Targeting of Autoantigens to DEC205+ Dendritic Cells In Vivo Suppresses Experimental Allergic Encephalomyelitis in Mice

Sabine Ring, Michael Maas, Dirk M. Nettelbeck, Alexander H. Enk and Karsten Mahnke
J Immunol September 15, 2013, 191 (6) 2938-2947; DOI: https://doi.org/10.4049/jimmunol.1202592

Abstract

The dendritic and epithelial cell receptor with a m.w. of 205 kDa (DEC205) is expressed by dendritic cells (DCs) and facilitates Ag presentation. After injection of Ags coupled to Abs specific for DEC205 into mice, Ag presentation occurs by nonactivated DCs, which leads to induction of regulatory T cells (Tregs). To test this system for tolerance induction in experimental allergic encephalomyelitis (EAE), we created single-chain fragment variables (scFv) specific for DEC205 and fused the scFv to the self-Ag myelin oligodendrocyte glycoprotein (MOG; scFv DEC:MOG). An anti–β-galactosidase scFv:MOG fusion protein (scFv GL117:MOG) served as isotype control. After staining of DCs in vitro with purified scFv DEC:MOG, binding to DCs and colocalization with MHC class II was apparent, whereas isotype controls did not bind. We next injected scFv DEC:MOG into mice and observed elevated numbers of highly activated, IL-10–producing CD4+CD25+Foxp3+ Tregs (17% of CD4) in spleens, as compared with isotype controls and uninjected mice (12% of CD4). Furthermore, DCs isolated from scFv DEC:MOG-injected animals produced significantly increased levels of TGF-β. Most importantly, when EAE was induced in scFv DEC:MOG-injected mice, 90% of the mice were protected from EAE, whereas all mice in the isotype controls (scFv GL117:MOG) experienced development of EAE. When applying scFv DEC:MOG to mice that had already experienced EAE symptoms, abrogation of the disease in 90% of the animals was apparent, whereas all animals in the control groups experienced development of severe EAE. Thus, these data indicate that targeting of MOG to “steady-state” DCs in vivo may provide a tool to prevent and to treat EAE by a DC/Treg-driven mechanism.

Introduction

Dendritic cells (DCs) are professional APCs equipped with all necessary means to take up, to digest, and to present Ags to T cells. After Ag uptake in peripheral organs, DCs migrate to regional lymph nodes (LNs) where they encounter T cells. Because activated and Ag-loaded DCs express several T cell costimulatory molecules such as CD80, CD86, and CD40 in large quantities, this DC–T cell interaction normally results in activation of T cells and in induction of immune responses (1).

DCs can also initiate tolerogenic responses by inducing regulatory T cells (Tregs) or by induction of T cell anergy (2). These tolerogenic effects are mainly driven by immature, nonactivated DCs entering the LNs in the steady-state. These steady-state DCs are devoid of critical T cell costimulatory molecules on their surface and produce substantial amounts of IL-10 and IL-6, whereas T cell activating cytokines such as IL-12 are missing (3, 4). Thus, depending on the subtype of DCs, the cognate MHC/peptide–TCR interactions may leave the T cells anergic and can induce T cell apoptosis, as well as Tregs. In vitro, the investigation of steady-state DCs is not feasible because manipulation of the DCs, that is, the isolation and the cultivation of DCs, activates DCs and obliterates the steady-state. To overcome the obstacles of DC purification and concomitant DC activation, we devised a system in which we coupled Ags to Abs specific for the Ag receptor dendritic and epithelial cell receptor with an m.w. of 205 kDa (DEC205), which is nearly exclusively expressed by DCs. Upon injection into mice, anti-DEC:Ag conjugates target DCs in situ without activating them, and the Ags are taken up and presented to T cells in vivo. Using this targeting system in mice harboring transgenic T cells specific for OVA, we could show that anti-DEC:Ag conjugates induced CD4+CD25+ Tregs, which prevented immune responses against OVA and hen egg lysozyme. Thus, we were able to tolerize animals against Ags coupled to anti-DEC Abs (5–7). To test this novel approach of tolerance induction in a nontransgenic autoimmune disease setting, we developed recombinant single-chain fragment variables (scFv) conjugated to Ags for testing in experimental allergic encephalomyelitis (EAE) (8). Murine EAE serves as a model for multiple sclerosis. In the EAE model, antigenic peptides such as myelin oligodendrocyte glycoprotein (MOG) and myeloblastic protein are defined and upon immunization of mice against these peptides, autoreactive T cells are developing. The T cells enter the brain, attack nerve cells, and cause symptoms of ataxia and paraplegia of extremities, all of which are known to be present in humans affected by multiple sclerosis (9).

