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Keywords:

  • Collagen-induced arthritis;
  • Forkhead box P3;
  • Rheumatoid arthritis;
  • Semi-mature DC;
  • Treg

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Semi-mature DC (smDC) have been shown to be tolerogenic and thus applicable to the treatment of autoimmune disease. However, in our repeated experiments, even the same batches of smDC were found to be profoundly immunogenic rather than tolerogenic when inoculated at high doses into arthritic mice. In a cytokine chip assay, smDC were characterized by remarkable production of IL-2, IL-3, IL-5, and IL-13 together with well-known Th2 cytokines. Low doses (2×105) of smDC showed excellent anti-arthritic activity in collagen-induced arthritis animals, whereas high doses (2×106) of smDC uniformly accelerated arthritic symptoms. SmDC, vaccinated at lower doses, markedly induced forkhead box P3 Treg, Th2 cytokines (IL-4/IL-10), and TGF-â in their immune deviation. Interestingly, however, as the number of smDC increased from 2×105 to 2×106 in the same assay, the Treg population, Th2 cytokines, and TGF-β were dramatically reduced. Our present study clearly indicates that smDC could induce either T-cell tolerance or T-cell activation, depending on the inoculum size. Special attention should be paid to the optimal range of smDC in DC-mediated immunotherapy for the treatment of rheumatoid arthritis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

DC are established T-cell immunity inducers and are also being viewed increasingly as T-cell tolerance mediators 1–3. They reside within peripheral tissues and secondary lymphoid organs at an immature stage. The natural function of immature DC (iDC) is to create conditions for self-tolerance either via the generation of Treg or via the induction of apoptosis or anergy of autoreactive effector cells 4, 5. Moreover, some reports have demonstrated that tolerance is observed when partial- or semi-maturation of DC (semi-mature DC, smDC) occurs, whereas only fully matured DC (mDC) are immunogenic 6, 7. It has also been reported that in vitro-generated smDC, which were matured by TNF-α and intravenously injected into mice, functioned in a tolerogenic fashion via the prevention of Th1-dependent EAE, by inducing CD4+ Treg 8.

CD4+CD25+ Treg have emerged as a unique population of suppressor T cells, which maintain peripheral immune tolerance 9, 10. Recently, the Foxp3 (forkhead box P3) transcription factor has been reported to be expressed specifically in naturally occurring CD4+CD25+ Treg, thereby distinguishing them from activated CD4+ T cells, which also express CD25 after activation 11. Using Foxp3 as a specific molecular marker for the detection and manipulation of naturally occurring Treg, an accumulating body of evidence has shown that the CD4+CD25+Foxp3+ Treg population is engaged actively in the negative control of a variety of physiological and pathological immune responses and can be exploited not only for the prevention or treatment of autoimmune diseases but also for the induction of immunological tolerance to non-self antigens (transplantation tolerance) and the negative control of aberrant immune responses (allergy and immunopathology) 12. It has also been reported that TGF-β not only inhibits the proliferation of T cells 13 but also blocks the differentiation of both CD4+ and CD8+ naïve T cells into effector cells 14–16. Moreover, TGF-β has been demonstrated to be essential for the induction and maintenance of murine and human CD4+CD25+ Treg in the periphery 17–21. Additionally, a cell-surface form of TGF-â appears to be important for the functions of Treg 22–24.

On the other hand, the majority of previous reports have shown that the maturation stage of DC, depending on the culture conditions/cytokine environments, and/or the nature/concentration of antigens, are likely to determine the immunogenic and tolerogenic fates of DC 2–7, 25, 26. However, no study has yet addressed the question of the fate of DC with regard to the density of DC in both in vitro and in vivo contexts.

This study is, to the best of our knowledge, the first to determine that smDC, only at low density, prominently induced the Foxp3+ Treg population and immunosuppressive cytokine secretion when co-cultured with naïve T cells and evidenced excellent anti-arthritic activity in collagen-induced arthritis (CIA) animals. However, even an identical batch of smDC was found to be profoundly immunogenic with a reverse propensity (accelerating the arthritic symptom) when vaccinated with a higher smDC density. Our results strongly indicate that the density of DC is another important factor for the efficacy of smDC-mediated immunotherapy.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

smDC induce Treg population and Th2 cytokines

On the basis of a previous report demonstrating that the TNF-α-modulated smDC has the capacity to prevent EAE in C57BL/6 mice 8, we first assessed the immune responses induced by smDC in vitro.

