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Therapeutic effect of tolerogenic dendritic cells in established collagen-induced arthritis is associated with a reduction in Th17 responses.

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ARTHRITIS & RHEUMATISM
Vol. 62, No. 12, December 2010, pp 3656–3665
DOI 10.1002/art.27756
© 2010, American College of Rheumatology
Therapeutic Effect of Tolerogenic Dendritic Cells in
Established Collagen-Induced Arthritis Is
Associated With a Reduction in Th17 Responses
Jeroen N. Stoop, Rachel A. Harry, Alexei von Delwig, John D. Isaacs,
John H. Robinson, and Catharien M. U. Hilkens
Objective. Tolerogenic dendritic cells (DCs) are
antigen-presenting cells with an immunosuppressive
function. They are a promising immunotherapeutic tool
for the attenuation of pathogenic T cell responses in
autoimmune arthritis. The aims of this study were to
determine the therapeutic action of tolerogenic DCs in a
type II collagen–induced arthritis model and to investigate their effects on Th17 cells and other T cell subsets
in mice with established arthritis.
Methods. Tolerogenic DCs were generated by
treating bone marrow–derived DCs with dexamethasone
and vitamin D3 during lipopolysaccharide-induced maturation. Mice with established arthritis received 3 intravenous injections of tolerogenic DCs, mature DCs, or
saline. Arthritis severity was monitored for up to 4
weeks after treatment. Fluorescence-labeled tolerogenic
DCs were used for in vivo trafficking studies. The in vivo
effect of tolerogenic DCs on splenic T cell populations
was determined by intracellular cytokine staining and
flow cytometry.
Results. Tolerogenic DCs displayed a semimature phenotype, produced low levels of inflammatory
cytokines, and exhibited low T cell stimulatory capacity.
Upon intravenous injection into arthritic mice, tolerogenic DCs migrated to the spleen, liver, lung, feet, and
draining lymph nodes. Treatment of arthritic mice with
type II collagen–pulsed tolerogenic DCs, but not unpulsed tolerogenic DCs or mature DCs, significantly
inhibited disease severity and progression. This improvement coincided with a significant decrease in the
number of Th17 cells and an increase in the number of
interleukin-10–producing CD4ⴙ T cells, whereas tolerogenic DC treatment had no detectable effect on Th1 cells
or interleukin-17–producing ␥/␦ T cells.
Conclusion. Treatment with type II collagen–
pulsed tolerogenic DCs decreases the proportion of
Th17 cells in arthritic mice and simultaneously reduces
the severity and progression of arthritis.
Collagen-induced arthritis (CIA) is a wellestablished mouse model of rheumatoid arthritis (RA),
a chronic inflammatory joint disease. This model is
frequently used to develop and test new therapies. Like
RA, CIA is characterized by severe inflammation and
cellular infiltration of synovial tissue, leading to cartilage
and bone destruction (1). Furthermore, T cells play a
pivotal role in the pathogenesis of both RA and CIA.
Therapeutic T cell costimulation blockade with CTLA4Ig reduces disease activity in patients with RA (2), and
depletion of cytokine-producing T cells inhibits the
progression and severity of established arthritis in the
CIA model (3).
Recent evidence suggests that Th17 cells are key
players in the pathogenesis of CIA (4). In mice deficient
for the Th17 cell–associated molecules interleukin-17
(IL-17), IL-17 receptor, or IL-23p19, arthritis is markedly suppressed compared with that in their wild-type
counterparts (5–7), and neutralizing antibodies to IL-17
have a therapeutic effect in CIA (8). In humans, some
evidence supports the involvement of Th17 cells in the
pathogenesis of RA. For instance, the proportion of
Th17 cells is increased in the peripheral blood of pa-
Supported by the Medical Research Council (grant 80245).
Jeroen N. Stoop, PhD, Rachel A. Harry, PhD, Alexei von
Delwig, PhD, John D. Isaacs, PhD, MBBS, FRCP, John H. Robinson,
PhD, Catharien M. U. Hilkens, PhD: Newcastle University, Newcastle
upon Tyne, UK.
Address correspondence and reprint requests to Catharien
Hilkens, PhD, Institute of Cellular Medicine, Musculoskeletal Research Group, Faculty of Medical Sciences, Newcastle University,
Framlington Place, Newcastle upon Tyne NE2 4HH, UK. E-mail:
catharien.hilkens@ncl.ac.uk.
Submitted for publication December 23, 2009; accepted in
revised form September 14, 2010.
3656
THERAPEUTIC EFFECT OF TOLEROGENIC DCs IN CIA
tients with RA compared with healthy control subjects
(9), and the expression of IL-17 messenger RNA in RA
synovial tissue is predictive of the progression of joint
damage (10). In addition to Th17 cells, ␥/␦ T cells are a
major source of IL-17, and it has been shown that ␥/␦ T
cell–derived IL-17 exacerbates the severity of CIA
(11,12). Contrary to observations in the CIA model,
however, there is no evidence to suggest a role for IL-17
production by ␥/␦ T cells in RA (11,12).
