Download Antigen-induced, tolerogenic CD11c+,CD11b+ dendritic cells are

ARTHRITIS & RHEUMATISM
Vol. 54, No. 3, March 2006, pp 887–898
DOI 10.1002/art.21647
© 2006, American College of Rheumatology
Antigen-Induced, Tolerogenic CD11c⫹,CD11b⫹ Dendritic
Cells Are Abundant in Peyer’s Patches During the
Induction of Oral Tolerance to Type II Collagen and
Suppress Experimental Collagen-Induced Arthritis
So-Youn Min,1 Kyung-Su Park,2 Mi-La Cho,1 Jung-Won Kang,1 Young-Gyu Cho,1
Sue-Yun Hwang,3 Min-Jung Park,1 Chong-Hyeon Yoon,2 Jun-Ki Min,2 Sang-Heon Lee,2
Sung-Hwan Park,2 and Ho-Youn Kim2
Objective. Although oral tolerance is a well-known
phenomenon, the role of dendritic cells (DCs) is not well
characterized. This study was conducted to better understand the differential role played by each Peyer’s
patch DC subset in the induction of oral tolerance to
type II collagen (CII) in murine collagen-induced arthritis (CIA).
Methods. CII was fed 6 times to DBA/1 mice
beginning 2 weeks before immunization, and the effect
on arthritis was assessed. We compared the proportion
of CD11cⴙ,CD11bⴙ DCs and CD11cⴙ,CD8␣ⴙ DCs in
the Peyer’s patches of CII-fed tolerized and phosphate
buffered saline–fed nontolerized mice after the induction of CIA. The immunosuppressive properties of each
DC subset were determined using fluorescenceactivated cell sorter analysis for intracellular
interleukin-10 (IL-10) and IL-12 and mixed lymphocyte
culture. The ability of each DC subset to induce
CD4ⴙ,CD25ⴙ T regulatory cells was also examined.
Mice were injected with CII-pulsed CD11cⴙ,CD11bⴙ
DCs isolated from Peyer’s patches of tolerized mice, and
the effect on CIA was examined.
Results. The severity of arthritis was significantly lower in tolerized mice. The proportion of
CD11cⴙ,CD11bⴙ DCs was increased in the Peyer’s
patches of tolerized mice and those DCs exhibited
immunosuppressive characteristics, such as increased
IL-10 production, inhibition of T cell proliferative responses to CII, and CD4ⴙ,CD25ⴙ regulatory T cell
induction. Furthermore, the CD11cⴙ,CD11bⴙ DCs
suppressed the severity of arthritis upon adoptive
transfer.
Conclusion. Our observations demonstrate that
CD11cⴙ,CD11bⴙ DCs, which are abundant in Peyer’s
patches during the induction of oral tolerance to CII,
are crucial for the suppression of CIA and could be
exploited for immunotherapy of autoimmune diseases.
Supported by the Rheumatism Research Center at the Catholic University of Korea (grant R11-2002-098-05001-0 from the Korea
Science and Engineering Foundation).
1
So-Youn Min, PhD, Mi-La Cho, PhD, Jung-Won Kang, MS,
Young-Gyu Cho, PhD, Min-Jung Park, BS: Rheumatism Research
Center, The Catholic University of Korea, Seoul, Korea; 2Kyung-Su
Park, MD, Chong-Hyeon Yoon, MD, Jun-Ki Min, MD, Sang-Heon
Lee, MD, Sung-Hwan Park, MD, Ho-Youn Kim, MD, PhD: Kangnam
St. Mary’s Hospital, The Catholic University of Korea, Seoul, Korea;
3
Sue-Yun Hwang, PhD: Hankyong National University, Ansung,
Kyunggi-Do, Korea.
Drs. So-Youn Min and Kyung-Su Park contributed equally to
this work.
Address correspondence and reprint requests to Ho-Youn
Kim, MD, PhD, Rheumatism Research Center, Catholic Institute of
Medical Science, 505 Banpo-Dong, Seocho-Ku, Seoul 137-040, Korea.
E-mail: [email protected].
Submitted for publication July 6, 2005; accepted in revised
form November 21, 2005.
Oral tolerance refers to the immunologic hyporesponsiveness provoked by repeated exposure of the
mucosal immune system to ingested protein antigens.
Oral administration of antigens or peptides that are
structurally similar to the autoantigen leads to local and
systemic priming and, usually, to systemic tolerance,
making this a promising approach for the treatment of
autoimmune diseases. In animal models, it has been
shown that oral tolerance effectively ameliorates experimental autoimmune encephalitis and collagen-induced
arthritis (CIA) (1–8). Repeated oral administration of
type II collagen (CII) induces peripheral immune tolerance, resulting in the suppression of CIA, a representative experimental model of human rheumatoid arthritis
887
888
(RA). Clinical trials of oral CII in RA have already been
performed (8,9).
Oral tolerance is initiated in the gut-associated
lymphoid tissue, a well-developed immune network in
the alimentary tract that comprises the mucosal epithelium, lamina propria, Peyer’s patches, and mesenteric
lymph nodes (1–3). Many reports have suggested that
Peyer’s patches, which are lymphoid nodules interspersed among the intestinal villi, are essential for
mucosal immune responses and oral tolerance to soluble
antigens (2,3,10,11). Peyer’s patches contain professional antigen-presenting cells (APCs) known as dendritic cells (DCs), which are known to play an important
role in both immune responses and immune tolerance in
the intestinal mucosa. It has been reported that freshly
isolated Peyer’s patch DCs are functionally distinct from
splenic DCs with regard to their capacity to induce T
helper cell differentiation in vitro. Peyer’s patch DCs
were shown to prime naive CD4⫹ antigen-specific T
cells to secrete IL-10 and IL-4, whereas splenic DCs
predominantly primed CD4⫹ T cells to secrete
interferon-␥ (12).
Although the mechanisms by which DCs induce
oral tolerance have yet to be elucidated, it has been
reported that feeding of high doses of antigen induces
anergy or deletion of antigen-specific T cells, while
repeated feeding of low doses of antigen favors the
induction of active immune regulation involving T regulatory cells, including transforming growth factor ␤
(TGF␤)–producing Th3 cells, IL-10–producing Tr1
cells, and CD4⫹,CD25⫹ T cells (1). In Peyer’s patches
and mesenteric lymph nodes, DCs can activate T cells
and trigger them to differentiate into T regulatory cells
following repeated exposure to antigen. Once activated,
T regulatory cells can engage in bystander suppression,
whereby they suppress immune responses in an antigenindependent manner via cell-to-cell contact or the secretion of inhibitory cytokines such as IL-10 and TGF␤.
There have been several reports about the specific DC subsets that can induce T regulatory cells.
Bilsborough et al (13) and Martin et al (14) have
reported that plasmacytoid DCs in Peyer’s patches can
induce IL-10–producing T regulatory cells, and
Wakkach et al found that the CD45RBhigh,CD11clow
subset of DCs can induce the differentiation of T
regulatory cells (15). Because Peyer’s patches are the
primary site for the induction of mucosal immune
responses, we attempted in the present study to identify
a tolerogenic DC subset in Peyer’s patches that can
induce T regulatory cells and play an important role in
the induction of oral tolerance.