In this study, manufactured CD11c+ smDC was very pure and well characterized by the lower expression of co-stimulatory molecules CD80/CD86/CD40 (as compared with mDC) (Fig. 1A) and by the remarkable production of Th2 cytokines (IL-3, IL-13, IL-5, IL-4, and IL-10) (as compared with mDC and iDC) (Fig. 1B). In the phagocytic analysis with PE-conjugated latex bead, smDC and mDC showed markedly reduced phagocytic capacity as compared with that of iDC (Fig. 1C). The smDC efficiently induced Th2 immunity and Treg population in co-culture experiments (Fig. 2). When co-cultured with smDC as stimulators, CD4+ T cells were inclined to generate Th2 cytokines (IL-4 and IL-10) and TGF-β, as previously reported 17, rather than the Th1 cytokine (IFN-γ), whereas mDC were inclined to induce a Th1 profile in the cytokine expression (Fig. 2A). In the case of IL-17, secreted by unique T-cell population Th17 27, 28, smDC were clearly less active in stimulating Th17 cells than mDC in co-cultured experiments (Fig. 2A). In addition, the CD4+ T-cell inclination toward Th2 by smDC was more obvious at a 1/10 than at a 1/5 DC/T-cell ratio in the co-culture experiments (Fig. 2A). We also examined the Treg generated via the co-culture experiments. As shown in Fig. 2B, substantial quantities of Treg were detected in the co-culture when stimulated with a low dose (2×105 cells) of smDC, whereas the Treg population was in the basal range when co-cultured with identical doses of mDC. In identical co-culture experiments with smDC, the Treg population increased more efficiently at a 1/10 ratio in the smDC/CD4+ T cell (or smDC/splenocytes) mixture than at a ratio of 1/5 (Fig. 2B). Furthermore, the Treg population was not increased at all when co-cultured with a high dose (2×106 cells) of smDC, even at a 1/10 DC/T-cell mixture ratio (Fig. 2B). These results imply that the smDC have a potential to induce tolerance only when vaccinated at low doses.

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Figure 1. Characterization of smDC. SmDC, iDC, and mDC were generated as described in the Materials and methods. (A) Directly conjugated antibodies were used to stain cells for flow cytometric analysis. Numerical values on the histogram were calculated based on the unstained isotype control. Data are representative of ten experiments. (B) Cytokine expression profiles of smDC. DC were cultured in the absence of cytokines (GM-CSF and TNF- α) for 24 h; after incubation, the supernatant of DC cultures was harvested and analyzed for cytokines using the Beadlyte Mouse 21-Plex Cytokine Detection System. Data are mean±SD obtained from single experiments performed in triplicate. (C) For assessment of phagocytic activity, DC were pulsed with PE-conjugated latex bead at 37°C for 90 min, followed by flow cytometric analysis. Data shown are representative of at least two independent experiments.

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Figure 2. Immunosuppressive characteristics of smDC. CD4+ T cells were isolated from splenocytes using a CD4 MicroBead mouse kit. Splenocytes or CD4+ T cells were co-cultured with collagen-pulsed smDC or mDC at 37°C for 72 h, at stimulator:responder ratios of 1:5 or 1:10. Cells were harvested for Treg analysis and cytokine detection by ELISA. (A) Quantitative analysis of Th1 cytokine IFN-γ and Th2 cytokine IL-4/IL-10 levels was conducted via ELISA on culture supernatants from 72 h-co-cultures. Additionally, TGF-β and IL-17 levels were also evaluated via ELISA in the samples above. Data shown are mean±SD of triplicates. *p<0.01 compared with mDC (Mann–Whitney's U test). (B) Cells from co-cultures in (A) were stained with Foxp3 and CD25 for Treg analysis. Data shown in (A) and (B) are representative of five experiments.

