April 2003
Volume 44, Issue 4
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Immunology and Microbiology  |   April 2003
Antigen-Specific Inhibition of Experimental Autoimmune Uveoretinitis by Bone Marrow-Derived Immature Dendritic Cells
Author Affiliations
  • Hui-Rong Jiang
    From the Department of Ophthalmology, University of Aberdeen Medical School, Foresterhill, Aberdeen, Scotland, United Kingdom.
  • Elizabeth Muckersie
    From the Department of Ophthalmology, University of Aberdeen Medical School, Foresterhill, Aberdeen, Scotland, United Kingdom.
  • Marie Robertson
    From the Department of Ophthalmology, University of Aberdeen Medical School, Foresterhill, Aberdeen, Scotland, United Kingdom.
  • John V. Forrester
    From the Department of Ophthalmology, University of Aberdeen Medical School, Foresterhill, Aberdeen, Scotland, United Kingdom.
Investigative Ophthalmology & Visual Science April 2003, Vol.44, 1598-1607. doi:10.1167/iovs.02-0427
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      Hui-Rong Jiang, Elizabeth Muckersie, Marie Robertson, John V. Forrester; Antigen-Specific Inhibition of Experimental Autoimmune Uveoretinitis by Bone Marrow-Derived Immature Dendritic Cells. Invest. Ophthalmol. Vis. Sci. 2003;44(4):1598-1607. doi: 10.1167/iovs.02-0427.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To investigate the effect of maturation status of bone marrow-derived dendritic cells (BMDCs) on the in vivo immune response to interphotoreceptor retinoid-binding protein (IRBP) 161-180 peptide in experimental autoimmune uveoretinitis (EAU).

methods. Immature and mature BMDCs were generated without or with the stimulation by lipopolysaccharide (LPS), and their mRNA cytokine profile and phenotype were analyzed by RNase protection assay and flow cytometry. The effect of immature and mature DCs in inducing antigen-specific T-cell proliferation and cytokine profile was further investigated in an IRBP peptide-induced model of EAU.

results. BMDCs generated in granulocyte-macrophage-colony-stimulating factor (GM-CSF) were relatively immature (i)DCs, as determined by flow cytometry and cytokine profile. However, stimulation with LPS induced these cells to become mature (m)DCs with higher levels of surface major histocompatibility complex (MHC)-II and costimulatory molecules and higher mRNA expression of IL-1α, -1β, -6, and -12. Subcutaneous administration of iDCs induced a state of relative tolerance to the peptide induced-EAU, and the effect was lost after the DCs underwent maturation induced by in vitro exposure to LPS. In vitro, both iDCs and mDCs induced typical peptide-specific T-cell proliferation, but IFN-γ production by uveitogenic T cells was markedly inhibited by iDCs. In vivo, peptide-loaded iDCs induced draining lymph node (DLN) cells to secrete a distinct pattern of cytokine: namely, increased IL-10 and IL-5 and decreased IFN-γ and IL-2, indicating an altered immune responses to a low T-helper (Th) cell type 1 profile and a high Th2 profile after uveitogenic challenge.

conclusions. The data suggest that induction of tolerance to an autoantigen by peptide-loaded DCs requires presentation of antigen by iDCs and involves the generation of a high-level IL-10 and IL-5 immune response in DLN cells.