To test whether targeting of EAE-related peptides to steady-state DCs induces Tregs and may rescue mice from EAE symptoms, we generated scFv specific for DEC205 fused to the MOG peptide (scFv DEC:MOG), relevant for EAE induction. In this report, we show that scFv DEC:MOG conjugates were able to bind to DCs and the MOG Ag was presented to T cells. Upon injection of scFv DEC:MOG, DCs from spleens produced increased levels of TGF-β and the frequency of CD4+CD25+ Foxp3 Tregs increased. Most importantly, scFv DEC:MOG prevented EAE symptoms nearly completely when injected before EAE induction and even after symptoms of EAE had already developed, injection of scFv DEC:MOG alleviated the symptoms significantly. Thus, these data indicate that targeting of MOG peptides to steady-state DCs prevents and even cures EAE by a TGF-β/Treg–driven mechanism.

Materials and Methods

Mice and standard reagents

C57/Bl6 mice were purchased from Charles River Germany. 2D2 mice were a gift from Dr. N. Garbi (German Cancer Research Center, Heidelberg). All animals were housed in the central animal facility of the University of Heidelberg, and experiments were carried out according to the guidelines for animal welfare (permission of the State of Baden Württemberg #35-9185.81/G-172/08).

Medium and supplements (Pen/Strep, HEPES, glutamine, FCS) were purchased from PAA (Cölbe, Germany). Standard chemicals for cloning and purification of the scFv fusion proteins were purchased (if not stated otherwise) from Carl Roth (Karlsruhe, Germany).

Cloning and production of scFv

The cloning is described elsewhere in detail (10). In brief, RNA from the hybridoma cell line NLDC-145 for murine DEC205 and hybridoma cell line GL117 for β-galactosidase were isolated and RT-PCR was performed using degenerative primers for the VH and VL domains. The VH and VL domains were subcloned into the cloning vector pHEN3 (kind gift from Roland Kontermann, Stuttgart, Germany). To yield fusion scFv constructs, we added a DNA sequence coding the MOG peptide (amino acid sequence: MEVGWYRSPFSRVVHLYRNGK) in-frame to the appropriate cloning sites. For controls, the sequence for a tyrosinase-related protein 2 (TRP2) was used. The completed scFv region was subcloned into the expression vector pAB1 and transformed into Escherichia coli strain TG1. One-liter cultures of TG1 were induced by the addition of 1 mM. After overnight culture at 22°C, the periplasma of the bacteria was isolated according to standard procedures (10), and the scFv were isolated by an Ni-NTA column (Invitrogen, Karlsruhe, Germany). Protein content of the elution fractions was determined by standard Bradford assay, and the positive fractions were pooled and dialyzed overnight at 4°C against PBS. Protein production and purification steps were routinely analyzed by SDS-PAGE gel electrophoresis and Western blotting.

Preparation and analysis of bone marrow–derived DCs

Bone marrow–derived DCs (BMDCs) were prepared according to standard procedures (5). In brief, bone marrow cells from C57/BL6 mice were cultured in RPMI 1640 supplemented with GM-CSF and IL-4 (10 ng/ml each; eBioscience, Frankfurt, Germany) for 6 d with intermittent feeding. For immunohistological staining, cells were seeded onto Alcian blue (Sigma, Deisenhofen, Germany)–coated glass slides and fixed with paraformaldehyde (4% w/v). Thereafter cells were stained with scFv DEC:MOG, scFv GL117:MOG (5 μl/ml), and appropriate secondary reagents (anti–c-myc biotin [Miltenyi, Bergisch Gladbach, Germany] and streptavidin PE [eBioscience]). For double labeling, anti-MHC class II Abs (eBioscience) followed by anti-rat FITC (Dianova, Hamburg, Germany) were used. Cells were examined with a Leica fluorescence microscope.