MIN ET AL
DCs have been reported to be heterogeneous in
their phenotype and their localization in lymphoid tissues (16). Iwasaki and Kelsall have recently identified
and characterized 3 distinct subsets of DCs in murine
Peyer’s patches (17,18): 1) CD11b⫹,CD8␣⫺ DCs with a
myeloid lineage, residing in the subepithelial region; 2)
CD11b⫺,CD8␣⫹ DCs with a lymphoid lineage, residing
in the T cell–rich interfollicular region; and 3) DCs lacking
expression of both CD11b and CD8␣ (double-negative
DCs), present in both the subepithelial and interfollicular
regions (17). They also demonstrated that lymphoidrelated or double-negative DCs induce Th1 cell differentiation, whereas myeloid DCs are better able to skew T cell
responses toward Th2 differentiation. Recently, a novel
subset of murine CD11c⫹ DCs that express high levels of
surface B220 has been identified. This subset, which has
been referred to as plasmacytoid DCs, produces
interferon-␣ when challenged with virus (19). However, the
precise function of each DC subset and the identity of the
subset responsible for the induction of oral tolerance
remain the subject of much debate (16,20–23).
To elucidate the role of the different DC subsets
in oral tolerance, we prepared and characterized DCs
from Peyer’s patches of DBA/1 mice after induction of
oral tolerance by repeated oral administration of CII
and subsequent induction of CIA. The biologic and
molecular characteristics of CD11c⫹,CD11b⫹ DCs and
CD11c⫹,CD8␣⫹ DCs were investigated both in vitro
and in vivo. Our results demonstrate that one unique DC
subset in Peyer’s patches, i.e., the CD11c⫹,CD11b⫹
subset, has tolerogenic characteristics, plays an essential
role in the induction of oral tolerance in autoimmune
inflammatory conditions, and is capable of suppressing
CIA upon adoptive transfer.
MATERIALS AND METHODS
Animals. Male DBA/1 mice (8–12 weeks old; The
Jackson Laboratory, Bar Harbor, ME) were maintained in
groups of 2–4 in polycarbonate cages in a specific pathogen–
free environment and provided with standard mouse chow
(Ralston Purina, St. Louis, MO) and water ad libitum. All
experimental procedures were approved by the Animal Research Ethics Committee at the Catholic University of Korea.
Preparation of type II collagen. Bovine CII was kindly
provided by Dr. Andrew Kang (University of Tennessee,
Memphis). CII was extracted in its native form from the
articular cartilage of fetal calves and purified as previously
described (24).
Induction of oral tolerance to CII in DBA/1 mice. Mice
were divided into 3 groups: a CII-fed tolerance group, a phosphate buffered saline (PBS)–fed CIA group, and an untreated
control group. Six times over the course of 2 weeks, beginning 2
weeks before immunization, 100 ␮g of CII dissolved in 0.05N
SUPPRESSION OF CIA BY CD11c⫹,CD11b⫹ DENDRITIC CELLS
acetic acid at 2 mg/ml was orally administered to the tolerance
group using an oral Zonde needle (Natsume, Tokyo, Japan).
Mice in the CIA group were fed an equal volume of PBS.
Induction and evaluation of arthritis. Arthritis was
induced and then evaluated as previously described (6).
Briefly, bovine CII was dissolved in 0.05N acetic acid at 2
mg/ml and emulsified (1:1 ratio) with Freund’s complete
adjuvant (CFA). As a primary immunization, 0.1 ml of the
emulsion containing 100 ␮g of CII was injected into the tails of
DBA/1 mice in the tolerance and CIA groups (n ⫽ 9 per
group). Two weeks later, a booster injection consisting of 100
␮g of CII similarly dissolved and emulsified 1:1 with incomplete Freund’s adjuvant was injected into the hind legs of the
mice. Beginning 18 days after primary immunization, the
degree of arthritis was evaluated by 3 independent observers 3
times a week for up to 11 weeks. The severity of arthritis was
expressed as a mean arthritis index on a 0–4 scale, as follows:
0 ⫽ no edema or swelling; 1 ⫽ slight edema and erythema
limited to the foot or ankle; 2 ⫽ slight edema and erythema
from the ankle to the tarsal bone; 3 ⫽ moderate edema and
erythema from the ankle to the tarsal bone; 4 ⫽ edema and
erythema from the ankle to the entire leg.
Flow cytometric analysis of DCs. Mice were killed 5
weeks after primary immunization. The Peyer’s patches were
removed and treated for 90 minutes at 37°C with media
containing dithiothreitol and EDTA to remove epithelial cells,
before being washed extensively with Hanks’ balanced salt
solution. Peyer’s patches were then digested with collagenase
D and DNase and incubated in the presence of 5 mM EDTA
for 5 minutes at 37°C. Prepared mononuclear cells were
double-stained with fluorescein isothiocyanate (FITC)–labeled
anti-CD11c and phycoerythrin (PE)–labeled anti-CD11b or
anti-CD8␣ monoclonal antibodies (mAb) (PharMingen, San
Diego, CA). Finally, cells were washed with PBS and analyzed
using a FACSCalibur (Becton Dickinson, San Jose, CA).
For the analysis of intracellular cytokines in DCs,
prepared mononuclear cells (2.5 ⫻ 105) were cultured for 3
days in the presence of CII (40 ␮g/ml). Cells were subsequently
washed and resuspended in fluorescence-activated cell sorter
(FACS) staining buffer and probed with FITC-labeled antiCD11b or anti-CD8␣ mAb for 30 minutes at 4°C. Next, cells
were fixed with Cytoperm/Cytofix (PharMingen) for 20 minutes and probed for intracellular cytokines for 30 minutes at
room temperature, using PE-labeled anti–IL-10 or anti–IL-12
mAb (PharMingen). Finally, cells were washed with PBS and
analyzed using a FACSCalibur. All flow cytometric analyses
were performed using appropriate isotype controls (Cedarlane, Hornby, Ontario, Canada).
Isolation of DCs. DCs from Peyer’s patches were
prepared as previously described (12,17). Briefly, mononuclear
cells from Peyer’s patches were incubated with anti-mouse
CD11c–coated magnetic beads (Miltenyi Biotec, Auburn, CA)
and then subjected to selection through MACS separation
columns. Cells selected on the basis of CD11c expression
routinely showed ⬎98% viable DCs. Isolated CD11c⫹ DCs
were then stained with PE-labeled anti-CD11b or anti-CD8␣
mAb. Cells that were positive for either lineage marker were
sorted with a FACStar (BD Biosciences, San Jose, CA) into
CD8␣⫺,CD11b⫹ and CD8␣⫹,CD11b⫺ DC fractions.
Mixed lymphocyte culture. Freshly prepared
CD11c⫹,CD11b⫹ or CD11c⫹,CD8␣⫹ DCs from Peyer’s
889
patches of tolerized mice were cultured for 3 days with
CII-reactive CD4⫹ T cells (1 ⫻ 105) and irradiated APCs (1 ⫻
105) obtained from Peyer’s patches of mice with CIA, in the
presence of CII (40 ␮g/ml), at various DC:CD4⫹ T cell ratios.
Before the final 18 hours of culture, 0.5 ␮Ci of 3H-thymidine
(New England Nuclear, Boston, MA) was added to each well.
After the cells were harvested, incorporated radioactivity was
counted in a scintillation counter. Data were presented as the
mean counts per minute of triplicate cultures.