Esperimental autoimmune uveitis (EAU) is an organ-specific, T-cell-mediated disease that targets the retina 1 and in many ways is similar to other models of autoimmunity, such as experimental autoimmune encephalomyelitis (EAE). EAU can be induced in many species with uveitogenic retinal antigens emulsified in CFA, 2 or by adoptive transfer of retina-specific syngeneic CD4+ T cells. 3 The disease of murine EAU closely resembles that of several human noninfectious uveitic diseases, and the animal model of EAU serves as an ideal model for human posterior uveitis, a major sight-threatening disease. 2 4 5 Because of shared immunopathogenic mechanisms between EAU and other tissue-specific T cell-mediated autoimmune disease models, detailed study of the mechanisms and therapeutic approaches to EAU shed light on pathogenetic mechanisms in other autoimmune diseases. EAU has, in addition, the considerable advantage that the degree of damage to the retina and the level of inflammatory cell infiltration at the site of attack (i.e., the photoreceptor) is readily apparent and that both features can be semiquantitatively evaluated by simple microscopy, because of the unique architecture of the retina. This has been shown in determining the effects of the TNF receptor fusion protein on target organ damage in EAU. 6 EAU in genetically unmanipulated animals is a Th1-mediated disease, because uveitogenic T cells show a Th1-like cytokine profile (high IFN-γ), and susceptible rodent strains typically mount a Th1-dominant response to the uveitogenic antigen. 7 8 9 The balance between Th1 and Th2 immune responses plays an important role in determining the outcome of a uveitogenic challenge. 7 8 10  
Dendritic cells (DCs) are well known as the professional antigen-presenting cells (APCs) with the capacity to stimulate naive T cells to initiate immune responses, including autoimmune diseases such as EAE 11 and EAU. 5 DCs are derived from hemopoietic stem cells in the bone marrow from which they emerge as immature precursor (i)DCs. iDCs expressing low levels of MHC-II molecules on their cell surface are able to capture particulate antigen through phagocytosis 12 and soluble antigens through macropinocytosis or receptor-mediated endocytosis. 13 iDCs require activation by stimuli that promote their maturation and migration to the T-cell areas of lymphoid tissues, where they become potent mature APCs. After maturation, major histocompatibility complex (MHC)-II molecules are delivered to the plasma membrane 14 and the expression of costimulatory molecules is increased, thus favoring T-cell activation. 15 Injection of antigen-bearing mature (m)DCs leads to rapid enhancement of CD4+ and CD8+ T-cell immunity in humans, 16 17 18 confirming the role of these cells in priming the immune response. However, recent evidence has also pointed toward a role for DCs in central and peripheral tolerance induction, as indicated by the involvement of thymic DCs in negative, but not positive, selection. 19 In vitro, treatment of DCs with IL-10 or TGF-β has been successful in inhibiting the maturation of DCs and converting them into tolerogenic cells. 20  
In the present study, we investigated the effect of the maturation status of bone marrow-derived dendritic cells (BMDCs) on the in vivo immune response to interphotoreceptor retinal-binding protein (IRBP) 161-180 peptide in EAU disease. In our study, iDCs had the capacity to inhibit completely the production of IFN-γ production by uveitogenic T cells in vitro. Moreover, that iDCs, but not mDCs, inhibited induction of EAU in vivo, and this correlated with the induction of draining lymph node (DLN) cells that secreted high levels of IL-10 and IL-5. 
Materials and Methods
Animals
Inbred male B10.RIII mice 7 to 12 weeks of age were obtained from the Biological Service Unit at the Medical School, University of Aberdeen. The procedures adopted conformed to the regulations of the Animal License Act (UK) and to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Antigens
IRBP peptide 161-180 (SGIPYIISYLHPGNTILHVD; purity >95%) was synthesized by Sigma Genosys Co. (Cambridge, UK). 
Generation of BMDCs
BMDCs were prepared by a modification of the procedure described by Inaba et al. 21 A single-cell suspension of bone marrow cells was depleted of MHC-II+ cells, B cells, and T cells by using rat anti-mouse monoclonal antibodies (mAbs) to MHC-II (clone p7/7; Serotec, Cambridge, UK), CD4 (clone GK1.5), CD8 (clone 53 to 6.7), CD45R/B220 (clone RA3-6B2; all from BD-PharMingen UK Ltd., Oxford, UK) followed by Dynabeads coated with sheep anti-rat IgG (Dynabeads; Dynal, Ltd., Merseyside, UK). The remaining cells were cultured at 7.5 × 105/mL in 12-well plates in complete RPMI-1640 (Gibco, Paisley, Scotland, UK) supplemented with 5% fetal calf serum (FCS), 2 mM l-glutamine, 50 IU/mL penicillin, 50 μg/mL streptomycin, 5 × 10−2 mM 2-mercaptoethanol (ME), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 7.5% granulocyte-macrophage-colony-stimulating factor (GM-CSF) supernatant. The GM-CSF supernatant was prepared from the Ag8653 myeloma cell line transfected with murine GM-CSF cDNA. The probe used to generate the GM-CSF-secreting cell line was kindly given by Brigitta Stockinger (Division of Molecular Immunology, National Institute for Medical Research, London, UK). 22 23 From day 2, the cultures were fed daily by gently swirling the plates, aspirating 75% of the medium, and adding fresh medium with GM-CSF. Usually, the swirling and changing medium removed nonadherent granulocytes, while clusters of developing DCs remained loosely attached on a bed of firmly adherent macrophages. Six days after the culture, nonadherent cells were removed and discarded. The loosely adherent clusters were collected, and the contaminating granulocytes were depleted with antimouse Gr-1 mAb (clone RB6-8C5, BD PharMingen) and Dynabeads (Dynal). A single-cell suspension of the remaining cells was prepared and used for further experiments. 
In Vitro Conditioning of BMDCs with Lipopolysaccharide
For in vitro conditioning, 106 cells/mL per well of purified DCs were cultured with GM-CSF-supplemented complete medium in 24-well plates (iDCs). mDCs were generated by incubating BMDCs with GM-CSF plus 1 μg/mL lipopolysaccharide (LPS; Sigma, St. Louis, MO). An additional 30 μg/mL of peptide was added to the cells, according to the experimental design, and cultured overnight. Cells were then harvested and washed for further in vitro and in vivo use. 
Immunohistochemical Staining
Cytospin preparations of BMDCs were prepared and stained for CD11c (clone HL3), MHC-II (clone p7/7), CD8α (clone 53-6.7), MOMA-2, and F4/80 (clone C1:A3-; CD8α was from BD-PharMingen, the remainder were from Serotec), by using procedures for the frozen eye sections described previously. 5 Both rabbit anti-rat biotinylated and streptavidin-alkaline phosphatase (AP) Abs were from Dako Ltd. (Cambridge, UK). The color was visualized with fast red substrate and naphthol AS-BI phosphate (Sigma) in TBS. 
Flow Cytometry
BMDC surface markers were evaluated by flow cytometry, with cells stained with the following mAbs: anti-MHC-II, anti-CD8α-FITC, anti-CD11c-FITC, anti-CD86-PE, and anti-CD40-PE (all from BD PharMingen, except anti-MHC-II Ab from Serotec). In brief, after the cells were washed, they were incubated at 4°C for 30 minutes with each mAb diluted to the optimal concentration. Cells were then washed twice and analyzed by flow cytometry (FACSCalibur with CellQuest software; BD PharMingen). Because MHC-II was a purified rat anti-mouse Ab, biotinylated secondary Ab (Dako) and streptavidin-allophycocyanin (ALPC)-conjugated Ab (BD PharMingen) were further added. Matched isotypes were used as the negative control. 
Apoptosis of T cells was assessed as previously described. 24 After staining with ALPC-conjugated CD4 and CD8 Abs, cells were further incubated with 7-annexin V and 7-amino-actinomycin D (7-AAD,ViaProbe; all four Abs were from BD PharMingen) in the binding buffer 15 minutes before analysis. Apoptotic cells were defined as AnnexinV+/7-AAD
RNase Protection Assay
Total RNA was extracted from 5 × 106 BMDCs with phenol-chloroform-guanidinium isothiocyanate 25 and quantified by determining the absorbance at 260 nm. The RNase protection assay was performed with the mCK-2 multiprobe set (nine cytokine probes, RiboQuant; BD PharMingen) according to the manufacturer’s instructions. Briefly, isolated RNA was hybridized for 17 hours at 56°C with 32P-labeled multiprobe template sets and then treated with RNase. Protected RNA fragments were resolved on polyacrylamide gels and developed on film. Precise quantification was determined by analyzing the films on computer (Gene Genius software; Syngene, Cambridge, UK). 
Treatment of Mice with Antigen-Loaded DCs
iDCs and mDCs, with or without peptide loading (30 μg/mL), were collected and washed twice with PBS, then 5 × 105 DCs in 100 μL PBS were injected subcutaneously in the neck region of the mice. Control mice received the same volume of PBS. In some experiments, injection of iDCs was repeated three times over 1 week. Nine days after the first injection, mice were immunized. On day 15 or 26 after immunization, the mice were killed and eyes removed for histologic grading of the level of retinal inflammation and damage. 
EAU Induction and Disease Evaluation
Mice were immunized subcutaneously in the back with 50 μg/50 μL IRBP peptide emulsified with an equal volume of CFA (Mycobacterium tuberculosis strain H37RA; Difco, Detroit, MI). On the day of the termination of the experiment, the mice were killed by asphyxiation in CO2, and their eyes were carefully dissected and fixed in 2.5% buffered glutaraldehyde and embedded in resin for standard hematoxylin-eosin (H-E) staining. The disease severity was scored in a masked fashion after we examined four sections of each globe cut at different levels for each eye. Severity of EAU for each eye was graded on a scale of 0 (no disease) to 4 (maximum disease) in half-point increments, according to a semiquantitative system described by Chan et al. 26 In this method, the severity of the disease is graded according to the level of inflammatory cell infiltration in conjunction with the extent of damage to the retinal neural and photoreceptor layers, with minimal disease represented by occasional inflammatory cells in the vitreous and retina with maintenance of normal retinal architecture through to severe disease, in which there is extensive loss of retinal architecture, loss and damage to photoreceptor cells, exudative retinal detachment, and the presence of granulomatous collections of inflammatory cells. 
Antigen-Activated T-Cell Enrichment and DC-T-Cell Cocultures In Vitro