A new promising immunotherapeutic strategy for
the attenuation of pathogenic T cell responses is treatment with autologous dendritic cells (DCs). DCs are
antigen-presenting cells that initiate immune responses
to invading pathogens while maintaining tolerance to
self antigens (13). DCs with potent and stable tolerogenic activity can be generated in vitro by genetic or
pharmacologic modification (14). For instance, DCs
transduced with FasL or IL-4 have been used to prevent
CIA and to inhibit arthritis symptoms in mice with
established disease (15–17). Tolerogenic DCs modified
by drugs (dexamethasone; Bay 11-7082) or cytokines
(tumor necrosis factor [TNF]) have been used successfully to prevent the onset of CIA (18–22) or to suppress
established arthritis in a different model, the antigeninduced arthritis model (23).
Our group is in the process of developing tolerogenic DC therapy for RA and has opted for pharmacologic modification of DCs, because it is a robust, simple,
and effective method that is ideal for clinical application. Treatment of DCs with 1␣,25-dihydroxyvitamin D3
(vitamin D3) in combination with dexamethasone has
been shown to synergistically inhibit lipopolysaccharide
(LPS)–induced maturation of DCs (24). Previously, we
used these immunosuppressive drugs to generate human
tolerogenic DCs with potent immunoregulatory function
in vitro (25,26). An important outstanding question is,
however, whether these pharmacologically modified
tolerogenic DCs can inhibit pathogenic IL-17–mediated
responses in vivo, and whether they will be effective at
reducing the progression and severity of arthritis when
administered after disease onset. The aim of this study
was to determine the therapeutic and immunomodulatory actions of murine dexamethasone/vitamin D3–
modified tolerogenic DCs in mice with established CIA.
MATERIALS AND METHODS
Mice. Male DBA/1 mice were purchased from Harlan
UK, and female BALB/c mice were purchased from Charles
River. The mice were kept in individually ventilated cages.
Water and food were provided ad libitum. The experiments
3657
were performed under the terms of the Animals (Scientific
Procedures) Act of 1986 and were authorized by the Home
Secretary, Home Office, UK.
Generation of bone marrow–derived DCs. Cells were
cultured in RPMI 1640 (Sigma-Aldrich) supplemented with
10% fetal calf serum (PAA Laboratories), 2 mM glutamine,
100 units/ml penicillin, 100 ␮g/ml streptomycin, and 50 ␮M
2-mercaptoethanol (all from Sigma-Aldrich) at 37°C with 5%
CO2. Bone marrow was derived from the femurs and tibia of
the mice. DCs were generated as described previously (27).
Briefly, bone marrow was cultured for 10 days in medium
containing 20 ng/ml granulocyte–macrophage colonystimulating factor (GM-CSF; PeproTech). Cultures were refreshed on days 3, 6, and 8 with medium supplemented with
GM-CSF. On day 10, DCs were matured with 0.1 ␮g/ml LPS
(Sigma-Aldrich) for 16 hours. Tolerogenic DCs were generated by adding dexamethasone (10–6M; Sigma-Aldrich) and
vitamin D3 (10–10M; LEO Pharma) during LPS-induced maturation. When indicated, DCs were pulsed with 10 ␮g/ml of
bovine type II collagen (Chondrex) during maturation.
DC migration experiments. For tracking experiments,
DCs were labeled for 10 minutes at 37°C with 4 ␮M/ml
5,6-carboxyfluorescein succinimidyl ester (CFSE; Molecular
Probes) in Hanks’ balanced salt solution (HBSS). After labeling, cells were washed in RPMI 1640 plus 10% fetal calf serum
and rested for 10 minutes at 37°C with 5% CO2. CFSE-labeled
DCs (2 ⫻ 106 ) were injected intravenously into arthritic mice.
Three days after the DC injection, the liver, spleen, lung,
popliteal lymph nodes (LNs), and feet of the mice were
harvested. The skin was removed from the feet. The lungs were
treated for 30 minutes with collagenase (Sigma-Aldrich). The
tissues were cut into small pieces and ground through a cell
strainer to prepare single-cell suspensions for subsequent flow
cytometric analysis.
Induction, monitoring, and treatment of arthritis.
Arthritis was induced in 8–10-week-old DBA/1 mice. On day 0,
mice were injected subcutaneously at the tail base with 100 ␮g
of type II collagen emulsified in Freund’s complete adjuvant
(containing 1 mg/ml Mycobacterium tuberculosis; Difco). On
day 21, the mice received a subcutaneous booster injection
with 100 ␮g of type II collagen in Freund’s incomplete
adjuvant (Sigma-Aldrich). For the synchronous onset of arthritis, 25 ␮g of ultrapure LPS (InvivoGen) was injected intraperitoneally on day 24. Arthritis severity was monitored using a
clinical score, as follows: 0 ⫽ no signs of inflammation, 1 ⫽
erythema or swelling, 2 ⫽ both erythema and swelling, and 3 ⫽
erythema and severe swelling. The clinical score per mouse
was the cumulative value for all paws. The thickness of the feet
(swelling) was measured with a spring-loaded Oditest caliper
(Kroeplin). Tolerogenic DCs were administered as specified,
by one or multiple injections via the intraperitoneal or intravenous route in a dose ranging from 1 ⫻ 104 to 2.5 ⫻ 106
cells per injection. Control mice were treated with HBSS.
Treatment was started on day 3 after CIA onset. DCs used
for treatment were pulsed with type II collagen unless
otherwise specified.
