than the number of BrdU⫹ Teff cells.
We did not, however, find any correlation between our
gross measurements of paw inflammation with the numbers of BrdU⫹ Treg cells or Teff cells. Similar results
were seen in the spleens and other LNs (data not
Given the higher rate of Treg cell proliferation,
the proportions of Treg cells should increase over time.
However, this was not the case (Figure 2C). To reconcile
this discrepancy, we hypothesized that the increased
turnover is balanced by increased death. In support of
this, we found that Treg cells from arthritic mice expressed significantly less antiapoptotic factor Bcl-2 (Figures 3C and D) and higher levels of apoptosis (annexin
V⫹ cells) than did Teff cells ex vivo (Figure 3E).
Different kinetics of proliferation and death of
FoxP3ⴙ Treg cells and FoxP3– Teff cells. To test our
hypothesis that Treg cells proliferated and died at a
faster rate than Teff cells, we directly examined the
kinetics of Treg cell and Teff cell proliferation and
attrition in vitro. KRNgfp CD4⫹ T cells were purified
from pooled spleens and LNs and were cultured with
NOD DCs. This approach allowed us to specifically
examine intrinsic differences between Treg cells and
Teff cells separately from the inflammatory milieu in
arthritic mice. We used naive T cells to synchronize the
exposure to antigenic stimuli. At various times, BrdU
was added to the cultures, and BrdU incorporation was
analyzed 12 hours later.
Figure 4. Kinetics of Treg cell and Teff cell proliferation and cell
death upon antigen stimulation. A, Bromodeoxyuridine (BrdU) incorporation by Treg cells (TR) and Teff cells (TE) analyzed at the
indicated times. Purified CD4⫹ T cells from KRNgfp mice were
cultured with NOD dendritic cells (DCs). BrdU (1 mM) was added at
various times, and incorporation was analyzed by flow cytometry 12
hours later. Values are the mean ⫾ SD of duplicate wells. Results are
representative of 3 experiments. B and C, Percentages of dead and
apoptotic Treg cells and Teff cells in arthritic and nonarthritic mice.
Purified CD4⫹ T cells from KRNgfp and arthritic K/BxNgfp mice were
cultured with NOD DCs, and dead and apoptotic CD4⫹ T cells were
analyzed with 7-aminoactinomycin D (7-AAD) and annexin V, respectively, at the indicated times. Values are the mean of duplicate wells
(SDs were ⬍15%). The experiment was performed 3 times, and the
results were similar.
In contrast to the in vitro anergy noted when Treg
cells were cultured alone, Treg cells showed significant
proliferation when cultured at a physiologic ratio with
Teff cells and stimulated with physiologic amounts of
GPI. By day 1, 20% of FoxP3⫹ Treg cells had entered
cell cycle and incorporated BrdU, as compared with
⬍2% of FoxP3– Teff cells. BrdU incorporation by Treg
cells peaked on day 2 and declined over days 3 and 4
(Figure 4A). In contrast, Teff cells did not proliferate to
any appreciable degree in the first 24 hours of culture
but proliferated vigorously in the subsequent days. Our
data showed that Teff cells and Treg cells proliferated
with distinct kinetics and magnitude. Treg cells proliferated earlier, but the response was short lived. Teff cell
proliferation, though delayed, was more sustained.
In a parallel experiment, cell death and apoptosis
of KRNgfp T cells were analyzed using 7-aminoactinomycin D and annexin V to identify dead cells and
apoptotic cells, respectively. As shown in Figure 4B, the
proportions of dead and apoptotic Treg cells were
roughly twice the proportions of dead and apoptotic Teff
cells observed during this time course. Interestingly, the
time course of Treg cell apoptosis correlated with Treg
cell proliferation: both peaked on day 2 and declined on
days 3 and 4. This finding contrasted with the kinetics of
Teff cell proliferation and apoptosis and provided support that Bcl-2 expression in Teff cells conferred protection from apoptosis.
To determine if prior activation conferred protection from apoptosis, Teff cells and Treg cells from
arthritic K/BxNgfp mice were also analyzed (Figure 4C).
Naive and activated Teff cells displayed similar rates of
cell death. Compared with naive Treg cells, Treg cells
from K/BxNgfp mice displayed less apoptosis and death.
However, relative to their Teff cell counterparts, both
naive and activated Treg cells still showed higher rates of
apoptosis. Together, the data confirmed our hypothesis
that increased Treg cell proliferation was offset by
increased Treg cell apoptosis, resulting in Teff cell/Treg
cell homeostasis.
