Therapeutic effect of tolerogenic dendritic cells in established collagen-induced arthritis is associated with a reduction in Th17 responses.код для вставкиСкачать
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: firstname.lastname@example.org. 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. REFERENCES 1. Luross JA, Williams NA. The genetic and immunopathological processes underlying collagen-induced arthritis [review]. Immunology 2001;103:407–16. 2. Kremer JM, Westhovens R, Leon M, Di Giorgio E, Alten R, Steinfeld S, et al. Treatment of rheumatoid arthritis by selective inhibition of T-cell activation with fusion protein CTLA4Ig. N Engl J Med 2003;349:1907–15. 3. Chiang EY, Kolumam GA, Yu X, Francesco M, Ivelja S, Peng I, et al. 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