Detection of cytokine production by enzyme-linked
immunosorbent assay (ELISA). CII-reactive CD4⫹ T cells
(1 ⫻ 105) from Peyer’s patches of mice with CIA were
cocultured with CD11c⫹,CD11b⫹ or CD11c⫹,CD8␣⫹ DCs
(1 ⫻ 104) from Peyer’s patches of tolerized mice in the
presence of CII (40 ␮g/ml). After 3 days, culture media from
each well were harvested and stored at ⫺70°C. The concentrations of IL-10, IL-12, and TGF␤ in the culture supernatants
were measured by sandwich ELISA.
In vitro assay of CII-induced CD4ⴙ,CD25ⴙ T cells.
CD4⫹ T cells (1 ⫻ 105) were isolated from the Peyer’s patches
of tolerized or control mice using CD4 MACS beads. They
were then incubated in 96-well culture plates, in the absence or
presence of CII (40 ␮g/ml), with CD11c⫹,CD11b⫹ or
CD11c⫹,CD8␣⫹ DCs (1 ⫻ 104 cells) that had been isolated
from the Peyer’s patches of tolerized mice. After 3 days of
culture, the proportion of CD4⫹,CD25⫹ T cells was analyzed
using PE-labeled anti-mouse CD25 mAb (PharMingen) and a
FACSCalibur.
Various numbers of CD4⫹,CD25⫹ T cells, which had
been expanded by exposure to CD11c⫹,CD11b⫹ DCs from
Peyer’s patches of tolerized mice in the presence of CII antigen
stimulation as described above, were cultured for 3 days, in the
presence of CII (40 ␮g/ml), with CII-reactive CD4⫹ T cells
(1 ⫻ 105) and irradiated APCs (1 ⫻ 105) obtained from mice
with CIA. Proliferative responses were measured based on the
degree of 3H-thymidine incorporation during the last 18 hours
of incubation.
Adoptive transfer studies. Mice were fed CII (100
␮g/mouse) 6 times over the course of 2 weeks and then killed.
CD11c⫹ DCs were isolated from the Peyer’s patches of 25
CII-fed mice and separated into CD11b⫹ and CD11b⫺ cells
with a FACSVantage, using FITC-conjugated anti-mouse
CD11b. CD11c⫹,CD11b⫹ and CD11c⫹,CD11b⫺ DCs (5 ⫻
104 cells/well) were then stimulated with 40 ␮g/ml CII for 18
hours and transferred intravenously into naive DBA/1 mice
(n ⫽ 5 per group). Three days after adoptive transfer, CIA was
induced as described above.
Assay for mononuclear cell proliferation against CII
antigen. Five weeks after primary immunization, mononuclear
cells prepared from spleens of DC-transferred mice (2 ⫻ 105
cells) were cultured for 3 days with CII (40 ␮g/ml) or phytohemagglutinin (PHA; 5 ␮g/ml). The proliferation of mononuclear cells in response to CII antigen or PHA was measured
based on 3H-thymidine incorporation.
Reverse transcriptase–polymerase chain reaction (RTPCR) analysis of Foxp3 expression. RNAzol B was used to
isolate messenger RNA (mRNA) according to the instructions
of the manufacturer (Tel-Test, Friendswood, TX), and PCR
(30 cycles at 94°C for 30 seconds, 58°C for 30 seconds, and
72°C for 30 seconds for Foxp3; and at 94°C for 15 seconds,
53°C for 15 seconds, and 72°C for 30 seconds for hypoxanthine
890
MIN ET AL
phosphoribosyltransferase [HPRT]) was used for semiquantitative assessment of message levels. HPRT expression was
used as an internal control to ensure equal loading of every
reaction. The primer pairs were as follows: for Foxp3, 5⬘CAGCTGCCTACAGTGCCCCTAG-3⬘ (forward) and 5⬘CATTTGCCAGCAGTGGGTAG-3⬘ (reverse); for HPRT,
5⬘-GTAATGATCAGTCAACGGGGGAC-3⬘ (forward) and
5⬘-CCAGCAAGCTTGCAACCTTAACCA-3⬘(reverse).
Statistical analysis. Student’s unpaired t-test, assuming
equal variances, was used to determine the statistical significance of the differences in mean cell numbers or mean
percentages obtained via flow cytometry. This test was also
used to analyze comparative data between groups. Mixed
lymphocyte culture data were analyzed using one-way analysis
of variance followed by the Newman-Keuls test. P values less
than 0.05 were considered significant.
RESULTS
Induction of immune tolerance and inhibition of
arthritis development with repeated oral administration
of CII. The arthritis index remained low in both the
tolerance and the CIA groups until 4 weeks after
primary immunization with CII/CFA. In the CIA group,
the arthritis index began to increase 4 weeks after
primary immunization, reached a peak between weeks 5
and 6, then started to decrease by week 8. In the
tolerance group, the arthritis index peaked between the
eighth and ninth weeks but levels were significantly
lower than those seen in the CIA group throughout the
examination period (Figure 1).
Increased proportion of CD11cⴙ,CD11bⴙ DCs
and CD4ⴙ,CD25ⴙ T cells in the Peyer’s patches of mice
with CII-induced tolerance. To determine which DC
subset plays the most important role in inducing oral
tolerance, we examined the relative populations of
CD11c⫹,CD11b⫹ and CD11c⫹,CD8␣⫹ DCs in Peyer’s
patches after repeated oral administration of CII and
subsequent CIA induction. Using a 2-color plot after
first gating on CD11c⫹ DCs, the relative proportions of
CD11c⫹,CD11b⫹ and CD11c⫹,CD8␣⫹ DCs were
measured in mononuclear cells that had been isolated
from Peyer’s patches 5 weeks after primary immunization. A higher proportion of CD11c⫹,CD11b⫹ DCs was
seen in the tolerized mice than in mice with CIA
(mean ⫾ SD 9.5 ⫾ 0.54% versus 4.3 ⫾ 2.1%; P ⬍ 0.05),
while a higher proportion of CD11c⫹,CD8␣⫹ DCs was
seen in mice with CIA than in tolerized mice (11.3 ⫾
0.43% versus 3.6 ⫾ 0.04%; P ⬍ 0.05) (Figure 2a). These
findings indicate that the CD11c⫹,CD11b⫹ DC subset
in Peyer’s patches plays the main role in the induction of
oral tolerance to CII.
To further elucidate the process of oral tolerance
induction, we sought to determine the proportion of
Figure 1. Inhibition of arthritis development among mice in the
tolerance group. Mice in the tolerance group were fed 100 ␮g of type
II collagen (CII) 6 times over 2 weeks, beginning 2 weeks before
immunization. For induction of collagen-induced arthritis (CIA), CII
emulsified with Freund’s complete adjuvant was injected into the tails
of mice in the tolerance and CIA groups as a primary immunization.
Two weeks later, CII emulsified with Freund’s incomplete adjuvant
was injected into the hind legs as a booster injection. The arthritis
index was significantly lower in the tolerance group than in the CIA
group throughout the examination period. Values are the mean and
SD.
CD4⫹,CD25⫹ T cells among Peyer’s patch mononuclear cells. Five weeks after primary immunization,
the proportion of CD4⫹,CD25⫹ T cells was higher in
tolerized mice than in mice with CIA (mean ⫾ SD
3.04 ⫾ 0.22% versus 1.88 ⫾ 0.41%; P ⬍ 0.05) (Figure
2b). When the Peyer’s patch mononuclear cells were
cultured for 3 days in the presence of CII, the proportion
of CD4⫹,CD25⫹ T cells increased markedly in tolerized
mice compared with mice with CIA (19.13 ⫾ 1.45%
versus 3.98 ⫾ 0.19%; P ⬍ 0.001) (Figure 2b). We also
found that the proportion of CD4⫹,CD25⫹ T cells in
splenic mononuclear cells was higher in tolerized mice
than in mice with CIA both before in vitro CII stimulation (2.2 ⫾ 1.72% versus 1.3 ⫾ 0.7%; P ⬍ 0.05) and after
CII stimulation (12.4 ⫾ 4.5% versus 5.6 ⫾ 0.63%; P ⬍
0.001) (data not shown).