Dextran phagocytosis. Fluorescence-labeled dextran
(fluorescein isothiocyanate [FITC]–dextran; Sigma-Aldrich)
was used to compare the mannose receptor–mediated phago-
3658
cytic capacity of tolerogenic DCs and mature DCs. DCs were
incubated with FITC–dextran and LPS at 37°C for 1–5 hours,
with or without dexamethasone/vitamin D3. Control DCs were
left on ice to exclude extracellular binding of FITC–dextran.
Cells were extensively washed, and intracellular FITC–dextran
was quantified by flow cytometry.
Flow cytometric analysis. Cells were labeled in phosphate buffered saline (PBS) containing 0.5% bovine serum
albumin (BSA). Prior to labeling, DCs were incubated with
anti-mouse CD16/CD32 (Fc Block, 2.4G2; BD PharMingen).
DCs were labeled with the following: allophycocyanin (APC)–
conjugated anti–class II major histocompatibility complex
(Miltenyi Biotech), FITC-conjugated anti-CD86 (GL1), phycoerythrin (PE)–conjugated anti-CD80 (16-10A1), and PEconjugated anti-CD40 (3/23) (all from BD PharMingen). For
in vivo tracking experiments with CFSE-labeled DCs, various
tissues were labeled with PE-Cy7–conjugated anti-CD45 (30F11) and anti-CD11c–biotin (both from BD PharMingen),
followed by Streptavidin eFluor 450 (eBioscience). DC viability was assessed with FITC-conjugated annexin V (BD PharMingen) and Via-Probe (BD PharMingen). FoxP3 labeling
was performed according to the manufacturer’s instructions.
Briefly, cells were labeled with peridinin chlorophyll A protein
(PerCP)–Cy5.5–conjugated anti-CD4 (RM4-5; BD PharMingen) and Alexa Fluor 488–conjugated anti-CD25 (M-A251;
eBioscience). The cells were fixed and permeabilized with Fix/
Perm buffer and permeabilization buffer (eBioscience). APCconjugated anti-FoxP3 (FJK-16a) and PE-conjugated anti–
CTLA-4 (4C10-4B90) (both from eBioscience) and 4% rat
serum (Sigma-Aldrich) were added during permeabilization.
For intracellular cytokine staining, cells were stimulated with phorbol myristate acetate (50 ng/ml; Sigma-Aldrich)
and ionomycin (500 ng/ml; Sigma-Aldrich) for 5 hours. After 1
hour, brefeldin A (10 ␮g/ml; Sigma-Aldrich) was added. Cell
surface staining was performed with PerCP-Cy5.5–conjugated
anti-CD4, Pacific Blue–conjugated anti-CD8a (53-6.7; BD
PharMingen), and biotin-conjugated anti-␥/␦ T cell receptor
(GL3; BioLegend) followed by staining with PE-Cy7–
conjugated streptavidin (eBioscience). Cells were fixed and
permeabilized as described previously. Intracellular staining
was performed in the presence of 4% rat serum using FITCconjugated anti–interferon-␥ (anti-IFN␥) (XMG1.2), PEconjugated anti–IL-17 (TC11-1H10.1), and APC-conjugated
anti–IL-10 (JES5-16E3; all from BD PharMingen).
Cells were acquired on a FACScan or an LSR II
(Becton Dickinson), and data were analyzed using FlowJo
software (Three Star) or Venturi One software (Applied
Cytometry), respectively.
Cytokine production of DCs. CD40L-transfected
J558L mouse cells were fixed with 1% paraformaldehyde
(Sigma-Aldrich) and cultured with tolerogenic DCs or mature
DCs at a 2:1 ratio. After 24 hours, supernatants were harvested, and cytokine levels were determined by sandwich
enzyme-linked immunosorbent assay (ELISA). IL-1␤, IL-12,
IL-23, TNF␣ (eBioscience), and IL-10 (R&D Systems)
ELISAs were performed according to the manufacturers’
instructions.
STOOP ET AL
Type II collagen–specific proliferation assays. Spleen
cells from treated arthritic mice were harvested 28 days after
the start of treatment and were cultured in triplicate at a
concentration of 3 ⫻ 105 cells per well in 200 ␮l of X-Vivo
Medium (Lonza) supplemented with 2 mM glutamine, 100
units/ml penicillin, 100 ␮g/ml streptomycin, and 50 ␮M
2-mercaptoethanol (all from Sigma-Aldrich). Cells were stimulated with 50 ␮g/ml type II collagen for 96 hours or were
left unstimulated. 3H-thymidine (10 kBq; specific activity
74.0 GBq/mmole) (PerkinElmer) was added for the last 18
hours. Radioactivity was quantitated using a MicroBeta TriLux
beta counter (PerkinElmer). The stimulation index was calculated as the counts per minute in the presence of type II
collagen divided by the counts per minute in the absence of
type II collagen.
Proteoglycan-specific proliferation assays. Proteoglycan was prepared as described previously (28). Female
BALB/c mice were immunized with 30 ␮l of a 1:1 mixture of
proteoglycan and TiterMax Gold (CytRx) injected into the
footpad. After 7 days, popliteal LNs were harvested, and
CD4⫹ T cells were isolated using anti-CD4 MicroBeads
(Miltenyi Biotech). Tolerogenic DCs or mature DCs were
pulsed with 10 ␮g/ml proteoglycan during maturation, and 2 ⫻
103 DCs were used to stimulate 1 ⫻ 105 T cells in 96-well plates
in a final volume of 200 ␮l (Sigma-Aldrich) and incubated for
3 days. CD3/CD28 Dynal expander beads (Invitrogen) were
used as positive control. Proliferation was determined by
3
H-thymidine incorporation as described above.