Increasingly suppressive Treg cells during arthritis. To interrogate Treg cell function during arthritis,
we sorted Teff cells and Treg cells from acutely and
chronically arthritic mice. In addition, to determine if
there were site-specific effects, Teff cells and Treg cells
from spleens and draining LNs were analyzed separately. We first compared the proliferation of Teff cells
from the spleens and LNs during acute and chronic
disease. LN Teff cells proliferated better than splenic
Teff cells in response to GPI, but there was no statistically significant difference in the proliferation of Teff
cells between acute and chronic disease (Figure 5A).
We next examined the ability of Treg cells to
suppress Teff cell proliferation. Because Teff cells from
chronically arthritic mice would be targets for Treg cell
immunotherapy, we used LN Teff cells from chronically
arthritic mice as responders. As shown in Figure 5B,
Treg cells obtained from different sites and during
different disease states suppressed Teff cell proliferation
with differing efficacy. While all Treg cells were effective
at a 1:1 ratio, splenic Treg cells from acutely arthritic
Figure 5. Dynamic changes in Treg cell and Teff cell activity during
arthritis. A, Teff cells (CD4⫹FoxP3–) were sorted from spleens (Spl)
or pooled lymph nodes (LNs) obtained from acutely or chronically
arthritic mice. Teff cells (1 ⫻ 105/well) were stimulated with irradiated
NOD dendritic cells (DCs), and cell proliferation was assayed by
H-thymidine incorporation during the last 16 hours of a 72-hour
culture. Values are the mean ⫾ SD of triplicate wells. B, Treg cells
(TR) from different sites and different disease states were analyzed for
their suppressive activity against Teff cells (TE). Values are the
mean ⫾ SD of triplicate wells. C and D, CD4⫹FoxP3–CD45RBlow
(memory) and CD4⫹FoxP3–CD45RBhigh (naive) Teff cells from
arthritic mice were cultured at varying ratios of Treg cells with NOD
DCs. Values are the mean ⫾ SD of either the cpm of triplicate wells
(C) or normalized as a fraction of the maximum cpm of Teff cells alone
(D). P ⬍ 0.02 for CD4⫹FoxP3–CD45RBhigh versus CD4⫹FoxP3–
CD45RBlow cells at each of the Teff cell–to–Treg cell ratios in C, by
Fisher’s t-test. NS ⫽ not significant. Results are representative of 3
similar experiments.
mice were the least suppressive. Conversely, LN Treg
cells from chronically arthritic mice were the most
effective. Thus, Treg cell activity increased during arthritis and at sites of active inflammation.
To determine if the Teff cell activation state
influences its susceptibility to suppression, we sorted
FoxP3–CD45RB high (naive) Teff cells, FoxP3–
CD45RBlow (memory) Teff cells, and FoxP3⫹ Treg cells
from pooled splenic and draining LN T cells obtained
from arthritic mice. Naive CD45RBhigh Teff cells were
Figure 6. Reduced Treg cell suppression in dendritic cells (DCs) and B cells from arthritic mice.
A and B, Dendritic cells and B cells were purified from NOD and K/BxN arthritic mice using microbeads. Teff cells (TE) and Treg cells (TR) from FoxP3gfp mice were sorted. Teff cells (1 ⫻ 105/well)
were cultured with either 1 ⫻ 104 DCs (A) or 2 ⫻ 105 B cells (B) and 5 ␮g/ml of anti-CD3. Treg
cells were added to Teff cell cultures at the indicated ratios. Proliferation was measured at 72 hours
by 3H-thymidine incorporation. Mean ⫾ SD proliferation of Teff cells in response to antigenpresenting cells was 6,874 ⫾ 752 cpm in NOD DCs, 17,455 ⫾ 788 cpm in K/BxN DCs, 4,059 ⫾ 298
cpm in NOD B cells, and 8,094 ⫾ 1,442 cpm in K/BxN B cells. Values are the mean ⫾ SD of
triplicate wells. Results are representative of 2 experiments. C, Teff cells and Treg cells were
cultured at a 4:1 ratio and stimulated with anti-CD3 and irradiated splenocytes. DCs and B cells
from NOD or arthritic mice were purified and activated by plate-bound anti-CD40 for 1 hour and
then washed. Activated antigen-presenting cells were added to the T cell cultures either directly
(coCX) or separated by a Transwell membrane (TW). In some T cell cultures, conditioned supernatants from 48-hour cultures of anti-CD40–activated DCs and B cells were added (1:1 volume/
volume). Bromodeoxyuridine (BrdU) incorporation was assayed at 72 hours. Values are the mean ⫾
SD of duplicate wells. D, Teff cells (1 ⫻ 105/well), with or without Treg cells (5 ⫻ 104/well), were
stimulated with anti-CD3 and DCs (1 ⫻ 104/well) from either NOD or K/BxN arthritic mice in the
presence of neutralizing anti–interleukin-6 (␣IL-6) antibodies (10 ␮g/ml) or tumor necrosis factor
receptor:Fc fusion protein (TNFR-Fc) (10 ␮g/ml). Proliferation was measured at 72 hours by 3Hthymidine incorporation. Values are the mean ⫾ SD of triplicate wells. Results are representative of 2
less proliferative and more easily suppressed than experienced CD45RBlow Teff cells (Figure 5C). To determine if this was due to differences in the magnitude of
the T cell response, we normalized the response against
proliferation in the absence of Treg cells. At Teff
cell–to–Treg cell ratios of 2:1 and 4:1, Treg cell suppres-
sion was 2-fold less effective with CD45RBlow Teff cells
(Figure 5D). Thus, during arthritis, both Teff cells and
Treg cells enhanced their response to GPI, such that
increased Treg cell suppression was met by increased
Teff cell resistance, resulting in an immunologic détente.