B10.RIII mice were immunized as described earlier. On day 10, single cells from the inguinal lymph nodes (iLNs) were collected and incubated in flasks for 1 hour, and nonadherent cells were harvested and incubated with nylon wool for 0.5 hour. Unbound cells were collected and further incubated with CD4-MACS beads (Miltenyi Biotec, Surry, UK), which was followed by passing cells through a separation column (MiniMACS; Miltenyi Biotec) in a magnetic field. The positively selected cells were collected as T cells, and the cell purity was examined by flow cytometry (>95%). For coculture, 104 DCs were added with 2 × 105 T cells per well of 96-well plates in triplicate, with or without peptide in a proliferation assay. At the same time, 2 × 105 DCs were cultured with 4 × 106 T cells in each well of 24-well plates in the presence of 50 μg/mL peptide, and the cell-free supernatant was collected at 48 hours for measurement of IL-2 and at 96 hours for IFN-γ. 
Analysis of DC Activation of Naïve T Cells In Vivo
iDCs and mDCs loaded with peptide in vitro were injected subcutaneously into B10.RIII mice (n ≥ 3), whereas control mice received the same volume of PBS. On day 6, mice were killed, cervical lymph nodes (cLNs) were collected and pooled within each group, and triplicate cultures of 2 × 105 cells/200 μL per well were cultured in 96-well plates for assay of cell proliferation. In addition, 4 × 106 cells per well in 2 mL medium were cultured in 24-well plates together with 50 μg/mL peptide, and supernatant was collected at 48 hours measurement of IL-2 and at 96 hours for analysis of cytokine production. 
Analysis of Immune Responses after Uveitogenic Challenge
PBS or iDCs and mDCs loaded with peptide in vitro were administered in a single injection subcutaneously into B10.RIII mice (n ≥ 3). Nine days after injection, mice were immunized with IRBP peptide in CFA. At day 15 after immunization, cLNs, iLNs, and spleens were collected and pooled within each group and cultured with peptide for proliferation and cytokine production, as described. 
Lymphocyte Proliferation Assay and Cytokine Measurement
For lymphocyte proliferation, triplicate cultures of cells were incubated in 96-well round-bottomed tissue culture plates in 200 μL complete RPMI medium per well. The cultures were incubated for 90 hours at 37°C in 5% CO2 in air and were pulsed with 0.5 μCi [3H] thymidine per well during the last 16-hour incubation. 
Cytokines in culture supernatants were measured by ELISA using kits for IFN-γ and IL-2, IL-4, IL-5, and IL-10 (OptEIA; BD PharMingen). Briefly, 96-well plates were coated with the appropriate anti-cytokine Abs overnight. After the plates were blocked with bovine serum albumin and a further 2-hour incubation with supernatant or standard, the plates were developed with biotin-conjugated anti-cytokine Abs. Horseradish peroxidase-conjugated streptavidin was added before development with ELISA substrate solution (TMB; BD PharMingen). 
Statistical Analysis
Statistical analysis was performed on computer (Statistical Package for the Social Sciences; SPSS, Chicago, IL). Analysis of the EAU grades (nonparametric) was performed by Mann-Whitney test. Analysis of lymphocyte proliferation responses and cytokine production was performed by independent Student’s t-test. P < 0.05 was considered statistically significant. 
Results
Generation of Bone Marrow-Derived iDCs with GM-CSF
To achieve a high yield of DCs, we generated DCs from bone marrow in medium supplemented with GM-CSF according to the methods of Inaba et al. 21 DC clusters began to form after 24 hours of culture (Fig. 1A) , becoming larger and more numerous in the following days. Immunohistochemical staining of the purified BMDCs on a cytospin slide preparation after 6 days of culture showed that BMDCs were CD11c positive (Fig. 1B) but CD8α negative (Fig. 1C) , as confirmed by flow cytometry. MHC-II staining showed that DCs at this stage expressed intracellular MHC class II (Fig. 1D) , suggesting that they were immature DC precursors or iDCs. Some of the DCs also expressed MOMA-2 (Fig. 1E) and F4/80 (Fig. 1F) macrophage associated-antigens. 
Induction of Maturation and Activation of BMDCs by LPS
It has been shown that DCs can be induced to mature by coincubation with bacterial products such as LPS. 13 27 After the Gr-1+ cells were depleted, the purified BMDCs were cultured in a 24-well plate in GM-CSF-supplemented complete medium with 1 μg/mL LPS added to some of the wells for overnight culture. The results of flow cytometry showed that LPS in the culture led to the maturation of DCs with a significant increase of MHC-II expression and costimulatory (CD86, CD40) molecule expression (Fig. 2) . Moreover, an RNase protection assay (RPA) showed that expression of IL-12p40, IL-6, IL-1α, and IL-1β, but not macrophage migration inhibitory factor (MIF) and IL-1Rα, mRNAs were markedly increased after treatment of the DCs with LPS (Fig. 3) . Pulsing of DCs with peptide alone did not induce any significant change in expression of cell surface markers or in the profile of cytokine secretion, possibly because of the small size of the peptide (data not shown). 
Effects of iDCs on Murine EAU
To investigate the effects of peptide-loaded DCs on EAU induction in vivo, we injected DCs subcutaneously into syngeneic B10.RIII mice 10 days before immunization. Development of EAU was evaluated 2 weeks later. The histologic data show that mice pretreated with peptide-loaded iDCs showed either no evidence of disease (Fig. 4A) or significantly more focal and restricted disease (Fig. 4E) , usually at the posterior pole of the retina around the optic nerve. In the peripheral retina, less inflammation was also noted, with inflammatory cells mostly observed in the vitreous (Fig. 4C) and not at the site of antigenic target (e.g., rod outer segment, ROS), thus accounting for the considerably reduced structural damage and preservation of a normal retina. A similar phenomenon was also observed in mice protected from EAU by induction of mucosal tolerance after intranasal administration of uveitogenic antigen. 28 In contrast, severe EAU developed in eyes from the PBS group, with inflammatory cells in the vitreous fluid, retina, and ROS layer and severe structural damage (Fig. 4B) . Retinal detachment and vasculitis were also frequently observed (Figs. 4D 4F) . To summarize, our results showed that a single injection of peptide-loaded iDCs (iDC+Pep) significantly inhibited EAU, as shown by the reduced severity grade (Fig. 5B ,P < 0.05 compared with the control PBS group in Fig. 5A ), whereas the result of three injections at intervals of 2 to 3 days of peptide-loaded iDCs (over a week) further confirmed the antigen-specific protection of mice from EAU (Fig. 5C , P < 0.05 compared with the PBS group in Fig. 5A ). Mice preinjected with non-peptide-loaded iDCs also exhibited somewhat less severe signs of EAU, but the effect was not statistically significant (Figs. 5B 5C) . In contrast, LPS-treated mDCs (mDC+Pep) failed to demonstrate a tolerogenic effect on EAU and instead showed slightly more severe signs of EAU (Fig. 5D) . The protection of mice from EAU was also observed at day 26 after immunization (Fig. 5E) , which indicates the protective effect was persistent, not simply due to a delay in onset. 
Inhibition of IFN-γ Production
To investigate the mechanisms whereby the maturation status of DCs modulates this Th1-mediated immune disease, we first evaluated the ability of mature and immature BMDCs to take up peptide in vitro and their effect on the activated uveitogenic T cells. Purified BMDCs were cultured with medium alone (Fig. 6A ; iDCs−Pep), with 30 μg/mL peptide (iDCs+Pep), 1 μg/mL of LPS (mDCs-pep), 1 μg/mL of LPS+30 μg/mL peptide (mDCs+Pep) as described in the Materials and Methods Section. DCs were collected and washed extensively before they were cocultured with enriched T cells (>95%) from the DLNs of peptide/CFA-immunized mice (see the Materials and Methods section). The results show that non-peptide-loaded iDCs (iDC−Pep) failed to induce T-cell proliferation, unless peptide was present in the coculture, whereas peptide-loaded iDCs (iDC+Pep) induced T cells to proliferate in the presence or absence of additional peptide in the culture (Fig. 6A) . Similar results were also observed with mDCs, although some degree of proliferation of T cells was induced by mDCs that had not been exposed to the peptide. These data indicate that both iDCs and mDCs were equally effective in antigen uptake and presentation to T cells, although incubation of iDC or LPS stimulated-mDCs with peptide (30 μg/mL) did not change the phenotype of the cells (data not shown). In addition, both peptide-loaded iDCs and mDCs induced high levels of IL-2 production in T cells (Fig. 6B) . However, only peptide-loaded mDCs were able to induce T cells to secrete high levels of IFN-γ, whereas peptide-loaded iDCs induced minimal secretion of IFN-γ by peptide-primed T cells (Fig. 6C)
Induction of IL-5 and IL-10 by Draining Lymphocytes
To further investigate the mechanisms of inhibition of EAU by iDCs, proliferative responses and cytokine production by DLN cells from mice primed with DCs were evaluated. Mice were inoculated with peptide-loaded iDCs and mDCs or PBS. After 6 days, DLN cells were cultured in 96-well plates and assayed for proliferation and cytokine production, as described in the Materials and Methods section. The data in Figure 7A show that priming of LN cells occurred with administration of DCs (compare PBS versus both DC groups). However, mDCs induced slightly higher levels of cell proliferation than did iDCs. 
Analysis of cytokine production by lymphocytes primed with DCs indicated that iDCs promoted higher levels of IL-4, IL-5, and IL-10 secretion by DLN cells, whereas mDCs primed lymphocytes secreting slightly higher levels of IL-2 and IFN-γ but significantly lower levels of IL-10, IL-5, and IL-4. Taken together, the data show that both iDCs and mDCs primed and expanded naïve lymphocytes in vivo. However, only peptide-loaded iDCs were able to skew the cytokine profile of the lymphocytes in vivo to higher levels of IL-4, IL-5, and IL-10, and this correlated with inhibition of EAU. Analysis of T cells for levels of apoptosis in the DLNs showed that both DC-treated groups induced higher levels of apoptosis of both CD4 and CD8 T cells compared with PBS-treated control mice (data not shown), but there appeared to be no difference between the two DC-treated groups. 
Altered Immune Responses after iDCs+Pep Injection and Uveitogenic Challenge