Isolation of CD11c+, CD4+, and CD4+CD25+ cells

Single-cell suspensions were prepared from spleens by a collagenase digest (Collagenase IV, 800 U/ml; Cell Systems, Troisdorf, Germany) followed by straining through a 70-μm cell strainer (BD Biosciences, Heidelberg, Germany). Cells were incubated with anti-CD11c beads and separated by a magnetic column (all steps were carried out according to the manufacturer’s instructions; Miltenyi). Purity of isolated CD11c+ DC was analyzed by flow cytometry, with a typical yield of 80% pure CD11c+ cells.

CD4+ T cells were isolated from spleen and LN either from MOG-specific 2D2 mice or BALB/c mice using the untouched T cell kit from Miltenyi. Purity averaged out >97%.

CD4+CD25+ Tregs were isolated from LN and spleen cells (average purity of 90–95%) using the CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s protocol.

Cytokine secretion

CD11c+ DCs of the differently treated mice were isolated and cultivated overnight in standard medium. Aliquots were stimulated with LPS (10 ng/ml; Sigma). Thereafter tissue culture supernatants were tested for presence of TGF-β and IL-10 by ELISA (eBioscience).

Tregs isolated from naive mice or mice treated with scFv DEC:MOG or scFv GL117:MOG 24 h before were stimulated with either anti-CD3/anti-CD28 (0.5 μg/ml each; Becton Dickinson) or with MOG-pulsed BMDCs. IL-10 was measured after 48 h in the tissue culture supernatants using ELISA.

Proliferation assays

BMDCs were incubated with either scFv DEC:MOG, scFv DEC:TRP2, or scFv GL117:MOG (100 and 10 ng/ml) or directly with the MOG peptide for 3 h. These differently prepulsed DCs were cocultured with MOG-specific CD4+ T cells isolated from 2D2 mice in 96-well round-bottom plates (50,000 CD4+ T cells + 10,000 CD11c+ DCs).

For Ag presentation assays, CD11c+ DCs were isolated from spleens of mice that were treated either with 10 μg scFv DEC-MOG, scFv DEC:TRP2, or scFv GL117:MOG in 100 μl PBS or with MOG peptide (3 μg/100 μl PBS) 24 h before. These amounts equal roughly comparable molar ratios of the Ag, as the scFv DEC:MOG contains the scFv and the MOG peptide. Isolated DCs were cocultured with the MOG-specific CD4+ T cells (50,000 CD4+ T cells + 25,000 CD11c+ DCs), and cell proliferation was determined by measuring [3H]thymidine (Hartmann, Braunschweig, Germany) incorporation using a PerkinElmer scintillation counter. Assays were performed in triplicates.

CD11c+ DCs were also isolated from spleens of the differently treated groups during the EAE experiments on day 15 after EAE induction and cocultured with allogeneic BALB/c CD4+ T cells. Cell proliferation was determined after 72 h of culture.

Injection of scFv and EAE induction

In preventive approaches, 10 μg of respective scFv was injected i.v. into C57/Bl6 mice 7 and 3 d before induction of EAE. In some experiments, anti–TGF-β (clone 1D11.16.8; BioXCell, West Lebanon, NH) was injected i.p. (15 mg/kg body weight) before, during, and after scFv DEC:MOG treatment. In therapeutic experiments, EAE was induced first and after all mice showed symptoms (score 1), scFv fusion proteins were injected 1 and 4 d later. EAE induction was carried out according to standard procedures (9). In brief, homogenized and lyophilized spinal cord from C57/Bl6 mice was dissolved in PBS 150 mg/ml and mixed thoroughly 1:1 with Mycobacterium tuberculosis (4 mg/ml in CFA; Sigma). A total of 50 μl of the mixture was injected s.c. on both sides of the tail. One hour and 48 h after injection, pertussis toxin 500 ng/100 μl PBS (Sigma) was injected i.p. Animals were examined daily. The score was as follows: 0 = no symptoms; 1 = gripping response of the tail slowed down; 2 = loss of tail tonus; 3 = righting reflex delayed; 4 = loss of righting reflex; 5 = mouse drags the hind legs; 6 = partial hind-limb paresis; 7 = complete hind-limb paresis; 8 = partial forelegs paresis; 9 = quadriplegia; and 10 = moribund or dead.