Contribution of inflammatory antigenpresenting cells to resistance to Treg cell suppression.
Proinflammatory cytokines produced by monocytes and
DCs can abrogate Treg cell function by increasing Treg
cell apoptosis or by increasing Teff cell resistance
(9,23,24). In K/BxN mice, DCs and B cells comprised 2
major antigen-presenting cells for the activation of KRN
T cells (20). DCs were the most efficacious at stimulating KRN T cells, whereas B cells were the most abundant. We therefore compared the efficacy of Treg cell
suppression in the presence of DCs and B cells from
chronically arthritic K/BxN mice or control NOD mice.
We have previously reported that antigen-presenting
cells from arthritic K/BxN mice showed greater presentation of GPI peptide compared with nonarthritic
antigen-presenting cells (20). To eliminate the role of
the GPI dose, we used polyclonal T cells from FoxP3gfp
mice on the H-2k background in which the antigenpresenting cells would provide a strong allostimulation
in addition to anti-CD3.
As expected, DCs from arthritic mice attenuated
the suppressive effect of Treg cells (Figure 6A). With
NOD antigen-presenting cells, Treg cells suppressed
Teff cell proliferation by 50% at a Teff cell–to–Treg cell
ratio of 8:1. Four-fold higher numbers of Treg cells were
necessary in the presence of arthritic DCs. Arthritic B
cells similarly inhibited the suppressive effect of Treg
cells (Figure 6B).
To determine the mechanism by which DCs and
B cells inhibit Treg cell suppression, we added antiCD40–activated DCs and B cells to the Teff cell/Treg
cell suppression assays, either in the same wells or
separated by Transwell membranes. Proliferation of Teff
cells was assayed by flow cytometry and BrdU incorporation. As shown in Figure 6C, anti-CD40–activated
DCs and B cells abrogated Treg cell suppression only
when they were cocultured with the Teff cells and Treg
cells. Restoration of Teff cell proliferation was absent
when DCs and B cells were separated by Transwell
membranes or when supernatants from DC and B cell
cultures were added.
The finding that inflammatory DCs can reverse
Treg cell suppression has been previously described
(9,24). However, those reports attributed the abrogation
of Treg cell activity to soluble factors such as tumor
necrosis factor ␣ (TNF␣) and interleukin-6 (IL-6). Here,
we found that neutralization of IL-6 or TNF␣ had no
effect on the suppressive activity of Treg cells (Figure
6D). Similar results were seen when we used B cells as
antigen-presenting cells (data not shown).
Together, our data demonstrated that both arthritic DCs and B cells decrease Treg cell activity by a
cell–cell contact–dependent or close-contact–dependent
mechanism. The finding that arthritic B cells can also
inhibit Treg cell suppression provides a potential mechanism for the efficacy of B cell depletion in controlling
There is considerable interest in harnessing
CD4⫹CD25⫹ Treg cells to control autoimmune diseases (25,26). In RA patients, the number of
CD4⫹CD25⫹ Treg cells is increased in inflamed joints,
and these cells show variable activity against their Teff
cell counterparts. Results have differed among studies
partly due to differences in the criteria used for Treg cell
identification, as well as the disease states and patient
populations studied. It is also not clear what factors
control their tropism, function, growth, and death during
In the present study, we used K/BxNgfp mice to
model the development and function of arthritogenic
Teff cells and Treg cells in a well-characterized murine
model of RA. The K/BxN mouse model has many
advantages with regard to RA in humans. The progression of arthritis has been extensively characterized and
follows a predictable course: K/BxN mice develop overt
joint inflammation at 4–5 weeks, which peaks at 7–8
weeks, and is followed by a chronic phase in which joint
destruction and deformity predominate (27). In addition, the arthritis shows regional involvement, with the
distal joints being more affected than the proximal and
axial joints. This feature allowed us to determine if there
is regional expansion of Treg cells at sites of disease.
Moreover, by examining GPI reactivity, we focused
specifically on the arthritogenic KRN Teff cells and Treg
cells in this system. Last, we used FoxP3gfp to unequivocally identify Treg cells.