To examine the effect of peptide-pulsed DCs injection on uveitogenic effector cells in vivo, we collected lymphocytes from the cLNs, the iLNs, and the spleen representing the sites of immune responses generated by the DC injection, the peptide immunization, and the systemic environment, respectively. T cells were assayed for proliferation and cytokine production as before. Our data suggest that DC-treated mice produced higher levels of cell proliferation than in the PBS control group, because of a primary effect (Fig. 8) of the administration of peptide-loaded DCs (Fig. 8A) . cLN cells from the PBS group did not respond to peptide, as we have found before. 29 Cytokine production data indicated lower Th1- but higher Th2-type responses (higher levels of IL-4 and IL-5 but lower levels of IL-2 and IFN-γ; Figs. 8B 8C 8D 8E ) particularly in spleen cells from mice that had received iDCs+Pep before immunization compared with the mice that had received mDCs+Pep, and this shift in Th1-Th2 balance may explain the reduced levels of EAU disease in the iDC+Pep group compared with mDC+Pep. However, how IL-5 and IL-10-producing cells in the cLNs induced by iDC+Pep interacted with uveitogenic T cells and inhibited the Th1 responses and therefore reduced EAU disease is not clear and is currently the focus of further studies. 
Discussion
The generation of iDCs from cultures of bone marrow progenitors in GM-CSF alone has been reported. 30 31 32 Maturation can be induced by high concentrations of GM-CSF and by addition of other cytokines such as TNF-α or by stimulation with bacterial products such as LPS. 13 In this study, we show that BMDCs cultured in the presence of GM-CSF expressed low levels of surface MHC-II, CD86, and CD40 cell surface molecules. High yields of iDCs were obtainable with rigorous attention to detail in the selection of clusters in the iDC-generating cultures and exclusion of free-floating, nonadherent, mature DCs (with higher MHC-II and costimulatory molecule expression, data not shown) and firmly adherent macrophages. Granulocytes were then removed from these populations using magnetic bead depletion, and the purity and yield were confirmed by flow cytometry. Overnight stimulation of iDCs with LPS not only induced DC maturation, as evidenced by upregulation of MHC-II and costimulatory molecules, but also activated DCs to express higher levels of IL-1, IL-6, and IL-12p40 mRNA. This is in agreement with the work of others, who showed that maturation is associated with changes in DC phenotype and function, as shown by upregulation of costimulatory molecules and cytokine expression after stimulation with LPS or CD40L. 33 34 35 36  
Immunostimulatory properties of DCs are currently under active experimental and clinical investigation, mostly in cancer and viral infections. 16 Recently, the maturation status of DCs in the context of their ability to induce an immune response has received attention, and the optimal DC maturation stage for cancer and vaccination studies has yet to be fully established. 37 Recent studies also show that BMDCs are capable of providing both immunizing and tolerizing signals for T cells, depending on their maturation state. 15 iDCs have been shown to prolong allograft survival in mice 38 and to inhibit antigen-specific effector T-cell function in humans. 39 In contrast, fully mature, antigen-loaded DCs have been used in various systems to boost or induce cellular immune responses, particularly in tumor vaccine protocols. 18 40 Our in vitro data have shown that both iDCs and mDCs had comparable ability to take up and present peptide sufficiently to induce peptide-primed T cell proliferation and to secrete IL-2; but iDCs, unlike mDCs, failed to induce such T cells to secrete significant levels of IFN-γ, an essential proinflammatory cytokine secreted by uveitogenic T cells from wild-type mice. 7 Dhodapkar et al. 39 have also reported that subcutaneous injection of antigen-bearing iDCs in humans is not immunologically silent but can lead to antigen-specific inhibition of preexisting effector T-cell function. These data indicate the potential therapeutic role of iDCs in established autoimmune diseases. In addition, Jonuleit et al. 41 have shown that iDCs do not inhibit the proliferation of primed alloreactive T cells or induce T cell death. A remarkable finding in our study was that even a single injection of peptide-loaded iDCs led to the inhibition of EAU, as evaluated by clear reduction in severity of tissue damage. As stated earlier, the use of EAU as a model system allows a clear evaluation of the extent of tissue damage and infiltration, because the retinal neuronal cells and photoreceptor cells are immediately identifiable on routine histology (see Fig. 4 ). Treatment of DCs with LPS (i.e., maturation induction) abrogates this inhibitory function (Fig. 5) , as also observed by Xiao et al. 42 that tolerance in EAE is mediated by an immature stage of DCs. Our data are in agreement with these studies that induction of tolerance mediated by BMDCs is mainly related to the immature developmental stage of the DCs, and the induction of tolerance versus immunity is determined by resting iDCs versus activated mDCs. 17 39  
The mechanisms of tolerance induction by iDCs are unclear, but several possibilities exist. Recently, new studies have identified IL-10 as a differentiation factor for a novel subset of immune suppressive regulatory T cells (Tr1). 43 Tr1 cells are immunosuppressive in vitro and in vivo, 44 and it is likely that Tr1 cells exist naturally within the CD4+CD45RBlow T cell population and are dependent on IL-10 and TGF-β. Jonuleit et al. 41 reported that IL-10-producing CD4+ T cells with regulatory properties can be generated by repetitive stimulation with iDCs in vitro, whereas Dhodapkar et al. 39 observed the induction of antigen-specific IL-10-producing CD8+ T cells in vivo. Feili-Hariri et al. 45 reported that BMDCs administered intravenously can prevent the development of spontaneous diabetes in the NOD mouse by inducing a specific Th2 response, and they further suggested that the balance between regulatory Th2 and effector Th1 cells may have been altered in these mice. In the present study we have shown that subcutaneous inoculation of peptide-loaded iDCs, as well as inhibiting EAU, induced a population of cells in the draining nodes that were high secretors of IL-10 and IL-5. This is in contrast to the effects of inoculation with peptide-loaded mDCs which induced DLN cells producing low levels of IL-10 and IL-5. These data are partially in agreement with the findings of Dhodapkar et al., 39 who report that a defined phenotype of T regulatory cells may be generated after injection of iDCs. As indicated earlier, EAU is a Th1-dependent disease and Th1 proinflammatory cytokines are important in the induction and pathogenesis of uveitis in genetically unmanipulated animals and in patients with uveitis. 46 Studies of different strains of rats and mice indicate that the EAU-susceptible mouse is likely to be a high Th1 responder, whereas the EAU-resistant mouse is likely to be a low Th1 responder (low IFN-γ and IL-12) in which a dominant Th2 response (high IL-4 and IL-5) is induced after uveitogen immunization. 8 In addition, there is evidence showing that if the response to antigens becomes skewed toward a Th2 phenotype, protection from EAU and other tissue-specific autoimmune disease models can be achieved. 7 In the present study, we have detected higher levels of IL-4, IL-5, and IL-10 in mice primed by peptide-loaded iDCs, but not by peptide-loaded mDCs. This suggests a skewing of the cytokine profile to a Th2-dominant type of regulatory response after subsequent uveitogenic challenge, and this may explain the inhibition of EAU. In contrast, peptide-loaded mDCs failed to skew the immune response (Fig. 7) . After injecting iDCs, Dhodapkar et al. 39 did not assay for IL-5 production level but suggested that both IL-10-dependent and -independent mechanisms play a role in the inhibition of CD8+ T cell function, in agreement with Menges et al., 47 although Dhodapkar et al. used differently defined DCs. 
Our further study of the immune responses of effector cells after injection of DCs and immunization indicates that iDCs+Pep induced IL-5 and IL-10-producing T cells that were able to suppress the Th1 and increase Th2 responses after immunization, which may explain the final disease inhibition. However, how and where IL-10 and IL-5-producing cells interact with T effector cells and alter the immune responses in vivo is still unclear and needs further investigation. Taken together, our and others’ data suggest that IL-10-dependent mechanisms are involved in the downregulation of pathogenic immune responses, such as EAU by priming with iDCs, but that other mechanisms may also be involved, either independently or as a consequence of IL-10 production. 
Alternative mechanisms involving anergy or activation-induced cell death (AICD) have also been suggested in tolerance induction. Lutz et al. 48 have suggested that iDC without costimulatory molecules induce alloantigen-specific CD4 T-cell anergy in vitro. We also investigated these possibilities. Our proliferation data did not provide support for the induction of anergy. The disagreement may be due to the different definition of the maturation status of iDCs. In addition, no evidence for preferentially increased levels of apoptosis in CD4 and CD8 T cells from the DLNs after the injection with iDCs compared with mDCs was found, suggesting that tolerance induced by iDCs is not due to T-cell deletion. Taken together, our data indicate that neither anergy nor deletion is likely to be responsible for disease inhibition. Rather, our data support the view that mechanisms including generation of Tr cells and/or skewing of Th1-Th2 cell balance are involved in the inhibition of EAU by iDCs. This is under further investigation. 
Finally, the data presented in this study have some parallels to naturally induced tolerance generated by antigen located in immune privileged sites such as the eye (termed by Streilein et al. 49 anterior chamber-associated immune deviation, ACAID). Tolerance manifested by reduced cellular immunity to systemic challenge by eye-located antigens appears to be mediated by CD8+ Tr cells through the action of NKT cells in the spleen, is considered to be induced by tolerizing eye-derived APCs that migrate to the spleen and requires IL-10. 50  
In summary, our data show that the capacity of DCs to initiate or modulate immune responses appears to depend on their phenotype and functional maturation. Bone marrow-derived myeloid DCs cultured in GM-CSF alone remain immature, and the stimulation of DCs by LPS induces maturation as well as activation. Moreover, our data have shown that, in an autoimmune disease model, peptide-bearing iDCs but not mDCs protected animals from developing uveitis, possibly by inducing high levels of IL-10 and IL-5 during the immune response. This result may have important implications for the application of iDCs for the treatment of human autoimmune diseases and organ transplantation. 
 