Immunosuppressive characteristics of CD11cⴙ,
CD11bⴙ DCs. DCs can drive either immunity or immune
tolerance, and the cytokines they secrete after antigen
stimulation can be taken as clues to which of the two
immunologic roles a given DC subset is playing. IL-12–
producing DCs tend to drive the Th1 response, while
IL-10–producing DCs have been known to drive the Th2
response and to play an important role in inducing T
regulatory cells and systemic immune tolerance (25,26). To
SUPPRESSION OF CIA BY CD11c⫹,CD11b⫹ DENDRITIC CELLS
891
Figure 2. Increased proportion of CD11c⫹,CD11b⫹ dendritic cells (DCs) and CD4⫹,CD25⫹ T cells in the Peyer’s patches of tolerized mice after
oral administration of type II collagen (CII). a, Mononuclear cells were isolated from Peyer’s patches 5 weeks after primary immunization, and the
proportions of CD11c⫹,CD11b⫹ DCs and CD11c⫹,CD8␣⫹ DCs were determined using flow cytometry. The proportion of CD11c⫹,CD11b⫹ DCs
in Peyer’s patches was higher in tolerized mice than in mice with collagen-induced arthritis (CIA) (mean ⫾ SD value in 3 experiments 9.5 ⫾ 0.54%
versus 4.3 ⫾ 2.1%; P ⬍ 0.05), while that of CD11c⫹,CD8␣⫹ DCs was higher in mice with CIA than in tolerized mice (11.3 ⫾ 0.43% versus 3.6 ⫾
0.04%; P ⬍ 0.05). One representative set of results from 3 independent experiments is shown. b, Mononuclear cells (2.5 ⫻ 105) isolated from Peyer’s
patches 5 weeks after primary immunization were cultured for 3 days in the presence or absence (nil) of CII (40 ␮g/ml), and the proportion of
CD4⫹,CD25⫹ T cells was determined using flow cytometry. Before CII stimulation, the proportion of CD4⫹,CD25⫹ T cells was significantly higher
in tolerized mice than in mice with CIA. In the presence of CII, the proportion of CD4⫹,CD25⫹ T cells in tolerized mice was increased further
compared with mice with CIA. Values are the mean from 4 independent experiments (individual symbols are the mean in individual animals; bars
show the group means).
892
MIN ET AL
Figure 3. Immunosuppressive characteristics of CD11c⫹,CD11b⫹ DCs. a, Mononuclear cells (2.5 ⫻ 105) from Peyer’s patches were cultured in the
presence of CII (40 ␮g/ml) for 3 days, and 3-color fluorescence-activated cell sorter analysis for intracellular interleukin-10 (IL-10) and IL-12 was
performed on CD11c⫹,CD11b⫹ and CD11c⫹,CD8␣⫹ DCs. Gray histograms represent background staining with an isotype-matched control. One
representative set of results from 5 independent experiments is shown. b, Freshly prepared CD11c⫹,CD11b⫹ or CD11c⫹,CD8␣⫹ DCs from Peyer’s
patches of tolerized mice were cultured with CII-reactive CD4⫹ T cells (1 ⫻ 105) and irradiated antigen-presenting cells (Irrad. APC; 1 ⫻ 105)
obtained from the Peyer’s patches of mice with CIA, for 3 days at different DC:CD4⫹ T cell ratios in the presence of CII (40 ␮g/ml). The
proliferative responses of CD4⫹ T cells to CII were determined. Values are the mean and SD from 3 independent experiments. ⴱ ⫽ P ⬍ 0.05. c,
CII-reactive CD4⫹ T cells (1 ⫻ 105) from mice with CIA were cocultured with 1 ⫻ 104 CD11c⫹,CD11b⫹ DCs or CD11c⫹,CD8␣⫹ DCs from
tolerized mice for 3 days in the presence of CII (40 ␮g/ml). Concentrations of IL-10, transforming growth factor ␤ (TGF␤), and IL-12 in the culture
supernatants were determined by sandwich enzyme-linked immunosorbent assay. The concentrations of IL-10 and TGF␤ were higher in the culture
supernatant of CD11c⫹,CD11b⫹DCs than in that of CD11c⫹,CD8␣⫹ DCs. Values are the mean and SD from 4 independent experiments. See
Figure 2 for other definitions.
characterize the cytokine profile of each DC subset, we
cultured Peyer’s patch mononuclear cells with CII for 3
days and performed 3-color FACS analysis for intracellular
IL-10 and IL-12 in CD11c⫹,CD11b⫹ and CD11c⫹,
CD8␣⫹ DCs. The proportion of IL-10–producing
CD11c⫹,CD11b⫹ DCs increased by up to 36% in the
SUPPRESSION OF CIA BY CD11c⫹,CD11b⫹ DENDRITIC CELLS
tolerized mice, while no such increase was noted for
CD11c⫹,CD8␣⫹ DCs (10%) (Figure 3a). In contrast, the
proportion of IL-12–secreting CD11c⫹,CD8␣⫹ DCs increased by up to 41.4% in the mice with CIA.
To analyze the functional properties of each DC
subset, freshly prepared CD11c⫹,CD11b⫹ or
CD11c⫹,CD8␣⫹ DCs from Peyer’s patches of tolerized
mice were cultured with CII-reactive CD4⫹ T cells and
irradiated APCs obtained from mice with CIA and were
exposed to CII at various DC:CD4⫹ T cell ratios. As the
number of CD11c⫹,CD11b⫹ DCs increased, the proliferative response of CD4⫹ T cells to CII protein declined; however, no such suppressive effect was exerted
by CD11c⫹,CD8␣⫹ DCs (Figure 3b).
Finally, to confirm induction of tolerance by
analyzing cytokine secretion by DCs, we cocultured
CD11c⫹,CD11b⫹ or CD11c⫹,CD8␣⫹ DCs from Peyer’s patches of tolerized mice with CD4⫹ T cells from
mice with CIA in the presence of CII and measured the
concentrations of IL-10, IL-12, and TGF␤ in culture
supernatants. The concentrations of IL-10 and TGF␤
were higher in the culture supernatant of CD11c⫹,
CD11b⫹ DCs than in that of CD11c⫹,CD8␣⫹ DCs
(mean ⫾ SD 712.12 ⫾ 16.54 pg/ml versus 340.87 ⫾ 53.24
pg/ml; P ⫽ 0.029 and 70.12 ⫾ 7.02 pg/ml versus 29.37 ⫾
8.43 pg/ml; P ⫽ 0.019). In contrast, the IL-12 concentration was higher in the culture supernatant of
CD11c⫹,CD8␣⫹ DCs than in that of CD11c⫹,CD11b⫹
DCs (266.23 ⫾ 11.03 pg/ml versus 116.45 ⫾ 5.12 pg/ml;
P ⫽ 0.014) (Figure 3c). Collectively, these findings
suggest that CD11c⫹,CD11b⫹ DCs induce tolerance
through the suppression of CII-reactive T cells.