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Low dose of smDC evidenced anti-arthritic activity while a high dose of smDC accelerated arthritis

The CIA model of arthritis is a well-established method for the evaluation of therapeutic interventions in cases of autoimmune arthritis. The incidence of the onset of chicken type II collagen (CII)-induced arthritis was almost 100% in the untreated groups. We first determined a therapeutically optimal concentration of smDC via smDC treatment and then measured the footpad thickness and arthritic index of the CIA-inducing mice, as described in the Materials and methods. In the present experiments, we injected CIA-inducing mice with two different doses of smDC (2×105 or 2×106 cells) and CII-unloaded smDC. Repeated experiments have demonstrated that the treatment of CIA-inducing mice with a low dose of smDC abrogates the onset of arthritis (Fig. 3). Arthritic symptoms were not observed at all in the animals injected with 2×105 smDC, while arthritic symptoms were accelerated in the mice vaccinated with 2×106 smDC, and the severity of the symptoms was similar to that observed in the untreated control CIA mice (Fig. 3A and C). Mice vaccinated with 2×105 smDC after CIA-induction kept arthritis scores in the normal range, as compared with what was observed in the 2×106 smDC-vaccinated mice or the untreated control CIA mice after 2 months (Fig. 3B). However, arthritic symptoms in the mice vaccinated with 2×105 CII-unloaded smDC were similar to those observed in the untreated control CIA mice (Fig. 3B). As a next step, we further assessed histological differences in the smDC-vaccinated and -untreated CIA mice. The mice vaccinated with 2×105 smDC evidenced no rheumatoid arthritis (RA) histopathology (Fig. 4C). In contrast, 2×106 smDC-vaccinated (Fig. 4D) and untreated control CIA mice (Fig. 4B) evidenced marked infiltrations of inflammatory cells (black arrows) and clear pannus formation (white arrows). These results were consistent with the data obtained in the in vitro experiments (Fig. 2) and imply that smDC are very potent for the inhibition of RA progression when vaccinated with an appropriate (relatively lower) dosage.

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Figure 3. Vaccination with a low dose of smDC suppressed CIA development in mice. See the Materials and methods for details on the induction of CIA. Mice were divided into seven groups (n=8–12 for each group); mice were vaccinated with 2×105 smDC (CII-loaded), 2×106 smDC (CII-loaded), 2×105 mDC (CII-loaded), 2×105 CII-unpulsed smDC, 2×106 CII-unpulsed smDC, and unvaccinated, and a control group of naïve mice. Twenty-one days after first CII-inoculation, animals were vaccinated (injected subcutaneously into the abdominal area) with smDC. (A) The footpad thickness of each mouse was measured twice a week. Data show mean footpad thickness±SD of eight mice per group. *p<0.01 compared with CIA/2×106 smDC (Mann–Whitney's U test). (B) Disease severity of the CIA mice was visually evaluated twice a week between days 27 and 61 by two blinded observers, who utilized a three-point scale for each paw. The arthritic severity was expressed as a mean arthritis index on a 0–3 scale (0, normal joint; 1, slight inflammation and redness; 2, severe erythematic and swelling affecting the entire paw; and 3, deformed paw or joint, with ankylosis, joint rigidity, and loss of function). The total score for clinical disease activity was based on all four paws, with a maximum score of 12 for each mouse. Data show mean arthritis score±SD of eight mice per group. (C) Mice from each group were selected randomly and photographed around the paw region using a digital camera. Data shown are representative of at least eight experiments.

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Figure 4. Histological analysis of the hind paws. (A–D) For histological studies, animals were sacrificed on day 41. Hind paws were removed from mice in all groups and fixed for 2 days in 10% phosphate-buffered formalin. Thereafter, fixed samples were decalcified for 18 days in 10% formic acid, dehydrated, and embedded in paraffin blocks. Sections (5 μm) were cut along a longitudinal axis, mounted, and stained with hematoxylin and eosin. The black and white arrows indicate inflammatory cell infiltration and pannus formation, respectively (original magnification ×200). The satellite pictures show toe swelling in each mouse (original magnification ×40). SY: synovial membrane. Data shown are representative of at least three independent experiments.

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Vaccination with a low dose of smDC induced Treg population and Th2 cytokines

Next, we assessed the Treg population in the CIA mice following vaccination with low doses of smDC. As shown in Fig. 5A, CD4+CD25+Foxp3+ Treg increased markedly in the spleens of mice vaccinated with 2×105 smDC, whereas this was not observed in the control CIA mice or 2×106 smDC-vaccinated ones. Additionally, Th1 cytokine IFN-γ and Th2 or the immune-suppressive cytokines IL-4/IL-10/TGF-β decreased and increased by significant levels, respectively, in the 2×105 smDC-vaccinated mice, whereas those cytokine expression profiles were reversed completely in CIA control mice and 2×106 smDC-vaccinated mice (Fig. 5B). These results are consistent with the data shown above (Figs. 3 and 4).