Preparation of tissue slices and cell suspensions from brains

Brains (cerebellum/brainstem) and spinal cords were mounted in Tissue-Tek mounting-freezing media (Miles, Torrance, CA). Cryosections (6 μm) were prepared using a cryostat (Leica, Solms, Germany), fixed with 4% paraformaldehyde, and incubated with anti-CD4 Ab (eBioscience) and TRITC-labeled goat anti-rat Ab (Dianova). Stainings with H&E were performed according to the standard protocol. For cell suspensions, brains were minced and digested in 800 U/ml collagenase IV followed by straining through a 70-μm cell strainer. Cells were further segregated by density gradient centrifugation (30 min, 400 × g, without break) using 40% and 70% Percoll (Biochrom, Berlin, Germany). For FACS analysis, the cells were stained in suspension with anti–CD4-PE (eBioscience). For detection of cytokines, cell suspensions were cultured for 48 h in standard medium. Thereafter tissue culture supernatants were tested for presence of IL-17 and IFN-γ by ELISA (eBioscience).

Flow cytometric analysis

Cell suspensions from spleen, LN, blood, and brain of the differently treated mice were prepared and diluted in PBS/3% FCS. The following Abs (all from eBioscience) were used for staining: anti–CD4-allophycocyanin, anti–CD25-PE, anti–CD69-PE, FITC Foxp3 staining set. For intracellular cytokine stainings, cells were stimulated with PMA/Ionomycin (2 and 400 ng/ml) and treated with brefeldin A (eBioscience) 4 h before cytokine detection. After staining of the surface molecules, cells were fixed and permeabilized before adding anti–IL-17–PE and anti–IFN-γ–FITC (eBioscience).

BMDCs were incubated with either scFv DEC:MOG or scFv GL117:MOG followed by staining with anti–c-myc biotin and streptavidin-FITC (Miltenyi and eBioscience).

Isolated CD11c+ DCs from the differently treated mice were stained with PE-labeled anti-B7H1, anti-B7H2, or anti-B7H3 (eBioscience). All FACS samples were analyzed on a FACSCanto (BD Biosciences).

Statistical analysis

Student t test was used as indicated in the figure legends.

Results

scFv specific for DEC205 bind to DCs in vivo and in vitro

scFv specific for DEC205 (clone NLDC-145) and control scFv specific for β-galactosidase (clone GL117) were cloned into expression vectors by standard procedures. A DNA sequence coding for the MOG peptide was cloned into the same vector, yielding a scFv MOG fusion protein (Fig. 1A). To test the scFv for binding to DC, we stained BMDCs with different scFv and parental anti-DEC Ab, respectively. In these experiments, the scFv DEC:MOG did bind to DCs and the fluorescence intensity was comparable with that obtained with parental anti-DEC205 Abs (Fig. 1B). All cells displayed a strong MHC class II stain, which even showed some partial overlap with the DEC staining, indicating a possible colocalization of DEC and MHC class II as described previously (11). Similar results were obtained when testing the scFv DEC:MOG by flow cytometry, as here binding of the scFv could also be observed (Fig. 1C). In contrast, the control scFv GL117:MOG, as well as the IgG isotype control, did not bind to BMDCs as assessed by immunofluorescence microscopy and FACS. To next test whether scFv DEC:MOG targets DCs in vivo, we i.v. injected 10 μg scFv DEC:MOG into C57/Bl6 mice. Twenty-four hours later, mice were sacrificed and cryosections of the spleens and LNs were stained with appropriate secondary reagents to demonstrate the presence of the scFv. Double labeling with CD11c Abs was used to identify DCs in the sections. As depicted in Fig. 1D, injected scFv DEC:MOG (green) was present in spleen and LNs, and colocalized with CD11c+ (red). Thus, these data indicate that scFv DEC:MOG is targeting specifically to DCs in vivo and can be used to load DCs in situ with respective Ags.