Generation of CD4ⴙ,CD25ⴙ T cells by
CD11cⴙ,CD11bⴙ DCs from Peyer’s patches of tolerized
mice in vitro. To determine which subset of DCs was
capable of triggering differentiation of CD4⫹ T cells
with a regulatory phenotype, CD11c⫹,CD11b⫹ or
CD11c⫹,CD8␣⫹ DCs from the Peyer’s patches of tolerized mice were cultured with CD4⫹ T cells obtained
from the Peyer’s patches of tolerized mice or of control
mice for 3 days, with or without CII. When the
CD11c⫹,CD11b⫹ DCs were cultured with CD4⫹ T
cells from tolerized mice in the presence of CII, the
proportion of CD4⫹,CD25⫹ T cells increased by as
much as 30% after 3 days of culture; no such increase
was seen with identically cultured CD11c⫹,CD8␣⫹ DCs
(Figure 4a). In addition, the generation of CD4⫹,
CD25⫹ T cells was more prominent in CD4⫹ T cells
from tolerized mice than in those from control mice.
These findings clearly show that CD4⫹,CD25⫹ T cells
893
Figure 4. Generation of CD4⫹,CD25⫹ T regulatory cells by
CD11c⫹,CD11b⫹ DCs from Peyer’s patches of tolerized mice
in vitro. a, Generation of CD4⫹,CD25⫹ T cells by CD11c⫹,
CD11b⫹ and CD11c⫹,CD8␣⫹ DCs. Sorted CD11c⫹,CD11b⫹
DCs (E) and CD11c⫹,CD8␣⫹ DCs (F) (1 ⫻ 104 cells) from the
Peyer’s patches of tolerized mice were cocultured with CD4⫹ T
cells (1 ⫻ 105) from control (Con) or tolerized (Tol) mice for 3 days
in the absence or presence of CII (40 ␮g/ml). The proportion of
CD4⫹,CD25⫹ T cells was examined using fluorescence-activated
cell sorter analysis. Values are the mean from 5 independent
experiments (individual symbols are the mean in individual animals; bars show the group means). b, Regulatory function of the
CII-induced CD4⫹,CD25⫹ T cells. After expansion by exposure
to CD11c⫹,CD11b⫹ DCs from the Peyer’s patches of tolerized
mice in the presence of CII antigen stimulation, varying numbers
of CD4⫹,CD25⫹ T cells were cultured for 3 days with CII-reactive
CD4⫹ T cells (1 ⫻ 105) and irradiated antigen-presenting cells
(1 ⫻ 105) obtained from mice with CIA, in the presence of CII
(40 ␮g/ml). Proliferative responses were measured based on the
degree of 3H-thymidine incorporation. Values are the mean and
SD from 3 independent experiments. See Figure 2 for other
definitions.
894
MIN ET AL
Figure 5. Suppression of CIA by adoptive transfer of CII-pulsed CD11c⫹,CD11b⫹ DCs. a, Mice were fed CII 6 times over the course of 2 weeks and
then killed. CD11c⫹ DCs were isolated from Peyer’s patch mononuclear cells using the MACS system and separated into CD11b⫹ and CD11b⫺ DCs
using a FACSVantage and fluorescein isothiocyanate–conjugated anti-mouse CD11b monoclonal antibody. Sorted CD11c⫹,CD11b⫹ and
CD11c⫹,CD11b⫺ DCs were cultured in the presence of CII (40 ␮g/ml) for 18 hours. CII-pulsed CD11c⫹,CD11b⫹ or CD11c⫹,CD11b⫺ DCs (5 ⫻
104–1 ⫻ 105) were transferred intravenously into normal DBA/1 mice (n ⫽ 5 per group). After adoptive transfer, CIA was induced by immunization with
CII and Freund’s complete adjuvant according to the conventional CIA protocol. The mean arthritis index was determined in the CD11c⫹,CD11b⫹
DC–transferred, CD11c⫹,CD11b⫺ DC–transferred, and nontransferred groups 18–52 days after primary immunization. b, Thirty-five days after primary
immunization, mononuclear cells from the spleens of DC-transferred mice were prepared and cultured for 3 days with CII (40 ␮g/ml) or
phytohemagglutinin (PHA; 5 ␮g/ml). Proliferative responses were measured based on the degree of 3H-thymidine incorporation. The proliferative
responses against CII were low in CD11c⫹,CD11b⫹ DC–transferred mice (solid bars), compared with CD11c⫹,CD11b⫺ DC–transferred mice (hatched
bars) or nontransferred mice (open bars). c, Mononuclear cells from spleens were cultured for 3 days with or without CII (40 ␮g/ml). Stimulation with CII
caused a significant increase in the proportion of CD4⫹,CD25⫹ T cells in spleen cells from CD11c⫹,CD11b⫹ DC–transferred mice (solid bars), compared
with CD11c⫹,CD11b⫺ DC–transferred mice (hatched bars) or nontransferred mice (open bars). Values in a–c are the mean and SD from 5 mice per
group in 2 independent experiments. d, Splenic mononuclear cells from CD11c⫹,CD11b⫹ DC–transferred mice (lane 1), CD11c⫹,CD11b⫺
DC–transferred mice (lane 2), or nontransferred mice (lane 3) were cultured for 3 days with or without CII (40 ␮g/ml), and CD4⫹,CD25⫹ T cells were
sorted using a FACSVantage. Expression of Foxp3 mRNA by CD4⫹,CD25⫹ T cells was examined by reverse transcriptase–polymerase chain reaction.
HPRT ⫽ hypoxanthine phosphoribosyltransferase (see Figure 2 for other definitions).
were generated upon contact with CD11c⫹,CD11b⫹
DCs from the Peyer’s patches of tolerized mice in the
presence of CII stimulation.
To verify that the increased population of
CD4⫹,CD25⫹ T cells retained suppressive function,
varying numbers of CD4⫹,CD25⫹ T cells that had been
expanded by exposure to CD11c⫹,CD11b⫹ DCs from
tolerized mice were cocultured for 3 days, in the pres-
ence of CII, with CII-reactive CD4⫹ T cells and irradiated APCs from mice with CIA. In this assay,
CD11c⫹,CD11b⫹ DC–induced CD4⫹,CD25⫹ T cells
inhibited the proliferation of CII-reactive CD4⫹ T cells
in a dose-dependent manner (Figure 4b). At a concentration of ⬃10 responder cells to 1 CD4⫹,CD25⫹ T cell,
CD4⫹,CD25⫹ T cells showed a suppression rate of up
to 45%.
SUPPRESSION OF CIA BY CD11c⫹,CD11b⫹ DENDRITIC CELLS
To further characterize the regulatory properties
of CD4⫹,CD25⫹ T cells expanded by exposure to
CD11c⫹,CD11b⫹ DCs, the expression of Foxp3 mRNA
by CD4⫹,CD25⫹ T cells was analyzed by RT-PCR.
CD4⫹,CD25⫹ T cells obtained by coculture with
CD11c⫹,CD11b⫹ DCs did not show a significant
change in Foxp3 expression in the absence of CII
stimulation. However, in the presence of CII stimulation, CD4⫹,CD25⫹ T cells obtained by coculture with
CD11c⫹,CD11b⫹ DCs expressed significantly higher
levels of Foxp3 mRNA than did those obtained by
coculture with CD11c⫹,CD8␣⫹ DCs (data not shown).