Figure 1.
 
Phenotype of in vitro-generated BMDCs. (A) DCs began to form clusters from day 2; (B-F) immunohistochemical staining of purified BMDCs with CDllc (B), CD8 α (C), MHC-II (D), MOMA-2 (E), and F4/80 (F). BMDCs were CD11c positive and CD8α negative with intracellular expression of MHC-II. Isotype controls were negative.
Figure 1.
 
Phenotype of in vitro-generated BMDCs. (A) DCs began to form clusters from day 2; (B-F) immunohistochemical staining of purified BMDCs with CDllc (B), CD8 α (C), MHC-II (D), MOMA-2 (E), and F4/80 (F). BMDCs were CD11c positive and CD8α negative with intracellular expression of MHC-II. Isotype controls were negative.
Figure 2.
 
Flow cytometric analysis of BMDC phenotype after stimulation with LPS. After 6 days in culture in GM-CSF-supplemented medium, the loosely attached clusters were collected. Gr-1-positive cells were depleted, and the remaining cells were stimulated with or without LPS overnight, and DCs were pooled for flow cytometry. Data of MHC-II (top), CD86 (middle), and CD40 (bottom) expression on non-LPS treated immature (thin trace) and LPS-treated mature (thick trace) DCs, showing LPS-induced DC maturation by upregulating MHC-II and costimulatory molecule expression (dotted trace: isotype control).
Figure 2.
 