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Figure 5. Vaccination with a low dose of smDC significantly enhanced Treg population and Th2 immunity in mice. For evaluation of immune status of smDC-vaccinated CIA mice, CD4+ T cells, isolated from the spleen of each mouse vaccinated with a low (2×105) or high (2×106) dose of collagen-pulsed smDC, were cultured in the presence of collagen (50 μg/mL) for 48 h, and the Treg population (A) and IFN-γ/IL-4/IL-10/TGF-β secretion (B) were analyzed by flow cytometry and ELISA, respectively. Data show mean±SD of triplicate samples. *p<0.01 compared with CIA (Mann–Whitney's U test). Data shown in (A) and (B) are representative of three experiments.

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Density (number) is likely an important factor in determining smDC-induced immune deviation

Finally, in order to elucidate the correlation between the number of smDC and the induced Treg population, co-culture experiments were conducted with varying numbers of smDC (2×105–2×106 cells). As shown in Fig. 6A, the Foxp3+CD25+ Treg population increased in reverse proportion to the number of smDC between 2×106 and 2×105 in the co-culture experiments. This result was further supported by the abundant existences of immunosuppressive cytokines, IL-10 and IL-4, in the supernatant of the co-cultures with 2×105 smDC (Fig. 6A, lower panel). Another interesting point is that the population of CD80+CD86+ cells (probably involved in T-cell immunity rather than tolerance) increased gradually as the number of smDC increased in the co-culture experiments (Fig. 6B). These results show that the density (number) of smDC is another important factor in determining the fate of smDC-mediated immune responses, specifically toward either immunogenic or tolerogenic fates.

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Figure 6. Correlations between the number of smDC and induced Treg (A) and CD80+CD86+ DC (B) populations in the co-culture with smDC and naïve CD4+ T cells. Varying numbers (2×105–2×106) of smDC were co-cultured with 1×107 CD4+ T cells for 72 h. (A) CD25+FoxP3+ Treg were assessed by flow cytometry after co-culture. IL-10 and IL-4 were also assessed from the supernatant of the co-cultures by ELISA. (B) After co-culture of smDC with splenic CD4+ T cells, the expression of B7 molecules (CD80 and CD86) was assessed on the surface of smDC. Data shown in (A) and (B) are representative of three experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Thus far, DC-based immunotherapy has been primarily a consideration in the field of cancer therapy 29. However, some reports have demonstrated that DC are able to induce not only immunity but also immune tolerance, depending on the stage of their maturation. In fact, some papers have demonstrated that certain types of DC have potential for the treatment of autoimmune diseases 6, 7, 30. Our results also indicated that TNF-α-mediated smDC might be applicable to the RA treatment at least in animal models, and this claim was further supported by our detailed analysis of smDC-mediated immune reactions both in vitro and in vivo.

smDC was well characterized by the lower expression of co-stimulatory molecules CD80/CD86/CD40 as compared with mDC (Fig. 1A). However, the level of PD-L1/2 (regulatory surface marker) was not discernible between smDC and mDC, and the level of Gr-1 (myeloid suppressor cell marker) was at an undetectable range in both populations (data not shown). In a cytokine chip assay, smDC were specially characterized by the remarkable expression of IL-3, IL-5, and IL-13 together with well-known Th2 cytokines, IL-4 and IL-10, as compared with mDC and iDC, thus implying that smDC were obviously tolerogenic rather than immunogenic (Fig. 1B). It is worth noting that smDC produced quite large amounts of IL-2 as compared with mDC and iDC (Fig. 1B), probably reasoning that the CD4+CD25+Foxp3+ Treg population was significantly augmented by smDC, possibly associated with the IL-2 receptor CD25.