Measurement of type II collagen–specific antibodies in
serum. Nunc Immuno MaxiSorp 96-well plates were coated
overnight at 4°C with 4 ␮g/ml bovine type II collagen (Chondrex). Plates were blocked with PBS/1% BSA. Mouse serum
diluted 1:5,000 was incubated for 2 hours. Plates were subsequently treated with one of the following detection antibodies:
horseradish peroxidase–conjugated anti-mouse immunoglobulin, IgG1 (X56), or IgG2a (R19-15) (all from BD PharMingen). Orthophenylenediamine (Sigma-Aldrich) was used as
substrate.
Statistical analysis. Statistical testing was performed
using Prism 4 (GraphPad Software).
RESULTS
Typical semi-mature phenotype of tolerogenic
DCs. DCs were generated from bone marrow and
were treated with dexamethasone and vitamin D3
during LPS-induced maturation to generate tolerogenic
DCs. LPS-activated DCs that were not exposed to
dexamethasone/vitamin D3 (mature DCs) and untreated
DCs (immature DCs) were used as control populations.
Tolerogenic DCs displayed a semi-mature phenotype.
They expressed higher levels of CD40 and the costimulatory molecules CD80 and CD86 as compared with
immature DCs; however, the expression of these 3
markers was lower compared with the expression by
THERAPEUTIC EFFECT OF TOLEROGENIC DCs IN CIA
3659
Figure 1. Phenotypic and functional characteristics of tolerogenic dendritic cells (DCs). A, The median fluorescence intensity (MFI) of CD40,
CD80, CD86, and class II major histocompatibility complex (MHC) expression by immature DCs (iDC), tolerogenic DCs (TolDC), or mature DCs
(mDC) (n ⫽ 6 samples) was determined. Statistical testing was performed using one-way analysis of variance with Bonferroni adjustment for multiple
testing. B, Tolerogenic DCs and mature DCs were extensively washed and stimulated with CD40L-expressing cells to mimic CD40-dependent
activation of DCs by activated T cells. Supernatants were harvested after 24 hours, and cytokine levels were determined by enzyme-linked
immunosorbent assay (n ⫽ 7 samples). Statistical testing was performed using Wilcoxon’s matched pairs signed rank sum test. C, CD4⫹ T cells were
isolated from the popliteal lymph nodes of proteoglycan-immunized mice and restimulated with proteoglycan-pulsed tolerogenic DCs in a DC:T cell
ratio of 1:50. Proliferation was determined after 3 days, using 3H-thymidine incorporation. The symbols represent individual mice, and the horizontal
lines represent the mean. Statistical analysis was performed using Student’s t-test. D, Immature DCs were incubated for the indicated times with
dextran–fluorescein isothiocyanate (FITC) and dexamethasone/vitamin D3/lipopolysaccharide (LPS) (tolerogenic DCs) or LPS (mature DCs). The
proportion of FITC-positive CD11c-positive cells was determined by flow cytometry (n ⫽ 5 samples). Values in A, B, and D are the mean and SEM.
ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01. IL-1␤ ⫽ interleukin-1␤; TNF␣ ⫽ tumor necrosis factor ␣.
mature DCs (Figure 1A). Because cytokine production
is an important mechanism by which DCs regulate the
immune response, the cytokine production profiles of
DCs upon CD40 ligation were determined. Tolerogenic
DCs produced lower levels of the inflammatory cytokines IL-1␤, TNF␣, IL-12p70, and IL-23 as compared
with mature DCs (Figure 1B); production of antiinflammatory IL-10 was also decreased but to a lesser extent
(Figure 1B). The semi-mature phenotype of tolerogenic
DCs was consistent with their low ability to stimulate
proteoglycan-specific CD4⫹ T cell proliferation (Figure
1C). Dexamethasone/vitamin D3 treatment of DCs did
not induce apoptosis or cell death (as determined by
annexin V and ViaProbe labeling; data not shown) and
did not inhibit their capacity to mediate mannose
receptor–dependent phagocytosis (Figure 1D).
Migration of tolerogenic DCs to the feet, liver,
and LNs in arthritic mice. To study the migratory
capacity of tolerogenic DCs in arthritic mice, cells were
labeled with CFSE and injected intravenously 3 days
after arthritis had developed. Mature DCs were used
as a control. Representative flow cytometry data for
different tissues from mice treated with CFSE-labeled
DCs and control mice are depicted in Figure 2A. All
CFSE-positive cells within the gated area were CD11c
positive (results not shown). CFSE-positive cells were
detected in the popliteal LNs, spleen, liver, lung, and,
interestingly, in arthritic feet 3 days after DC injection
(Figure 2B).
Because endogenous DCs are potentially involved in disease pathogenesis and may counteract the
suppressive effects of tolerogenic DCs, we also calculated the percentage of tolerogenic DCs (CFSE⫹
CD11c⫹) of the total CD11c⫹ DC population within
the different tissues. Interestingly, in arthritic feet, popliteal LNs, and liver, ⬎50% of the DCs present were
CFSE positive, which is indicative of a predominance of
tolerogenic DCs. This percentage was substantially
lower in the spleen and lungs (Figure 2C). These data
indicate that adoptively transferred tolerogenic DCs
migrate to various tissues and become one of the main
DC populations in arthritic feet. We did not observe a
difference in the migratory capacity between mature
DCs and tolerogenic DCs.