FIGURE 1.
FIGURE 1.

scFv specific for DEC205 bind to DCs in vivo and in vitro. (A) Scheme of scFv:MOG fusion proteins. (B) BMDCs were double stained with anti–MHC class II and either anti-DEC Ab, scFv GL117:MOG, or scFv DEC:MOG followed by fluorescence-labeled corresponding secondary Abs. Merged pictures show binding of anti-DEC Ab and scFv DEC-MOG to the DCs. (C) Flow cytometric analysis of binding of scFv GL117:MOG and scFv DEC:MOG to BMDCs. (D) scFv DEC-MOG was injected i.v. into C57/Bl6 mice, and 24 h later cryosections of spleen and LN were prepared and stained with anti-CD11c and anti–c-myc biotin followed by the corresponding PE- and FITC-labeled secondary Ab. Binding of injected scFv DEC-MOG to CD11c+ DCs was detected in spleen and LNs. (B–D) Data show one exemplary result from three (C) or two (B, D) experiments.

ScFv DEC:MOG is taken up by DCs in vitro and in vivo, and mediate activation of MOG-specific T cells

Because this system of Ag targeting was originally devised to allow loading of DCs in vivo with the MOG Ag, we next pulsed BMDCs with different concentrations of scFv DEC:MOG, scFv GL117:MOG, and MOG peptide, and added 2D2 T cells. To rule out that binding of a DEC-specific scFv per se activates DCs and causes unspecific T cell proliferation, we included a control by pulsing DCs with scFv DEC:TRP2. This conjugate contains the EAE-unrelated tumor peptide TRP2 (10). When analyzing the proliferation of the MOG-specific 2D2 T cells, we could show (Fig. 2A) that scFv DEC:MOG-pulsed DCs induced strong T cell proliferation in contrast with their scFv GL117:MOG or scFv DEC-TRP2–pulsed counterparts. Proliferation was even induced after pulsing with the low 10-ng amount of scFv DEC:MOG. Moreover, the proliferation observed in scFv DEC:MOG-pulsed samples was higher as compared with the peptide-pulsed samples, which indicate that the DEC targeting may help to concentrate the Ag by receptor-mediated endocytosis within the cells and/or targets the Ag intracellular to highly effective Ag processing compartments. To further test whether scFv DEC:MOG is also able to load DCs with Ags in vivo, we injected mice with scFv DEC:MOG, scFv GL117:MOG, scFv DEC:TRP2, and MOG peptide, respectively, isolated splenic DCs 24 h later and set up cocultures with 2D2 T cells. Fig. 2B shows that DCs from scFv DEC:MOG-injected mice were able to induce robust T cell proliferation, which again was higher than that obtained after peptide injection. In contrast, T cells from scFv GL117:MOG-injected mice, as well as from mice injected with scFv DEC:TRP2, failed to induce any substantial T cell proliferation. Likewise, CD11+ and CD4+ cells alone did not proliferate. Thus, these data indicate that DEC:MOG fusion scFv is superior in loading DCs with the MOG Ag in vivo and facilitates access of the antigenic cargo to Ag loading compartments.

FIGURE 2.
FIGURE 2.

scFv DEC:MOG is taken up by DCs in vitro and in vivo, and mediates activation of MOG-specific T cells. Proliferation of MOG-specific CD4+ T cells isolated from 2D2 mice induced by (A) differently pulsed BMDCs or by (B) CD11c+ DCs isolated from spleen of differently treated mice. Data shown represent the mean ± SD from triplicates of one characteristic experiment out of four (*p < 0.005) in (A) and the mean ± SD of four experiments (*p < 0.001) in (B). Asterisks indicate significant differences compared with the respective scFv GL117:DEC-treated groups.

Injection of scFv DEC:MOG induces activated, IL-10–producing CD4+CD25+Foxp3+ Tregs

Because nonactivated, so-called steady-state DCs induce tolerance by means of induction of Tregs (12), we next asked what are the immunological consequences of targeting steady-state DCs in situ by scFv DEC:MOG.

At first we tested whether injection of different scFv conjugates affects the frequency of CD25+Foxp3+ Tregs within the CD4+ T cell compartment in different lymphoid organs and in blood. Exemplary FACS results, as well as a summary of the data, are depicted in Fig. 3A. We found that the total amount of CD4+Foxp3− T cells did not change upon injection of the respective scFv conjugates. In contrast, we recorded a substantially increased frequency of Tregs in scFv DEC:MOG-injected animals (Fig. 3A) in all organs tested, whereby the highest increase was observed in spleens. In comparison, injection of scFv GL117:MOG did not alter the number of Tregs as compared with controls. Moreover, when analyzing the total number of Foxp3+ cells in a representative spleen, in pooled peripheral LNs, and in 300 μl blood, we found elevated quantities of Tregs in scFv DEC:MOG-injected animals, as compared with controls. Thus, these results indicate that an increase of absolute numbers of Tregs in lymphoid organs after scFv DEC:MOG injection may be involved in mediating the suppressive effects observed after scFv DEC:MOG injection.