As expected, CII-pulsed smDC was very effective to treat the arthritis when delivered subcutaneously into CIA mice (Figs. 3 and 4). However, CII-unloaded smDC did not show any anti-arthritic activity in CIA mice. These results indicate that the anti-arthritic activity of smDC is an antigen-specific mode of action rather than a non-specific general suppression. In parallel with this therapeutic effect, smDC vaccination significantly increased CD4+CD25+Foxp3+ Treg populations, as well as Th2 and immunosuppressive cytokines, in CIA mice (Fig. 5). Recent studies have addressed that IL-17-producing Th17 cells play an essential role in the induction of autoimmune arthritis 31, 32. In the present study, CD4+ T cells co-cultured with mDC produced quite large amounts of IL-17 while those co-cultured with smDC did not (Fig. 2A), implying that suppression of arthritis by smDC vaccination is likely, to some extent, due to the smDC-mediated Th17 inhibition. These results imply that smDC vaccination is quite functional for the induction of immune tolerance, thereby resulting in the inhibition of disease progression in RA pathogenesis.

However, we found another interesting point in terms of the inoculation size (number) of smDC and the fate of immune reactions. In our repeated experiments, smDC evidenced potent anti-arthritic activity in CIA mice only when inoculated with low doses (2×105/mice) of smDC. However, anti-arthritic smDC activity was not detected in CIA mice when inoculated with high doses of smDC. Arthritic symptoms in the mice vaccinated with 2×106 smDC were rather accelerated, and the severity was getting worse than that observed in the untreated control CIA mice with no exception (Figs. 3 and 4). These results were further supported by the data resulting from a series of immunological analysis. Vaccination with 2×105 smDC markedly induced the CD4+CD25+Foxp3+ Treg population in the spleens of CIA mice (Fig. 5). In Fig. 5A, the large populations of Foxp3+ CD25- cells (the lower right section of the FACS data) seemed to be a natural or adaptive Treg population that has nothing to do with type II collagen (CII) in its antigen specificity, so that it remains inactive (CD25-) even after activation of CD4+ T cells with type II collagen. It was reported that the Foxp3 expression was also detected in CD25low or negative CD4+ T cells under certain conditions 33. The Th2 cytokines IL-4/IL-10 and the immunosuppressive TGF-β were also induced prominently in the culture supernatant of the splenocytes acquired from the 2×105 smDC-vaccinated mice. However, those phenomena observed in the 2×105 smDC-vaccinated mice were not observed in the 2×106 smDC-vaccinated mice (Fig. 5). It was elucidated via in vitro co-culture experiments with varying numbers of smDC and a fixed number of CD4+ T cells that the fate of smDC-mediated immune reactions between these opposite propensities is dependent on the density (or number) of smDC (Fig. 6). This is, to the best of our knowledge, the first study to demonstrate that the density (number) of smDC is another important factor in the determination of the fate of DC-mediated immune reactions. Our finding that smDC at high densities are immunogenic would be explained as follows; DC in the semi-mature stage (smDC) might be further maturated via vivid DC-DC interactions at high densities in the presence of T cells after injection, thereby resulting in the adoption of immunogenic, rather than tolerogenic qualities. It has been established that DC acquire antigens shed from other cells and, particularly, from other DC 34, 35. However, we could not detect any surface phenotype changes of smDC even in the high density of smDC before co-culturing with CD4+ T cells or inoculation into CIA mice. The capacity of DC both to shed antigens and to acquire shed antigens allows for effective antigen transfer between DC, thereby suggesting that high densities of smDC may possibly stimulate each other, followed by the induction of immunity rather than tolerance in the immune system environment.

In conclusion, our current results clearly demonstrate that smDC are potentially applicable to RA immunotherapy via the induction of T-cell tolerance. However, even identical batches of smDC become immunogenic when treated at high dosages. This means that smDC cannot be applied to the treatment of autoimmune arthritis until the optimal number of smDC can be determined. Special attention should be paid to smDC-based RA immunotherapy with regard to the number or inocula of smDC.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Mice and reagents