Flow cytometric analysis of BMDC phenotype after stimulation with LPS. After 6 days in culture in GM-CSF-supplemented medium, the loosely attached clusters were collected. Gr-1-positive cells were depleted, and the remaining cells were stimulated with or without LPS overnight, and DCs were pooled for flow cytometry. Data of MHC-II (top), CD86 (middle), and CD40 (bottom) expression on non-LPS treated immature (thin trace) and LPS-treated mature (thick trace) DCs, showing LPS-induced DC maturation by upregulating MHC-II and costimulatory molecule expression (dotted trace: isotype control).
Figure 3.
 
Cytokine mRNA expression by iDCs and LPS-induced mDCs detected by RPA, using a multiprobe template set mCK2. IL-12p40, IL-1α, IL-1β, and IL-6 were upregulated after overnight stimulation with LPS. Lane 1: template; lane 2: LPS-treated mDCs; lane 3: iDCs.
Figure 3.
 
Cytokine mRNA expression by iDCs and LPS-induced mDCs detected by RPA, using a multiprobe template set mCK2. IL-12p40, IL-1α, IL-1β, and IL-6 were upregulated after overnight stimulation with LPS. Lane 1: template; lane 2: LPS-treated mDCs; lane 3: iDCs.
Figure 4.
 
Histopathology of EAU in mice pretreated with peptide-pulsed iDCs (A, C, E) and PBS (B, D, F) before immunization. (A) Intact retina with identified layers from a mouse pretreated with peptide-pulsed iDCs. V, vitreous; ILM, inner limiting membrane layer; INL, inner nuclear layer; ONL, outer nuclear layer; ROS, photoreceptor outer segment layer; Ch, choroid; Sc, sclera. (C, E) Retina from B10RIII mice treated with peptide-pulsed iDCs before immunization, also showing that the disease was restricted to a single focal lesion at the posterior pole of the eye (E), and that most of the infiltrating cells appeared in the vitreous but not in the target tissue ROS layer (C). (B, D, F) Retina from the mice that were treated with PBS before immunization showed extensive disease in the entire retina (F), massive levels of infiltrating cells in the vitreous fluid and ROS layer (B, D, arrows), vasculitis (B, arrowhead), severe retinal detachment and structural damage to the retina.
Figure 4.
 
Histopathology of EAU in mice pretreated with peptide-pulsed iDCs (A, C, E) and PBS (B, D, F) before immunization. (A) Intact retina with identified layers from a mouse pretreated with peptide-pulsed iDCs. V, vitreous; ILM, inner limiting membrane layer; INL, inner nuclear layer; ONL, outer nuclear layer; ROS, photoreceptor outer segment layer; Ch, choroid; Sc, sclera. (C, E) Retina from B10RIII mice treated with peptide-pulsed iDCs before immunization, also showing that the disease was restricted to a single focal lesion at the posterior pole of the eye (E), and that most of the infiltrating cells appeared in the vitreous but not in the target tissue ROS layer (C). (B, D, F) Retina from the mice that were treated with PBS before immunization showed extensive disease in the entire retina (F), massive levels of infiltrating cells in the vitreous fluid and ROS layer (B, D, arrows), vasculitis (B, arrowhead), severe retinal detachment and structural damage to the retina.
Figure 5.
 
Immature, but not mature, peptide-pulsed DCs inhibited EAU. B10RIII mice received a single or triple subcutaneous injection of peptide-loaded or unloaded iDCs or mDCs 10 days before the immunization. Then eyes were harvested for histopathology on day 15 and were graded on a scale of 0 (no disease) to 4 (maximum disease). Each point represents one mouse. Horizontal bar: Average score of each group. (A) A single injection or triple subcutaneous injections of PBS before immunization in the control group showed a high grade of EAU. (B) A single injection of peptide-loaded iDCs inhibited development of EAU, but non-peptide-loaded DCs had a minimal effect (*P < 0.05 compared with the PBS group). (C) Triple injections of peptide-loaded iDCs confirmed the effect of iDCs+Pep in inhibiting EAU (*P < 0.05 compared with the PBS group). (D) LPS-treated mDCs failed to inhibit EAU. (E) The protection persisted at day 26 after immunization (*P < 0.05 compared with the PBS group).
Figure 5.
 
Immature, but not mature, peptide-pulsed DCs inhibited EAU. B10RIII mice received a single or triple subcutaneous injection of peptide-loaded or unloaded iDCs or mDCs 10 days before the immunization. Then eyes were harvested for histopathology on day 15 and were graded on a scale of 0 (no disease) to 4 (maximum disease). Each point represents one mouse. Horizontal bar: Average score of each group. (A) A single injection or triple subcutaneous injections of PBS before immunization in the control group showed a high grade of EAU. (B) A single injection of peptide-loaded iDCs inhibited development of EAU, but non-peptide-loaded DCs had a minimal effect (*P < 0.05 compared with the PBS group). (C) Triple injections of peptide-loaded iDCs confirmed the effect of iDCs+Pep in inhibiting EAU (*P < 0.05 compared with the PBS group). (D) LPS-treated mDCs failed to inhibit EAU. (E) The protection persisted at day 26 after immunization (*P < 0.05 compared with the PBS group).
Figure 6.
 
Effect of LPS-induced DC maturation on peptide presentation to peptide-activated T cells. (A) iDCs and mDCs were either loaded with peptide (+Pep) or not loaded with peptide (−Pep) and then peptide was washed out before incubation of the cells with purified T cells isolated from DLNs of mice immunized with peptide+CFA (peptide-activated T cells). Cells were cultured in the presence or absence of an additional 20 μg/mL of peptide. The proliferation data show that non-peptide-loaded iDCs, but less so mDCs, failed to induce significant responses unless peptide was added to the culture, whereas peptide-loaded iDCs and mDCs induced good T-cell proliferation even in the absence of additional peptide in the culture. (B, C) Peptide-loaded iDCs and mDCs were incubated with purified peptide-activated T cells in the presence of 50 μg/mL of peptide and the supernatant assayed for the secretion of IL-2 (B) and IFN-γ (C). The data show that both iDCs and mDCs released significant quantities of IL-2, but only mDCs induced production of IFN-γ, a proinflammatory cytokine secreted by uveitogenic T cells. 5 Error bars, standard deviation of triplicate cultures from a representative of four experiments (*P < 0.01).
Figure 6.
 
Effect of LPS-induced DC maturation on peptide presentation to peptide-activated T cells. (A) iDCs and mDCs were either loaded with peptide (+Pep) or not loaded with peptide (−Pep) and then peptide was washed out before incubation of the cells with purified T cells isolated from DLNs of mice immunized with peptide+CFA (peptide-activated T cells). Cells were cultured in the presence or absence of an additional 20 μg/mL of peptide. The proliferation data show that non-peptide-loaded iDCs, but less so mDCs, failed to induce significant responses unless peptide was added to the culture, whereas peptide-loaded iDCs and mDCs induced good T-cell proliferation even in the absence of additional peptide in the culture. (B, C) Peptide-loaded iDCs and mDCs were incubated with purified peptide-activated T cells in the presence of 50 μg/mL of peptide and the supernatant assayed for the secretion of IL-2 (B) and IFN-γ (C). The data show that both iDCs and mDCs released significant quantities of IL-2, but only mDCs induced production of IFN-γ, a proinflammatory cytokine secreted by uveitogenic T cells. 5 Error bars, standard deviation of triplicate cultures from a representative of four experiments (*P < 0.01).
Figure 7.
 