Effect of treatment with type II collagen–pulsed
tolerogenic DCs in established arthritis. To determine
whether tolerogenic DCs have a therapeutic effect in
established arthritis, mice were injected 3 times (on days
3, 7, and 11 after arthritis onset) with 1 ⫻ 106 type II
collagen–pulsed tolerogenic DCs, 1 ⫻ 106 type II
collagen–pulsed mature DCs, or saline (HBSS). Tolerogenic DC treatment resulted in a rapid and sharp decline
3660
STOOP ET AL
the therapeutic effects of unpulsed tolerogenic DCs and
type II collagen–pulsed tolerogenic DCs were compared. Treatment with type II collagen–pulsed tolerogenic DCs did reduce the progression of arthritis,
whereas treatment with unpulsed tolerogenic DCs had
no therapeutic effect (Figure 3B).
We next investigated whether the therapeutic
effect of tolerogenic DC treatment could be further
optimized by varying the dose, route of administration,
and number of injections. Increasing the dose of tolero-
Figure 2. Migration of tolerogenic DCs after injection into arthritic
mice. Collagen-induced arthritis was induced in DBA/1 mice by
immunization with type II collagen in adjuvant. Arthritic mice were
injected intravenously with 2 ⫻ 106 5,6-carboxyfluorescein succinimidyl
ester (CFSE)–labeled DCs (tolerogenic or mature). After 3 days, the
spleen, liver, lung, lymph nodes (LNs), and arthritic feet were harvested. A, Representative examples of CFSE expression of CD45⫹
cells in liver, spleen, lung, LNs, and feet of untreated arthritic mice
(top) or arthritic mice treated with CFSE-positive tolerogenic DCs
(bottom), in a live gate are shown. All CFSE-positive cells within the
gated area were CD11c⫹ (not shown). B, The total number of
CFSE-positive mature DCs and tolerogenic DCs was determined by
multiplying the percentage of CD45⫹CFSE⫹CD11c⫹ cells as established by flow cytometry with the total number of harvested cells from
each tissue (n ⫽ 3 mice). C, The proportion of CFSE-positive cells as
a percentage of CD11c⫹ cells was determined by flow cytometry.
Values in B and C are the mean and SEM. PE-Cy7 ⫽ phycoerythrin–
Cy7 (see Figure 1 for other definitions).
in the severity of arthritis symptoms (Figures 3A and B),
whereas injection with mature DCs exacerbated arthritis
(Figure 3A and Table 1). Although the tolerogenic
DC–induced decrease in arthritis severity was followed
by a slow worsening of the clinical score, arthritis
severity never reached the same level as that in untreated or mature DC–treated mice (Figure 3A and
Table 1). At the end of followup (i.e., 28 days after the
start of tolerogenic DC therapy), the clinical scores in
tolerogenic DC–treated mice were similar to the clinical
scores at the start of treatment (Table 1). In contrast,
arthritis progressed significantly in the HBSS- and mature DC–treated control groups during the 28-day followup (Table 1). The values for swelling of the feet
followed a trend similar to that of the clinical score (data
not shown).
Because it is still a matter of debate whether
tolerogenic DCs need to be pulsed with an autoantigen,
Figure 3. Therapeutic effects of tolerogenic DCs in arthritic mice.
Collagen-induced arthritis was induced in DBA/1 mice by immunization with type II collagen (CII) in adjuvant. Mice were treated with
DCs intravenously on days 3, 7, and 11 after arthritis onset. Arrows
indicate the days on which mice received treatment. Arthritis severity
is depicted as the mean ⫾ SEM clinical score for each group or the
change in clinical score (the clinical score on day 28 after the start of
therapy minus the clinical score at the start of therapy). In the right
panels, the symbols represent individual mice, and the horizontal lines
represent the median. Statistical testing was performed using the
Kruskal-Wallis test with Dunn’s post hoc test. The mice were treated
with 1 ⫻ 106 type II collagen–pulsed tolerogenic DCs, 1 ⫻ 106 type II
collagen–pulsed mature DCs, or Hanks’ balanced salt solution (HBSS)
(A), 1 ⫻ 106 type II collagen–pulsed tolerogenic DCs, 1 ⫻ 106
unpulsed tolerogenic DCs, or HBSS (B), or 1 ⫻ 106 or 2.5 ⫻ 106 type
II collagen–pulsed tolerogenic DCs or HBSS (C). ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽
P ⬍ 0.01. See Figure 1 for other definitions.
THERAPEUTIC EFFECT OF TOLEROGENIC DCs IN CIA
Table 1.