Pathogen-free female DBA/1 mice, 6–7 wk of age, were purchased from Orient Bio (Gyeonggi, Republic of Korea) and maintained in the Animal Maintenance Facility of the CreaGene Research Institute (Gyeonggi, Republic of Korea). Five or six mice were housed per cage under standard temperature and light conditions and were fed on standard laboratory chow and water ad libitum. All experiments with mice were conducted in accordance with local animal ethics guidelines. Recombinant mouse (rm) TNF-α was purchased from R&D Systems (Abington, OX, UK), and rmGM-CSF and rmIL-4 were purchased from CreaGene (Gyeonggi, Republic of Korea). CFA, incomplete Freund's adjuvant, collagen (type II collagen, chicken), and LPS were obtained from Sigma-Aldrich (St Louis, MO, USA). The culture medium utilized was RPMI 1640 (GIBCO Laboratories, Grand Island, NY, USA) supplemented with 10% FBS (GIBCO Laboratories), 50 μM 2-mercaptoethanol (Life technologies, Gaithersburg, MD, USA), 50 μg/mL streptomycin, 50 U/mL penicillin, and 25 μg/mL amphotericin B (GIBCO Laboratories). For immunofluorescence staining, PE-conjugated anti-mouse CD25, CD11c, and CD8 and FITC-conjugated anti-mouse MHC class II, CD80, CD86, CD40, CD54, CD14, CD4, CD19, and CD3 were purchased from BD Pharmingen (San Diego, CA, USA). FITC-conjugated anti-mouse Foxp3 was purchased from eBioscience (San Diego, CA, USA). For the assessment of DC phagocytosis, PE-conjugated latex bead was purchased from Sigma-Aldrich.

DC generation

DC were generated from BM progenitors of DBA/1 mice as described previously with some modifications 36. In brief, BM cell suspensions were cultured in bacterial plates in the presence of 20 ng/mL of GM-CSF and 2 ng/mL of IL-4. At day 3, non-adherent cells were washed and re-fed in the same culture condition. As at days 6 and 8, 50% of the culture medium was replaced with fresh medium containing the same concentrations of GM-CSF/IL-4. After 10 days of culture, the smDC and mDC were generated by additional incubation with 500 U/mL of TNF-α and 50 μg/mL of collagen for 4 h, and with 1 μg/mL of LPS and 50 μg/mL of collagen for 24 h, respectively.

Co-cultures with smDC

Splenocytes were isolated from the spleens of DBA/1 mice and disaggregated into RPMI 1640 medium. Additionally, CD4+ T cells were isolated from splenocytes using a CD4 MicroBead mouse kit (Miltenyi Biotec, Auburn, CA, USA). In brief, CD4+ T cells were separated by passing the cell suspension over a magnetic-activated cell sorter MS column held in a MACS magnetic separator (Miltenyi Biotec). The CD4+ T cells adhering to the column were then utilized for this assay. Splenocytes or purified CD4+ T cells were employed as responders (1×106/well, 2×106/well, 1×107/well, and 2×107/well). Collagen-pulsed smDC (2×105/well and 2×106/well) or mDC (2×105/well and 2×106/well) were added as stimulators into plates containing responder cells. The co-culture was conducted at a ratio of 1 stimulator to 5 or 10 responders (2×105 stimulators: 1×106 or 2×106 responders, 2×106 stimulators: 1×107 or 2×107 responders). The mixed cells were cultured for 72 h at 37°C in 2 mL of RPMI 1640 supplemented with 10% FBS. Finally, the cells were harvested in order to measure the Treg population and the culture supernatants were collected for cytokine ELISA. However, in order to investigate changes in the Treg population and the extent of DC maturation (by using CD80 and CD86 markers) according to the change of DC numbers in response to CD4+ T cells, 2×105–2×106 smDC were co-cultured for 72 h with 1×107 CD4+ T cells.

FACS analysis

For phenotypic analysis, direct immunofluorescence was utilized for cell surface staining of smDC, which were stained in FACS buffer (0.2 BSA, 0.02 sodium azide in PBS) at 1×105 cells per staining. Antibody incubation was conducted for 20 min at 4°C. Data were reported as a histogram or dot plot using FACSCalibur (BD Bioscience, Mountain View, CA, USA) with CellQuest software. The cells were gated in accordance with their forward and side-scattering patterns. For each marker, 104 cells were counted in the gate. Additionally, FITC-conjugated Foxp3 and PE-conjugated CD25 markers were utilized in order to determine the Treg population. CD80+CD86+ DC were assessed among CD11c+ DC isolated by use of the Dyna Beads Mouse DC Enrichment kit (Invitrogen Dynal, Oslo, Norway). For the assessment of DC phagocytosis, cells (2×105 cells) were pulsed with PE-latex bead at 37°C for 90 min, and then the cells were washed with FACS buffer and analyzed by FACScan.