Effect of DC maturation on priming naïve lymphocytes in vivo. Cells from the draining cLNs of mice injected with peptide-loaded immature (□) and mature DCs (▪) or PBS 6 days earlier were cultured with peptide for cell proliferation and cytokine production assay. mDCs induced higher levels of cell proliferation (A) and of production of IL-2 (C) and IFN-γ (E). In contrast, iDCs induced higher levels of IL-10 (B), IL-4 (D), and IL-5 (F). Error bars, standard deviation of triplicate cultures from a representative of two experiments (*P < 0.05 compared with the mDC group).
Figure 7.
 
Effect of DC maturation on priming naïve lymphocytes in vivo. Cells from the draining cLNs of mice injected with peptide-loaded immature (□) and mature DCs (▪) or PBS 6 days earlier were cultured with peptide for cell proliferation and cytokine production assay. mDCs induced higher levels of cell proliferation (A) and of production of IL-2 (C) and IFN-γ (E). In contrast, iDCs induced higher levels of IL-10 (B), IL-4 (D), and IL-5 (F). Error bars, standard deviation of triplicate cultures from a representative of two experiments (*P < 0.05 compared with the mDC group).
Figure 8.
 
Effect of injection of peptide-pulsed DCs on uveitogenic effector cells in vivo. Cells from the cLNs, iLNs, and spleen of mice injected in the neck area with peptide-loaded immature and mature DCs or PBS and then immunized with peptide subcutaneously in the back, were cultured with peptide for cell proliferation and cytokine production assay. Injection of DCs plus immunization induced higher levels of cell proliferation than did PBS alone (A). However, iDCs induced higher levels of IL-4 (C) and IL-5 (E) but lower levels of IL-2 (B) and IFN-γ (D). Error bars, standard deviation of triplicate cultures from a representative of two experiments (*P < 0.05 compared with the mDC+Pep group).
Figure 8.
 
Effect of injection of peptide-pulsed DCs on uveitogenic effector cells in vivo. Cells from the cLNs, iLNs, and spleen of mice injected in the neck area with peptide-loaded immature and mature DCs or PBS and then immunized with peptide subcutaneously in the back, were cultured with peptide for cell proliferation and cytokine production assay. Injection of DCs plus immunization induced higher levels of cell proliferation than did PBS alone (A). However, iDCs induced higher levels of IL-4 (C) and IL-5 (E) but lower levels of IL-2 (B) and IFN-γ (D). Error bars, standard deviation of triplicate cultures from a representative of two experiments (*P < 0.05 compared with the mDC+Pep group).
The authors thank Janet Liversidge for helpful discussion and critical reading of the manuscript. 
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Figure 1.
 
Phenotype of in vitro-generated BMDCs. (A) DCs began to form clusters from day 2; (B-F) immunohistochemical staining of purified BMDCs with CDllc (B), CD8 α (C), MHC-II (D), MOMA-2 (E), and F4/80 (F). BMDCs were CD11c positive and CD8α negative with intracellular expression of MHC-II. Isotype controls were negative.
Figure 1.
 
Phenotype of in vitro-generated BMDCs. (A) DCs began to form clusters from day 2; (B-F) immunohistochemical staining of purified BMDCs with CDllc (B), CD8 α (C), MHC-II (D), MOMA-2 (E), and F4/80 (F). BMDCs were CD11c positive and CD8α negative with intracellular expression of MHC-II. Isotype controls were negative.
Figure 2.
 
Flow cytometric analysis of BMDC phenotype after stimulation with LPS. After 6 days in culture in GM-CSF-supplemented medium, the loosely attached clusters were collected. Gr-1-positive cells were depleted, and the remaining cells were stimulated with or without LPS overnight, and DCs were pooled for flow cytometry. Data of MHC-II (top), CD86 (middle), and CD40 (bottom) expression on non-LPS treated immature (thin trace) and LPS-treated mature (thick trace) DCs, showing LPS-induced DC maturation by upregulating MHC-II and costimulatory molecule expression (dotted trace: isotype control).
Figure 2.
 
Flow cytometric analysis of BMDC phenotype after stimulation with LPS. After 6 days in culture in GM-CSF-supplemented medium, the loosely attached clusters were collected. Gr-1-positive cells were depleted, and the remaining cells were stimulated with or without LPS overnight, and DCs were pooled for flow cytometry. Data of MHC-II (top), CD86 (middle), and CD40 (bottom) expression on non-LPS treated immature (thin trace) and LPS-treated mature (thick trace) DCs, showing LPS-induced DC maturation by upregulating MHC-II and costimulatory molecule expression (dotted trace: isotype control).
Figure 3.
 
Cytokine mRNA expression by iDCs and LPS-induced mDCs detected by RPA, using a multiprobe template set mCK2. IL-12p40, IL-1α, IL-1β, and IL-6 were upregulated after overnight stimulation with LPS. Lane 1: template; lane 2: LPS-treated mDCs; lane 3: iDCs.
Figure 3.
 
Cytokine mRNA expression by iDCs and LPS-induced mDCs detected by RPA, using a multiprobe template set mCK2. IL-12p40, IL-1α, IL-1β, and IL-6 were upregulated after overnight stimulation with LPS. Lane 1: template; lane 2: LPS-treated mDCs; lane 3: iDCs.
Figure 4.
 
Histopathology of EAU in mice pretreated with peptide-pulsed iDCs (A, C, E) and PBS (B, D, F) before immunization. (A) Intact retina with identified layers from a mouse pretreated with peptide-pulsed iDCs. V, vitreous; ILM, inner limiting membrane layer; INL, inner nuclear layer; ONL, outer nuclear layer; ROS, photoreceptor outer segment layer; Ch, choroid; Sc, sclera. (C, E) Retina from B10RIII mice treated with peptide-pulsed iDCs before immunization, also showing that the disease was restricted to a single focal lesion at the posterior pole of the eye (E), and that most of the infiltrating cells appeared in the vitreous but not in the target tissue ROS layer (C). (B, D, F) Retina from the mice that were treated with PBS before immunization showed extensive disease in the entire retina (F), massive levels of infiltrating cells in the vitreous fluid and ROS layer (B, D, arrows), vasculitis (B, arrowhead), severe retinal detachment and structural damage to the retina.
Figure 4.
 
Histopathology of EAU in mice pretreated with peptide-pulsed iDCs (A, C, E) and PBS (B, D, F) before immunization. (A) Intact retina with identified layers from a mouse pretreated with peptide-pulsed iDCs. V, vitreous; ILM, inner limiting membrane layer; INL, inner nuclear layer; ONL, outer nuclear layer; ROS, photoreceptor outer segment layer; Ch, choroid; Sc, sclera. (C, E) Retina from B10RIII mice treated with peptide-pulsed iDCs before immunization, also showing that the disease was restricted to a single focal lesion at the posterior pole of the eye (E), and that most of the infiltrating cells appeared in the vitreous but not in the target tissue ROS layer (C). (B, D, F) Retina from the mice that were treated with PBS before immunization showed extensive disease in the entire retina (F), massive levels of infiltrating cells in the vitreous fluid and ROS layer (B, D, arrows), vasculitis (B, arrowhead), severe retinal detachment and structural damage to the retina.
Figure 5.
 