3661
Effects of different treatment regimens on collagen-induced arthritis*
Treatment/
no. and route
of injections
HBSS/3 IV (n ⫽ 30)
Mature DCs/3 IV (n ⫽ 5)
Tolerogenic DCs
1 ⫻ 106/3 IV (n ⫽ 30)
2.5 ⫻ 106/3 IP (n ⫽ 5)
1 ⫻ 106/1 IV (n ⫽ 10)
2 ⫻ 105/3 IV (n ⫽ 5)
4 ⫻ 104/3 IV (n ⫽ 5)
Baseline
Day 7
End of
followup
5.5 ⫾ 0.4
6.0 ⫾ 1.3
6.5 ⫾ 0.5
7.4 ⫾ 1.0
9.0 ⫾ 0.4
12.0 ⫾ 0
6.0 ⫾ 0.5
5.4 ⫾ 1.2
6.2 ⫾ 0.8
7.0 ⫾ 0.9
7.6 ⫾ 0.6
3.4 ⫾ 0.7†
5.0 ⫾ 0.7
3.6 ⫾ 1.0
7.2 ⫾ 1.0
8.6 ⫾ 0.9
5.7 ⫾ 0.7†
7.8 ⫾ 1.0
7.7 ⫾ 0.9
9.0 ⫾ 0.8
9.4 ⫾ 1.1
* Values are the mean ⫾ SEM clinical score (see Materials and Methods). Baseline is day 31 after
immunization (before the first injection of dendritic cells [DCs]), day 7 is 7 days after the first DC injection
(day 38 after the first immunization), and the end of followup is 28 days after the first DC injection (day
59 after the first immunization). Statistical analysis for each time point was performed using the
Kruskal-Wallis test with Dunn’s post hoc test. IV ⫽ intravenous; IP ⫽ intraperitoneal.
† P ⬍ 0.05 versus Hanks’ balanced salt solution (HBSS).
genic DCs from 1 ⫻ 106 to 2.5 ⫻ 106 cells did not
improve the therapeutic effect; both doses of type II
collagen–pulsed tolerogenic DCs significantly improved
the clinical score to the same extent (Figure 3C). In
contrast, lower doses of tolerogenic DCs (2 ⫻ 105 and
1 ⫻ 104) were not effective in reducing the severity of
arthritis. Furthermore, lowering the number of tolerogenic DC injections (a single injection of 1 ⫻ 106 cells)
abrogated the beneficial effect of tolerogenic DC therapy (Table 1). A change in the route of administration
also abolished the therapeutic effect of tolerogenic DCs:
intraperitoneal injections did not reduce arthritis severity (Table 1). Thus, at least 3 intravenous injections of
⬎2 ⫻ 105 tolerogenic DCs are needed for an optimal
therapeutic effect.
Enhancement of IL-10–positive CD4ⴙ T cells
and inhibition of Th17 cells in tolerogenic DC–treated
mice. To study the in vivo effect of tolerogenic DC
treatment on the T cell response, splenocytes from
treated mice were harvested 28 days after the first
tolerogenic DC injection. No significant differences
were observed in tolerogenic DC–treated versus control
mice for spleen size (results not shown), percentages of
CD4⫹ T cells (Figure 4B), CD8⫹ T cells (Figure 4C),
␥/␦ T cells (Figure 4D), and FoxP3-positive Treg cells
(Figure 5B). We also measured the expression of
CTLA-4 by Treg cells, because this molecule plays an
important role in their mechanism of suppression (29).
However, tolerogenic DC treatment did not result in an
increase in the proportion of CTLA-4–positive Treg
cells (Figure 5B) or the level of CTLA-4 expressed by
Treg cells (mean ⫾ SEM median fluorescence intensity
1,711.9 ⫾ 74.0 in control mice versus 1,627.9 ⫾ 33.1 in
tolerogenic DC–treated mice [P ⫽ 0.85]; n ⫽ 10 per
group). Importantly, tolerogenic DC therapy did decrease the collagen-specific proliferation of spleen cells
(Figure 5C).
Type II collagen–specific antibodies in the sera of
mice with CIA are often used to examine the class of the
type II collagen–specific immune response (17–19,30).
Type II collagen–specific IgG2a antibodies are associated with a Th1 cell–driven immune response, whereas
type II collagen–specific IgG1 antibodies are associated
with a Th2 cell–driven response (18). Sera from tolerogenic DC– and HBSS-treated mice were collected 14
days after the first tolerogenic DC injection. Tolerogenic
DC treatment did not result in a decrease in the
expression of type II collagen–specific immunoglobulins
overall (Figure 5D) or IgG2a or IgG1 levels (data not
shown) and did not alter the type II collagen–specific
IgG2a:IgG1 ratio (Figure 5D).
Interestingly, tolerogenic DC treatment altered
the proportion of various cytokine-producing T cell
populations. The treatment resulted in an increase in
the number of IL-10–producing CD4⫹ T cells that did
not produce detectable levels of IL-17 and IFN␥ and a
decrease in the percentage of IL-17–producing CD4⫹
T cells, while the proportions of IFN␥-producing CD4⫹
T cells and IL-17–producing ␥/␦ T cells were not affected by tolerogenic DC therapy (Figures 4B and D).
No CD4⫹ T cells producing both IL-17 and IFN␥ were
detected. Tolerogenic DC therapy increased the percentage of IFN␥-producing CD8⫹ T cells (Figure 4C).
Thus, clinical improvement observed after tolerogenic
DC therapy is accompanied by an increase in the
3662
Figure 4. Effect of tolerogenic DC therapy on splenic T cell populations and cytokine production. Arthritic mice were treated intravenously with 3 injections of 1 ⫻ 106 type II collagen–pulsed tolerogenic
DCs; spleens were harvested 28 days after the first injection. The
proportions of T cell subsets and cytokine-producing cells following
phorbol myristate acetate/ionomycin stimulation were determined by
flow cytometry. A, Representative dot plots for intracellular cytokines
depicting the gating strategy. The top left and top center dot plots
depict cells in a live gate based on forward scatter and side scatter.