Suppression of the severity of CIA by adoptive
transfer of CII-pulsed CD11cⴙ,CD11bⴙ DCs. To determine whether CD11c⫹,CD11b⫹ DCs have functional
activity in vivo, DCs were separated from Peyer’s
patches of CII-fed mice, pulsed with CII in vitro, and
then transferred into naive recipients. Three days later,
mice were immunized with CII/CFA according to the
conventional CIA protocol.
Four weeks after primary immunization, the
mean arthritis index was lower in CD11c⫹,CD11b⫹
DC–transferred mice than in CD11c⫹,CD11b⫺ DC–
transferred or nontransferred mice (Figure 5a). When
spleen cells were isolated 5 weeks after primary immunization, the proliferative responses to CII stimulation
were significantly lower in those from CD11c⫹,CD11b⫹
DC–transferred mice than in those from CD11c⫹,
CD11b⫺ DC–transferred or nontransferred mice (Figure 5b). When PHA was applied instead of CII as a
control, no difference was observed in the proliferative
responses among the 3 groups of spleen cells (Figure
5b), suggesting that adoptive transfer of CII-pulsed
CD11c⫹,CD11b⫹ DCs induced CII-specific immune
tolerance.
Based on our assumption that oral tolerance to
CII was mediated by CII-induced CD4⫹,CD25⫹ T
cells, we next sought to compare the proportion of
CD4⫹,CD25⫹ T cells among spleen cells from CIIpulsed CD11c⫹,CD11b⫹ DC–transferred mice before
and after CII stimulation. Stimulation with CII increased the proportion of CD4⫹,CD25⫹ T cells in
spleen cells from CD11c⫹,CD11b⫹ DC–transferred
mice significantly more than that in cells from CD11c⫹,
CD11b⫺ DC–transferred or nontransferred mice (Figure 5c). Foxp3 mRNA expression by CD4⫹,CD25⫹ T
cells was also higher in the spleen cells of CD11c⫹,
CD11b⫹ DC–transferred mice than in those of
CD11c⫹,CD11b⫺ DC–transferred or nontransferred
mice (Figure 5d).
895
DISCUSSION
DCs are professional APCs that play a decisive
role in determining whether immunity or immune tolerance is induced in response to antigen. That determination is widely believed to be based on the maturation/
activation state and subset of DCs and cytokines in the
microenvironment at the time of antigen uptake (24).
Because Peyer’s patches are essential sites for the induction of mucosal immune responses and oral tolerance to
soluble antigen, Peyer’s patch DCs have been thought to
play an important part in mucosal immunity (1,2). There
are at least 3 subsets of DCs in the Peyer’s patches of
mice: CD11c⫹,CD11b⫹ DCs, CD11c⫹,CD8␣⫹ DCs,
and double-negative DCs. Each DC subset seems to
have a distinct lineage and set of functional characteristics (17,18).
In order to determine which subset of DCs plays
a major role in the induction of oral tolerance, we first
examined the change in the population of
CD11c⫹,CD11b⫹ DCs and CD11c⫹,CD8␣⫹ DCs in
Peyer’s patches after repeated oral administration of CII
and subsequent induction of CIA. In freshly isolated
Peyer’s patches of tolerized mice, the proportion of
CD11c⫹,CD11b⫹ DCs was higher than that of
CD11c⫹,CD8␣⫹ DCs. In contrast, the Peyer’s patches
of mice with CIA were enriched in CD11c⫹,CD8␣⫹
DCs. It is of interest that CII feeding influenced the
distribution of DC subsets in Peyer’s patches of tolerized
mice and mice with CIA in our study. Recently, it was
reported that T regulatory cells develop in response to
chronic antigenic stimulation and act directly on APCs,
rendering them tolerogenic and capable of eliciting the
differentiation of CD4⫹ T cells with suppressive activity
(27). This implies that interaction between DCs and T
cells could play an important role in the change in the
population of DC subsets. However, the linkage between
the change in DC subset proportions and the induction
of oral tolerance has yet to be definitively demonstrated,
and the underlying mechanism remains to be elucidated.
IL-10 has been reported to have a major part in
immune tolerance, and its production by DCs is critical
for the differentiation of T regulatory cells (28–30). In
our study, increased levels of IL-10–positive cells were
seen in CD11c⫹,CD11b⫹ DCs from tolerized mice.
Furthermore, after coculture with CII-reactive CD4⫹ T
cells in the presence of CII, CD11c⫹,CD11b⫹ DCs
obtained from Peyer’s patches of tolerized mice produced high levels of IL-10 and TGF␤, but low levels of
IL-12. In contrast, CD11c⫹,CD8␣⫹ DCs produced high
levels of IL-12. Furthermore, CD11c⫹,CD11b⫹ DCs
896
inhibited the proliferative responses of CII-reactive
CD4⫹ T cells to CII stimulation in a dose-dependent
manner, but CD11c⫹,CD8␣⫹ DCs did not. Taken
together, these findings suggest that CD11c⫹,CD11b⫹
DCs may play a crucial role in oral tolerance to CII in
the murine CIA model.
In the resting state, autoreactive T cells residing
in the periphery are effectively suppressed by T regulatory cells, which are thought to prevent the development
of autoimmune diseases. Three kinds of T regulatory
cells have been reported: CD4⫹,CD25⫹ T regulatory
cells, IL-10–producing Tr1 cells, and TGF␤-producing
Th3 cells. Of those, CD4⫹,CD25⫹ T cells are known to
arise spontaneously during ontogeny and are believed
to be essential for oral tolerance (31). However, it has
been reported that CD4⫹,CD25⫹ T cells also can be
induced, or their proportion increased, by DCs in the
periphery. Zhang and colleagues found that oral administration of ovalbumin to ovalbumin T cell receptor–
transgenic mice resulted in an increase in IL-10– and
TGF␤-secreting CD4⫹,CD25⫹ T cells (32). Yamazaki
et al reported that the proportion of immunoregulatory CD4⫹,CD25⫹ T cells can be increased by antigenprocessing DCs (33). Our group previously demonstrated that, after repeated oral administration of CII
and subsequent CIA induction, the proportion of IL-10–
producing CD4⫹,CD25⫹ T cells increased more in the
Peyer’s patches and spleens of tolerized mice than in
those of nontolerized mice (34).
Based on those findings, we sought to determine
which subset of DCs in Peyer’s patches was involved
in generating and increasing the proportion of antigenspecific CD4⫹,CD25⫹ T regulatory cells. We found
that CD11c⫹,CD11b⫹ DCs induced the differentiation of CD4⫹ T cells into CD4⫹,CD25⫹ T regulatory
cells in the presence of CII. In addition, the release
of IL-10 by CD11c⫹,CD11b⫹ DCs from Peyer’s patches
of tolerized mice could play a role in inducing CD4⫹,
CD25⫹ T regulatory cells to differentiate. Several welldesigned studies have demonstrated that immature DCs,
semi-immature DCs, fully mature DCs, or granulocyte–
macrophage colony-stimulating factor–pulsed DCs could
cause naive T cells to differentiate into CD4⫹,CD25⫹ T
regulatory cells in the periphery (35–38). However, the
specialized subset of tolerogenic DCs generated under
physiologic conditions, such as those seen in the nontransgenic autoimmune arthritis animal model, is yet to be
identified. In our study, tolerogenic CD11c⫹,CD11b⫹
DCs were physiologically induced by repeated oral administration of CII in the animal model, without artifi-
MIN ET AL
cial administration of IL-10 or other DC-modifying
agents.