Immature, but not mature, peptide-pulsed DCs inhibited EAU. B10RIII mice received a single or triple subcutaneous injection of peptide-loaded or unloaded iDCs or mDCs 10 days before the immunization. Then eyes were harvested for histopathology on day 15 and were graded on a scale of 0 (no disease) to 4 (maximum disease). Each point represents one mouse. Horizontal bar: Average score of each group. (A) A single injection or triple subcutaneous injections of PBS before immunization in the control group showed a high grade of EAU. (B) A single injection of peptide-loaded iDCs inhibited development of EAU, but non-peptide-loaded DCs had a minimal effect (*P < 0.05 compared with the PBS group). (C) Triple injections of peptide-loaded iDCs confirmed the effect of iDCs+Pep in inhibiting EAU (*P < 0.05 compared with the PBS group). (D) LPS-treated mDCs failed to inhibit EAU. (E) The protection persisted at day 26 after immunization (*P < 0.05 compared with the PBS group).
Figure 5.
 
Immature, but not mature, peptide-pulsed DCs inhibited EAU. B10RIII mice received a single or triple subcutaneous injection of peptide-loaded or unloaded iDCs or mDCs 10 days before the immunization. Then eyes were harvested for histopathology on day 15 and were graded on a scale of 0 (no disease) to 4 (maximum disease). Each point represents one mouse. Horizontal bar: Average score of each group. (A) A single injection or triple subcutaneous injections of PBS before immunization in the control group showed a high grade of EAU. (B) A single injection of peptide-loaded iDCs inhibited development of EAU, but non-peptide-loaded DCs had a minimal effect (*P < 0.05 compared with the PBS group). (C) Triple injections of peptide-loaded iDCs confirmed the effect of iDCs+Pep in inhibiting EAU (*P < 0.05 compared with the PBS group). (D) LPS-treated mDCs failed to inhibit EAU. (E) The protection persisted at day 26 after immunization (*P < 0.05 compared with the PBS group).
Figure 6.
 
Effect of LPS-induced DC maturation on peptide presentation to peptide-activated T cells. (A) iDCs and mDCs were either loaded with peptide (+Pep) or not loaded with peptide (−Pep) and then peptide was washed out before incubation of the cells with purified T cells isolated from DLNs of mice immunized with peptide+CFA (peptide-activated T cells). Cells were cultured in the presence or absence of an additional 20 μg/mL of peptide. The proliferation data show that non-peptide-loaded iDCs, but less so mDCs, failed to induce significant responses unless peptide was added to the culture, whereas peptide-loaded iDCs and mDCs induced good T-cell proliferation even in the absence of additional peptide in the culture. (B, C) Peptide-loaded iDCs and mDCs were incubated with purified peptide-activated T cells in the presence of 50 μg/mL of peptide and the supernatant assayed for the secretion of IL-2 (B) and IFN-γ (C). The data show that both iDCs and mDCs released significant quantities of IL-2, but only mDCs induced production of IFN-γ, a proinflammatory cytokine secreted by uveitogenic T cells. 5 Error bars, standard deviation of triplicate cultures from a representative of four experiments (*P < 0.01).
Figure 6.
 
Effect of LPS-induced DC maturation on peptide presentation to peptide-activated T cells. (A) iDCs and mDCs were either loaded with peptide (+Pep) or not loaded with peptide (−Pep) and then peptide was washed out before incubation of the cells with purified T cells isolated from DLNs of mice immunized with peptide+CFA (peptide-activated T cells). Cells were cultured in the presence or absence of an additional 20 μg/mL of peptide. The proliferation data show that non-peptide-loaded iDCs, but less so mDCs, failed to induce significant responses unless peptide was added to the culture, whereas peptide-loaded iDCs and mDCs induced good T-cell proliferation even in the absence of additional peptide in the culture. (B, C) Peptide-loaded iDCs and mDCs were incubated with purified peptide-activated T cells in the presence of 50 μg/mL of peptide and the supernatant assayed for the secretion of IL-2 (B) and IFN-γ (C). The data show that both iDCs and mDCs released significant quantities of IL-2, but only mDCs induced production of IFN-γ, a proinflammatory cytokine secreted by uveitogenic T cells. 5 Error bars, standard deviation of triplicate cultures from a representative of four experiments (*P < 0.01).
Figure 7.
 
Effect of DC maturation on priming naïve lymphocytes in vivo. Cells from the draining cLNs of mice injected with peptide-loaded immature (□) and mature DCs (▪) or PBS 6 days earlier were cultured with peptide for cell proliferation and cytokine production assay. mDCs induced higher levels of cell proliferation (A) and of production of IL-2 (C) and IFN-γ (E). In contrast, iDCs induced higher levels of IL-10 (B), IL-4 (D), and IL-5 (F). Error bars, standard deviation of triplicate cultures from a representative of two experiments (*P < 0.05 compared with the mDC group).
Figure 7.
 
Effect of DC maturation on priming naïve lymphocytes in vivo. Cells from the draining cLNs of mice injected with peptide-loaded immature (□) and mature DCs (▪) or PBS 6 days earlier were cultured with peptide for cell proliferation and cytokine production assay. mDCs induced higher levels of cell proliferation (A) and of production of IL-2 (C) and IFN-γ (E). In contrast, iDCs induced higher levels of IL-10 (B), IL-4 (D), and IL-5 (F). Error bars, standard deviation of triplicate cultures from a representative of two experiments (*P < 0.05 compared with the mDC group).
Figure 8.
 
Effect of injection of peptide-pulsed DCs on uveitogenic effector cells in vivo. Cells from the cLNs, iLNs, and spleen of mice injected in the neck area with peptide-loaded immature and mature DCs or PBS and then immunized with peptide subcutaneously in the back, were cultured with peptide for cell proliferation and cytokine production assay. Injection of DCs plus immunization induced higher levels of cell proliferation than did PBS alone (A). However, iDCs induced higher levels of IL-4 (C) and IL-5 (E) but lower levels of IL-2 (B) and IFN-γ (D). Error bars, standard deviation of triplicate cultures from a representative of two experiments (*P < 0.05 compared with the mDC+Pep group).
Figure 8.
 
Effect of injection of peptide-pulsed DCs on uveitogenic effector cells in vivo. Cells from the cLNs, iLNs, and spleen of mice injected in the neck area with peptide-loaded immature and mature DCs or PBS and then immunized with peptide subcutaneously in the back, were cultured with peptide for cell proliferation and cytokine production assay. Injection of DCs plus immunization induced higher levels of cell proliferation than did PBS alone (A). However, iDCs induced higher levels of IL-4 (C) and IL-5 (E) but lower levels of IL-2 (B) and IFN-γ (D). Error bars, standard deviation of triplicate cultures from a representative of two experiments (*P < 0.05 compared with the mDC+Pep group).
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