These experiments are the first to demonstrate
the expansion and function of antigen-specific Treg cells
during the initiation and progression of spontaneous
arthritis. In these experiments, we confirmed our previous findings that incomplete deletion of KRN thymocytes by endogenous GPI resulted in an increased frequency of GPI-specific Treg cells in K/BxNgfp mice
through selective deletion of V␤6bright FoxP3– Teff cells
and induction of FoxP3⫹ Treg cells in the thymus (14).
In contrast to other models of autoimmune disease in
which Treg cell deficits resulted in pathologic changes,
K/BxN mice displayed enhanced populations of GPIspecific Treg cells (14,15,28–30). These Treg cells proliferated quite well, both in vitro and in vivo, in response
to endogenously presented GPI, they were functional,
and they suppressed Teff cell proliferation in vitro. Treg
cell numbers expanded in parallel with Teff cell numbers
in the spleens and LNs during arthritis. In K/BxN mice
with a ubiquitous self antigen, both Teff cell and Treg
cell responses were initiated in the spleen, with subsequent extension to the LNs and with a preference for the
draining LNs, indicating selective tropism to inflamed
joints from the increased inflammation and/or GPI
presentation during disease. However, this enhanced
activity was offset by an increased resistance of GPIspecific Teff cells and antigen-presenting cells to suppression.
A recent study described similar dynamic changes
in the spatiotemporal expansion of Teff cells and Treg
cells in the draining LNs during the induction and
progression of adjuvant-induced arthritis that presumably reflected changes in the concentration of the inciting antigen (31). Those findings complement the findings of our studies, with the exception that their waxing/
waning Treg cell course likely reflected the monophasic
course in adjuvant-induced arthritis compared with the
progressive expansion of Teff cells and Treg cells in the
K/BxN model.
Despite the enrichment in Treg cell activity,
K/BxN mice nevertheless developed arthritis. Our in
vitro studies demonstrate effective Teff cell suppression
at ratios that approximate the physiologic ratio found in
vivo. Why, then, are Treg cells ineffective in vivo? There
are several possible explanations. First, Treg cells and
Teff cells are differentially regulated during ontogeny.
CD4⫹FoxP3– single-positive thymocytes reached mature levels during the first week of life. In contrast,
CD4⫹FoxP3⫹ single-positive thymocyte development
was substantially delayed and did not achieve mature
levels until 21 days of age (32). The delayed appearance
of Treg cells in K/BxN mice would result in unopposed
Teff cell reactivity during the initiation of arthritis.
Second, the precursor frequency of GPI-specific T cells
is supraphysiologic in K/BxN mice. The self antigen is
also ubiquitously expressed and can be presented by all
antigen-presenting cells, including B cells. Under such
conditions, autoreactive B cells can be driven into memory B cells despite abundant Treg cells (33). Thus, for a
humorally driven arthritis, Treg cells may be less effective than for a T cell–driven disease such as colitis. We
propose that the developmental regulation and numeric
superiority of KRN Teff cells overcome Treg cell sup-
pression at the initiation of disease. Treg cells might
simply arrive too late and in too few numbers.
During the ensuing arthritis, Treg cells are highly
activated, with ⬎30% of Treg cells proliferating in a
12-hour period. However, this vigorous Treg cell activity
is attenuated by 3 factors: Treg cells undergo shorter
proliferative bursts and higher apoptosis rates than Teff
cells upon antigen stimulation, Teff cells become more
refractory to Treg cell suppression as they differentiate
into memory T cells, and antigen-presenting cells from
arthritic mice abrogate Treg cell suppression. This inhibition of Treg cell suppression required close contact,
but it is unknown whether this is due to increased
resistance of antigen-presenting cells to Treg cells or to
enhanced antigen presentation to Teff cells. It is also
unclear whether these features are due to changes in the
costimulatory molecules or to secretion of paracrine
cytokines in activated antigen-presenting cells from arthritic mice. Studies to dissect these mechanisms are
Our findings parallel observations in human RA
and provide an explanation for the paradoxical occurrence of disease in the presence of abundant Treg cells.
By elucidating the factors and mechanisms that augment
Treg cell activity, it may be possible to achieve control of
disease. K/BxNgfp mice thereby provide a valuable model
in which to examine Teff cell/Treg cell homeostasis
during disease and therapy.
We are grateful to Drs. D. Mathis and C. Benoist and
to INSERM/IGBMC for the KRN mice. We thank Dr. A.
Rudensky for the FoxP3gfp mice. We are grateful to Drs. P.
Tarr, C. Pham, and J. P. Atkinson for critical review of the
Dr. Shih had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Shih.
Acquisition of data. Monte, Wilson.
Analysis and interpretation of data. Shih.
Manuscript preparation. Shih.
Statistical analysis. Shih.
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