Cytokine assays

For characteristic analysis of smDC, cytokine assays were performed using the Beadlyte Mouse 21-Plex Cytokine Detection System (Upstate Biotechnology, Lake Placid, NY, USA) according to the manufacturer's instruction. Briefly, DC (1×106 cells/2 mL) generated were cultured in the absence of cytokines (GM-CSF and TNF- α) for 24 h. After incubation, the supernatant of DC cultures was harvested and then incubated for 2 h with the Beadlyte Mouse 21-Plex Multi-Cytokine beads in individual wells of a 96-well plate. Each sample was plated in duplicate. Next, 25 μL Beadlyte Mouse 21-Plex Multi-Cytokine biotin was added to each well and the plate was incubated for 1.5 h. Diluted Beadlyte Streptavidin–Phycoerythrin was added to each well (25 μL per well), incubated for 30 min, and the plate was then read on a Luminex xMAP 100 analyser (Upstate Biotechnology). The concentration of cytokine was extrapolated from a standard curve using BeadView Data Analysis software (Upstate Biotechnology).

Evaluation of Th1/Th2/Th17 response

Th1 cytokine IFN-γ, Th2 cytokine IL-4/IL-10, and Th17 cytokine IL-17 levels were analyzed via ELISA on supernatants from 72 h-co-cultures using 2×105 DC and CD4+ T cells (per 2 mL). Additionally, quantitative analyses of TGF-β levels were conducted via ELISA on the samples described above. Commercially available ELISA kits (R&D system) were utilized as indicated by the manufacturer.

Induction of arthritis in DBA/1 mice

Arthritis was induced as reported previously 37. In brief, 6-wk-old female DBA/1 mice were immunized into the basal region of the tail with 200 μg of CII dissolved in 100 μL of 0.05 M acetic acid and mixed with an equal volume (100 μL) of CFA. Collagen (2 mg/mL) was dissolved by stirring overnight at 4°C. On day 21, mice were boosted with a subcutaneous injection of CII mixed in incomplete Freund's adjuvant. The booster injection was necessary to induce reproducible CIA, which normally developed at approximately day 34.

Animal experiments

The experimental mice were divided into seven groups of CIA mice; CIA mice vaccinated with 2×105 CII-pulsed smDC, with 2×106 CII-pulsed smDC, with 2×105 CII-pulsed mDC, with 2×105 CII-unpulsed smDC, with 2×106 CII-unpulsed smDC, unvaccinated CIA mice, and a control group of naïve mice. CIA DBA/1 mice were vaccinated with DC (subcutaneously injected in the abdominal area) at days 21 and 31 after collagen immunization. For histological studies and immune status evaluation, animals were sacrificed on day 41 via cervical dislocation. Additionally, for immune status evaluations, CD4+ T cells isolated from the spleens of mice were cultured for 48 h in the presence of 50 μg/mL of collagen, and the Treg population from the cell pellets and IFN-γ/IL-4/IL-10/TGF-β secretion from the culture supernatant were determined via FACS analysis and ELISA, respectively.

Arthritic severity and histopathology

The disease activity of the CIA was visually assessed twice a week, between days 27 and 61, by two blinded observers using a three-point scale for each paw. The severity of arthritis was expressed on a mean arthritis index graded on a scale of 0–3 (0, normal joint; 1, slight inflammation and redness; 2, severe erythema and swelling affecting the entire paw; and 3, deformed paw or joint, with ankylosis, joint rigidity, and loss of function). The total clinical disease activity score was based on all four paws, with a maximum score of 12 for each mouse 38. Additionally, footpad thickness was measured twice a week with a caliper. For histological studies, the hind paws were removed from mice in all groups on day 41 and fixed for 2 days in 10% phosphate-buffered formalin. Thereafter, fixed samples were decalcified for 18 days in 10% formic acid, dehydrated, and embedded in paraffin blocks. Sections (5 μm) were cut along a longitudinal axis, mounted, and stained with hematoxylin and eosin as described previously 39.

Statistical analysis

The results were expressed as the mean±SD. Mann–Whitney's U test was utilized for all statistical analyses. A p-value of less than 0.05 was considered to be significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

We are grateful to Michael S. Rabaa for his kind editorial assistance. This work was supported by a government grant (S0703222-E0841750-10000013) from The Ministry of Korean Small and Medium Business Administration.

Conflict of interest: The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
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