In this study, the population of CD4⫹,CD25⫹
T cells was greater in the Peyer’s patches of tolerized
mice than in those of mice with CIA. Although the
increase in CD4⫹,CD25⫹ T cells in tolerized mice was
modest, we believe it is a meaningful change. Dieckmann
et al demonstrated that CD4⫹,CD25⫹ T cells induce
long-lasting anergy and production of IL-10 in
CD4⫹C25⫺ T cells, which, like the so-called Tr1 cells,
then suppress syngeneic CD4⫹ T cells via IL-10 (39).
Although they represent only a small fraction of
CD4⫹ T cells, CD4⫹,CD25⫹ T cells are thought to
mediate immunosuppression by inducing Tr1-like cells.
Furthermore, in the present study there was a marked
increase in the proportion of CD4⫹,CD25⫹ T cells
when mononuclear cells from Peyer’s patches of tolerized mice were stimulated with CII in vitro, and
CD11c⫹,CD11b⫹ DCs induced the differentiation of
CD4⫹ T cells into CD4⫹,CD25⫹ T regulatory cells.
This means that CD4⫹,CD25⫹ T cells could be generated through interaction between tolerogenic DCs
and T cells in vivo. Considering that IL-10 produced by
CD4⫹,CD25⫹ T cell–induced Tr1-like cells can affect
the generation of tolerogenic DCs, we believe the
interaction between T regulatory cells and tolerogenic
DCs created an immunoregulatory loop and contributed
to the suppression of CIA. In addition, we believe
TGF␤-producing Th3 cells and IL-10–producing Tr1
cells also played an important role in the oral tolerance
to CII in our study, although we did not examine their
involvement.
Although the expression of CD25 can be upregulated by activated CD4⫹ T cells as well as T
regulatory cells, the CD4⫹,CD25⫹ T cells expanded by
exposure to CD11c⫹,CD11b⫹ DCs in our study are
thought to be T regulatory cells, because they inhibited
the proliferation of CII-reactive CD4⫹ T cells in a
dose-dependent manner and expressed high levels of
Foxp3 mRNA in response to CII. Foxp3 is known to play
a crucial role in the development and functioning of
CD4⫹,CD25⫹ T regulatory cells. Foxp3-transduced
cells act like regulatory cells and suppress CD4⫹ T cell
proliferation in response to CD3-specific mAb (40).
Finally, we confirmed that the CD11c⫹,CD11b⫹
DCs in the Peyer’s patches of tolerized mice were
tolerogenic by showing that the severity of arthritis was
significantly lower in CD11c⫹,CD11b⫹ DC–transferred
mice than in CD11c⫹,CD11b⫺ DC–transferred or nontransferred mice. Adoptive transfer of CII-pulsed
CD11c⫹,CD11b⫹ DCs induced CII-specific immune
SUPPRESSION OF CIA BY CD11c⫹,CD11b⫹ DENDRITIC CELLS
tolerance and a significant increase in the number of
splenic CD4⫹,CD25⫹ T regulatory cells, whose expression of Foxp3 mRNA was up-regulated in response to
CII stimulation in vitro.
DCs are currently the subject of active investigation for their immunoregulatory properties in experimental autoimmune diseases such as CIA (41–44). In
those studies, DCs were modulated with tumor necrosis
factor or were genetically modified to express IL-4 and
Fas ligand. However, we have shown that tolerogenic
DCs can be generated under physiologic conditions of
oral tolerance, without IL-10 treatment or genetic modification.
Taken together, these findings suggest that the
immune tolerance induced by repeated feeding with oral
antigen is initiated by an increase in tolerogenic
CD11c⫹,CD11b⫹ DCs in Peyer’s patches. These
CD11c⫹,CD11b⫹ DCs in the Peyer’s patches interact
with and differentiate antigen-reactive CD4⫹ T cells
into antigen-induced CD4⫹,CD25⫹ T regulatory cells,
which are involved in the induction of systemic tolerance. Our findings provide evidence of a new cellular
mechanism underlying oral tolerance. In addition, the
CD11c⫹,CD11b⫹ DCs generated as a part of this oral
tolerance inhibit the development of arthritis and somewhat lessen its severity, and thereby should be further
investigated for their potential use in novel therapeutic
approaches.
ACKNOWLEDGMENTS
We would like to thank Prof. Yong won Choi (University of Pennsylvania) and Dr. Andrew H. Kang (University
of Tennessee) for their critical comments on the manuscript,
and Jae-Seon Lee for technical assistance.
REFERENCES
1. Mayer L, Shao L. Therapeutic potential of oral tolerance. Nat Rev
Immunol 2004;4:407–19.
2. Mowat AM. Anatomical basis of tolerance and immunity to
intestinal antigens. Nat Rev Immunol 2003;3:331–41.
3. Neurath MF, Finotto S, Glimcher LH. The role of Th1/Th2
polarization in mucosal immunity. Nat Med 2002;8:567–73.
4. Bitar DM, Whitacre CC. Suppression of experimental autoimmune encephalomyelitis by the oral administration of myelin
basic protein. Cell Immunol 1988;112:364–70.
5. Whitacre CC, Gienapp IE, Orosz CG, Bitar DM. Oral tolerance in
experimental autoimmune encephalomyelitis. III. Evidence for
clonal anergy. J Immunol 1991;147:2155–63.
6. Kim WU, Lee WK, Ryoo JW, Kim SH, Kim J, Youn J, et al.
Suppression of collagen-induced arthritis by single administration
of poly(lactic-co-glycolic acid) nanoparticles entrapping type II
collagen: a novel treatment strategy for induction of oral tolerance. Arthritis Rheum 2002;46:1109–20.
7. Nagler-Anderson C, Bober LA, Robinson ME, Siskind GW,
897
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
Thorbecke GJ. Suppression of type II collagen-induced arthritis by
intragastric administration of soluble type II collagen. Proc Natl
Acad Sci U S A 1986;83:7443–6.
Barnett ML, Kremer JM, StClair EW, Clegg DO, Furst D,
Weisman M, et al. Treatment of rheumatoid arthritis with oral
type II collagen: results of a multicenter, double-blind, placebocontrolled trial. Arthritis Rheum 1998;41:290–7.
Trentham DE, Dynesius-Trentham RA, Orav EJ, Combitchi D,
Lorenzo C, Sewell KL, et al. Effects of oral administration of type
II collagen on rheumatoid arthritis. Science 1993;261:1727–30.
Tsuji NM, Mizumachi K, Kurisaki J. Interleukin-10-secreting
Peyer’s patch cells are responsible for active suppression in
low-dose oral tolerance. Immunology 2001;103:458–64.
Fujihashi K, Dohi T, Rennert PD, Yamamoto M, Koga T, Kiyono
H, et al. Peyer’s patches are required for oral tolerance to proteins.
Proc Natl Acad Sci U S A 2001;98:3310–5.
Iwasaki A, Kelsall BL. Freshly isolated Peyer’s patch, but not
spleen, dendritic cells produce interleukin 10 and induce the
differentiation of T helper type 2 cells. J Exp Med 1999;190:
229–39.
Bilsborough J, George TC, Norment A, Viney JL. Mucosal
CD8␣⫹ DC, with a plasmacytoid phenotype, induce differentiation and support function of T cells with regulatory properties.
Immunology 2003;108:481–92.