Cytokine production by ␥/␦ T cells is shown in the bottom left dot plot,
cytokine production by CD8⫹ T cells is shown in the top right dot plot,
and cytokine production by CD4⫹ T cells is shown in the bottom
center and bottom right dot plots. B, Proportions of CD4⫹ T cells and
interferon-␥ (IFN␥)–, interleukin-17 (IL-17)–, and IL-10–producing
CD4⫹ T cells. C, Proportions of CD8⫹ T cells and IFN␥-producing
CD8⫹ T cells. D, Proportions of ␥/␦ T cells and IL-17–producing ␥/␦
T cells. The symbols represent individual mice, and the horizontal lines
represent the median. Statistical testing was performed with the
Mann-Whitney U test. ⴱ ⫽ P ⬍ 0.05. PerCP-Cy5.5 ⫽ peridinin
chlorophyll A protein–Cy5.5; PE ⫽ phycoerythrin; APC ⫽ allophycocyanin; TCR ⫽ T cell receptor; HBSS ⫽ Hanks’ balanced salt solution
(see Figure 1 for other definitions).
STOOP ET AL
Figure 5. Effect of tolerogenic DC therapy on splenic T cell populations and serum type II collagen (CII)–specific antibody levels. Arthritic mice were treated intravenously with 3 injections of 1 ⫻ 106 type
II collagen–pulsed tolerogenic DCs; spleens were harvested 28 days
after the first injection. A, Representative dot plots for FoxP3, CD25,
and CTLA-4 expression are shown. The dot plots are gated on CD4⫹
cells. B, The proportions of CD25⫹FoxP3⫹ cells and CTLA-4–
positive FoxP3⫹ cells as percentages of CD4⫹ cells and CTLA-4–
positive cells as a percentage of FoxP3⫹ cells were determined by flow
cytometry. C, Spleen cells were restimulated with type II collagen for
96 hours, and proliferation was determined by 3H-thymidine incorporation. Statistical testing was performed with the Mann-Whitney U
test. D, Type II collagen–specific antibody levels after tolerogenic DC
therapy are shown. Mice were bled 14 days after the first injection with
1 ⫻ 106 tolerogenic DCs or Hanks’ balanced salt solution (HBSS).
Type II collagen–specific Ig, IgG1, and IgG2a antibody levels in sera
were determined by enzyme-linked immunosorbent assay. The left
panel shows type II collagen–specific antibody levels indicated by
optical density (OD), and the right panel shows the IgG2a:IgG1 ratio
(n ⫽ 20). The symbols represent individual mice, and the horizontal
lines represent the median. ⴱⴱ ⫽ P ⬍ 0.01. See Figure 1 for other
definitions.
number of immunoregulatory IL-10–producing T cells
and a decrease in the number of pathogenic Th17 cells.
DISCUSSION
Our results show that tolerogenic DCs generated
by pharmacologic modulation with dexamethasone and
THERAPEUTIC EFFECT OF TOLEROGENIC DCs IN CIA
vitamin D3 have a clear therapeutic effect in established
arthritis in the CIA mouse model. Injecting tolerogenic
DCs after the onset of disease significantly reduced the
severity and progression of arthritis, whereas treatment
with mature DCs exacerbated arthritis. The beneficial
effects of tolerogenic DCs required loading with type II
collagen, suggesting that tolerogenic DCs modulated the
immune response in a type II collagen–specific manner.
Tolerogenic DCs did not promote the expansion of
FoxP3-positive Treg cells. However, tolerogenic DC
treatment resulted in a decrease in type II collagen–
specific T cell proliferation and a decrease in the proportion of Th17 cells. Tolerogenic DC therapy also
increased the proportion of IL-10–producing T cells,
suggesting that a shift from pathogenic toward suppressive T cells may contribute to the suppression of
arthritis.
It has been shown that TNF-treated DCs prevent
experimental autoimmune encephalitis through the
enrichment of IL-10–producing T cells in vivo (31).
Previously, our group demonstrated that human
dexamethasone/vitamin D3–treated tolerogenic DCs polarize naive T cells toward high IL-10 production in vitro
(25). Here, we show for the first time that tolerogenic
DCs can promote IL-10–producing T cells in vivo in the
setting of inflammation, such as that associated with
severe arthritis. These IL-10–producing CD4⫹ T cells
did not produce detectable IFN␥ or IL-17 and resemble
T regulatory type 1 (Tr1) cells. Tr1 cells are phenotypically different from FoxP3⫹ Treg cells but have comparable regulatory function and are capable of inhibiting
pathogenic T cell responses (32,33).
Although it has been shown that treatment with
recombinant IL-10 or IL-10–producing T cells is effective for preventing arthritis in mouse models (34,35),
contradictory observations have been made regarding
the therapeutic effect of IL-10 in established arthritis.
Intraarticular administration of IL-10 did not reduce the
severity of arthritis (34), whereas systemic injection with
recombinant IL-10 effectively inhibited disease (36). We
detected a higher proportion of IL-10–producing T cells
in the spleen after tolerogenic DC treatment, suggesting
a systemic increase in IL-10. This increased IL-10 production could therefore be one of the mechanisms by
which tolerogenic DCs inhibit the progression and/or
severity of established arthritis.