Martin P, del Hoyo GM, Anjuere F, Arias CF, Vargas HH,
Fernandez LA, et al. Characterization of a new subpopulation of
mouse CD8␣⫹ B220⫹ dendritic cells endowed with type 1 interferon production capacity and tolerogenic potential. Blood 2002;
100:383–90.
Wakkach A, Fournier N, Brun V, Breittmayer JP, Cottrez F,
Groux H. Characterization of dendritic cells that induce tolerance
and T regulatory 1 cell differentiation in vivo. Immunity 2003;18:
605–17.
Shortman K, Liu YJ. Mouse and human dendritic cell subtypes.
Nat Rev Immunol 2002;2:151–61.
Iwasaki A, Kelsall BL. Localization of distinct Peyer’s patch
dendritic cell subsets and their recruitment by chemokines macrophage inflammatory protein (MIP)-3␣, MIP-3␤, and secondary
lymphoid organ chemokine. J Exp Med 2000;191:1381–94.
Iwasaki A, Kelsall BL. Unique functions of CD11b⫹, CD8␣⫹, and
double-negative Peyer’s patch dendritic cells. J Immunol 2001;166:
4884–90.
Castellaneta A, Abe M, Morelli AE, Thomson AW. Identification
and characterization of intestinal Peyer’s patch interferon-␣ producing (plasmacytoid) dendritic cells. Hum Immunol 2004;65:
104–13.
Pulendran B, Lingappa J, Kennedy MK, Smith J, Teepe M,
Rudensky A, et al. Developmental pathways of dendritic cells in
vivo: distinct function, phenotype, and localization of dendritic cell
subsets in FLT3 ligand-treated mice. J Immunol 1997;159:
2222–31.
Maldonado-Lopez R, de Smedt T, Michel P, Godfroid J, Pajak B,
Heirman C, et al. CD8␣⫹ and CD8␣⫺ subclasses of dendritic cells
direct the development of distinct T helper cells in vivo. J Exp Med
1999;189:587–92.
Pulendran B, Smith JL, Caspary G, Brasel K, Pettit D, Maraskovsky E, et al. Distinct dendritic cell subsets differentially regulate
the class of immune response in vivo. Proc Natl Acad Sci U S A
1999;96:1036–41.
Voisine C, Hubert FX, Trinite B, Heslan M, Josien R. Two
phenotypically distinct subsets of spleen dendritic cells in rats
exhibit different cytokine production and T cell stimulatory activity. J Immunol 2002;169:2284–91.
Stuart JM, Cremer MA, Kang AH, Townes AS. Collagen-induced
arthritis in rats: evaluation of early immunologic events. Arthritis
Rheum 1979;22:1344–51.
898
25. Moser M. Dendritic cells in immunity and tolerance—do they
display opposite functions? Immunity 2003;19:5–8.
26. Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells. Annu Rev Immunol 2003;21:685–711.
27. Vlad G, Cortesini R, Suciu-Foca N. License to heal: bidirectional
interaction of antigen-specific regulatory T cells and tolerogenic
APC. J Immunol 2005;174:5907–14.
28. Banchereau J, Steinman RM. Dendritic cells and the control of
immunity. Nature 1998;392:245–52.
29. Jonuleit H, Schmitt E, Schuler G, Knop J, Enk AH. Induction of
interleukin 10-producing, nonproliferating CD4(⫹) T cells with
regulatory properties by repetitive stimulation with allogeneic
immature human dendritic cells. J Exp Med 2000;192:1213–22.
30. Menges M, Rossner S, Voigtlander C, Schindler H, Kukutsch NA,
Bogdan C, et al. Repetitive injections of dendritic cells matured
with tumor necrosis factor ␣ induce antigen-specific protection of
mice from autoimmunity. J Exp Med 2002;195:15–21.
31. Dubois B, Chapat L, Goubier A, Papiernik M, Nicolas JF,
Kaiserlian D. Innate CD4⫹CD25⫹ regulatory T cells are required
for oral tolerance and inhibition of CD8⫹ T cells mediating skin
inflammation. Blood 2003;102:3295–301.
32. Zhang X, Izikson L, Liu L, Weiner HL. Activation of
CD25(⫹)CD4(⫹) regulatory T cells by oral antigen administration. J Immunol 2001;167:4245–53.
33. Yamazaki S, Iyoda T, Tarbell K, Olson K, Velinzon K, Inaba K, et
al. Direct expansion of functional CD25⫹ CD4⫹ regulatory T
cells by antigen-processing dendritic cells. J Exp Med 2003;198:
235–47.
34. Min SY, Hwang SY, Park KS, Lee JS, Lee KE, Kim KW, et al.
Induction of IL-10-producing CD4⫹CD25⫹ T cells in animal
model of collagen-induced arthritis by oral administration of type
II collagen. Arthritis Res Ther 2004;6:R213–9.
35. Dhodapkar MV, Steinman RM, Krasovsky J, Munz C, Bhardwaj
N. Antigen-specific inhibition of effector T cell function in humans
after injection of immature dendritic cells. J Exp Med 2001;193:
233–8.
MIN ET AL
36. Verhasselt V, Vosters O, Beuneu C, Nicaise C, Stordeur P,
Goldman M. Induction of FOXP3-expressing regulatory CD4pos
T cells by human mature autologous dendritic cells. Eur J Immunol 2004;34:762–72.
37. Akbari O, DeKruyff RH, Umetsu DT. Pulmonary dendritic cells
producing IL-10 mediate tolerance induced by respiratory exposure to antigen. Nat Immunol 2001;2:725–31.
38. Vasu C, Dogan RN, Holterman MJ, Prabhakar BS. Selective
induction of dendritic cells using granulocyte macrophage-colony
stimulating factor, but not fms-like tyrosine kinase receptor 3-ligand, activates thyroglobulin-specific CD4⫹/CD25⫹ T cells and
suppresses experimental autoimmune thyroiditis. J Immunol 2003;
170:5511–22.
39. Dieckmann D, Bruett CH, Ploettner H, Lutz MB, Schuler G.
Human CD4(⫹)CD25(⫹) regulatory, contact-dependent T cells
induce interleukin 10-producing, contact-independent type 1-like
regulatory T cells [correction] [published erratum appears in J Exp
Med 2002;196:559]. J Exp Med 2002;196:247–53.
40. Coffer PJ, Burgering BM. Forkhead-box transcription factors and
their role in the immune system. Nat Rev Immunol 2004;4:889–99.
41. Morita Y, Yang J, Gupta R, Shimizu K, Shelden EA, Endres J, et
al. Dendritic cells genetically engineered to express IL-4 inhibit
murine collagen-induced arthritis. J Clin Invest 2001;107:1275–84.
42. Kim SH, Kim S, Evans CH, Ghivizzani SC, Oligino T, Robbins
PD. Effective treatment of established murine collagen-induced
arthritis by systemic administration of dendritic cells genetically
modified to express IL-4. J Immunol 2001;166:3499–505.
43. Kim SH, Kim S, Oligino TJ, Robbins PD. Effective treatment of
established mouse collagen-induced arthritis by systemic administration of dendritic cells genetically modified to express FasL. Mol
Ther 2002;6:584–90.
44. Van Duivenvoorde LM, Louis-Plence P, Apparailly F, van der
Voort EI, Huizinga TW, Jorgensen C, et al. Antigen-specific
immunomodulation of collagen-induced arthritis with tumor necrosis factor–stimulated dendritic cells. Arthritis Rheum 2004;50:
3354–64.