Th17 cells play an important role in CIA (4). Our
study is the first to show a decrease in the number of
Th17 cells after tolerogenic DC therapy in mice with
established CIA. However, tolerogenic DC treatment
did not reduce the proportion of IL-17–producing ␥/␦ T
3663
cells. Because ␥/␦ T cells are known to exacerbate the
severity of CIA (11,12), a different strategy may therefore be needed to inhibit this pathogenic T cell subset.
Previous studies focused on the ability of tolerogenic DCs to inhibit Th1 cell responses (15,17–19,30). In
contrast, we observed an improvement in arthritis without an effect on Th1 cell responses. We did, however,
also observe a significant increase in IFN␥ production by
CD8⫹ T cells. Interestingly, there is some evidence for
a regulatory role of CD8⫹ T cells in arthritis. CD8knockout mice are more susceptible to a second induction of arthritis after remission of disease (37), and type
II collagen–specific CD8⫹ T cell hybridomas inhibited
established disease in a CIA model (38). It has also been
shown that CIA develops more readily in IFN␥
receptor–knockout mice, and that IFN␥ inhibits the
development of Th17 cells from naive precursor T cells
(39,40). Therefore, IFN␥-producing CD8⫹ T cells could
contribute to the inhibition of CIA progression by
decreasing de novo induction of Th17 cells.
Several studies have shown that prophylactic
treatment with tolerogenic DCs (e.g., IL-4–transduced
DCs and TNF-treated DCs) is associated with a reduced
type II collagen–specific IgG2a:IgG1 ratio, indicative of
a switch from a Th1 cell–driven toward a Th2 cell–driven
type II collagen–specific immune response (17,18). In
contrast, we did not observe such a change in the type II
collagen–specific antibody isotype ratio after tolerogenic
DC treatment. A possible explanation for this discrepancy is that our tolerogenic DCs had no inhibitory effect
on Th1 cells. Salazar et al have reported similar results
in this respect. The tolerogenic DCs used by those
investigators, which were generated by short-term LPS
treatment, also inhibited established arthritis without
changing the IgG2a:IgG1 ratio; however, their tolerogenic DC treatment did inhibit IFN␥ production (30).
Earlier studies of anti-TNF treatment of established
arthritis also did not show a change in the IgG2a:IgG1
ratio (41,42). It could therefore be contended that a
reduction of type II collagen–specific IgG2a is important
for the prevention of arthritis but may be of less
importance after disease onset.
We have clearly shown that for tolerogenic DCs
to be effective, it is necessary to pulse them with type II
collagen, suggesting that the targeting of type II
collagen–specific T cells by tolerogenic DCs is important
for their therapeutic effect. This is consistent with results
published by Salazar et al (30) but in contrast to the
results of other studies using IL-4–transduced DCs
(15,16). A possible explanation for this discrepancy is
3664
that the IL-4–transduced DCs were not matured with
LPS. Because immature DCs are likely to have higher
endocytic capacity than mature DCs, it is possible that
these DCs were effective at taking up relevant antigen in
vivo (43).
It has been shown previously that after DCs are
injected intravenously, they migrate to the spleen,
liver, and lungs (44). In the setting of inflammation, as in
CIA, DCs are likely to migrate to draining LNs as well
(15). Here, we show that intravenously injected DCs can
also migrate to arthritic feet. TNF␣ and IL-1␤, which
are major mediators of chronic inflammation, could play
a role in recruiting DCs to arthritic feet. These cytokines
increase the expression of cellular adhesion molecules,
enhancing leukocyte–endothelial cell interactions (45).
As expected, because both tolerogenic DCs and mature
DCs can modulate disease severity, we did not observe a
difference in migratory capacity between these DC
types. Future studies, tracking tolerogenic DCs in vivo in
real time, will determine in more detail the location of
tolerogenic DCs within arthritic feet and other tissues, as
well as their interactions with other immune cells. Such
studies would be helpful for elucidating where and how
tolerogenic DCs exert their inhibitory action(s).
Because vaccination with 1 ⫻ 106 tolerogenic
DCs was not sufficient to cure disease, we increased the
dose of tolerogenic DCs to 2.5 ⫻ 106 per vaccination, a
dose that is sufficient to prevent CIA using TNF-treated
tolerogenic DCs (18–20). However, there is a risk involved in increasing the dose of tolerogenic DCs: another study showed that a dose of 2.5 ⫻ 106 TNF-treated
DCs was pathogenic (22). Although our study shows that
increasing the dose of tolerogenic DCs does not enhance
the therapeutic effect, no adverse effects of the higher
dose were observed, indicating that our dexamethasone/
vitamin D3–treated tolerogenic DCs are safe to use even
at higher doses. The data shown in Table 1 demonstrate
that, based on the various doses, routes, and number of
injections tested, 3 intravenous injections with 1 ⫻ 106
tolerogenic DCs was the optimal tolerogenic DC treatment regimen. However, data from other studies indicate that optimal treatment regimens are different for
other types of tolerogenic DCs (15,16,23,30).
In conclusion, this study is the first to show that
the therapeutic effect of pharmacologically modified
tolerogenic DCs in CIA requires pulsing with type II
collagen and is associated with a decrease in the number
of Th17 cells and an increase in the number of IL-10–
producing T cells in arthritic mice.
STOOP ET AL
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Hilkens 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 conception and design. Stoop, von Delwig, Isaacs, Robinson,
Hilkens.
Acquisition of data. Stoop, Harry.
Analysis and interpretation of data. Stoop, Robinson, Hilkens.
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