FIGURE 3.
FIGURE 3.

Injection of scFv DEC:MOG induces activated CD4+CD25+Foxp3+ Tregs. Mice were i.v. injected with 10 μg scFv DEC:MOG or scFv GL117:MOG, or left untreated (naive). After 24 h, mice were sacrificed and cell suspensions were prepared from spleens, pooled LNs, and 300 μl blood. (A) FACS analysis of CD4+Foxp3− and CD4+CD25+Foxp3+ T cells. Dot plots show one exemplary FACS result from four experiments. Graphs show the mean percentages of CD4+Foxp3− T cells and the Foxp3+ T cells among CD4+ T cells in different organs ± SD of four independent experiments, each with three mice per group. (B) One characteristic example of the absolute amount of Foxp3+ T cells/organ/mouse. (C) FACS analysis of CD4+ Foxp3+CD69+ T cells. Dot plots show one exemplary FACS result from four experiments. Graph shows the mean percentages of CD69+ T cells of CD4+Foxp3+ T cells in the different organs ± SD of four independent experiments, each with three mice per group. (D) Concentrations of IL-10 in cell culture supernatants of isolated Tregs from spleen and LN of the three different groups. Tregs were either stimulated with anti-CD3/anti-CD28 or with MOG-pulsed DCs. Data shown represent the mean ± SD of three independent experiments (*p < 0.001).

Because Tregs have to be activated to exert suppressive activity (13), we next assessed the activation status of CD4+Foxp3+ T cells by quantifying the surface expression of the activation marker CD69. In this study, we show (Fig. 3C) that injection of scFv DEC:MOG upregulated CD69 in Tregs, indicating an activated phenotype of Tregs in vivo, whereas no substantial changes in CD69 expression in untreated and scFv GL117:MOG-injected mice were apparent. Moreover, isolated Tregs from all groups were subjected to in vitro suppression assays, and equal suppression was recorded (data not shown).

In contrast with the ex vivo suppressive capacity, the secretion of immunosuppressive IL-10 differed. Fig. 3D shows that Tregs isolated from scFv DEC:MOG-injected animals produced significantly enhanced amounts of IL-10 after unspecific restimulation by anti-CD3/CD28 as compared with the respective controls. Of note, the IL-10 production was even further increased after restimulating Tregs with MOG peptide-pulsed DCs, indicating that the cognate MHC-peptide–TCR interaction is potently able to induce IL-10 production in scFv DEC:MOG “primed” Tregs. However, production of TGF-β by Tregs was not altered. Thus, these data indicate that scFv DEC:MOG targeting of DCs does act in two ways on Tregs: first, by inducing elevated numbers; and second, by activating them and stimulating IL-10 production.

Injection of scFv DEC:MOG induces immunomodulatory molecules in DCs

In contrast with T cell costimulatory molecules mainly expressed by mature DCs, steady-state DCs express T cell regulatory molecules, such as molecules of the B7H family or suppressive cytokines. To test whether immunoregulatory surface molecules and/or cytokines are expressed by DCs after DEC targeting, we first analyzed the expression (B7H1, B7H2, and B7H3) by splenic DCs. Fig. 4A shows that after injection of scFv DEC:MOG, expression of B7H1 was elevated in DCs. However, the expression of other B7H family members, that is, B7H2 and B7H3, did not change. Moreover, the observed effect on B7H1 expression was specific for scFv DEC:MOG targeting, because respective controls left the surface expression of B7H1 unchanged. Other surface molecules known to affect T cell activation, such as CD40, CD80, CD86, and CTLA-4, did not change substantially (data not shown).

FIGURE 4.
FIGURE 4.

Injection of scFv DEC:MOG induces immunomodulatory molecules in DC. Mice were i.v. injected with 10 μg scFv DEC:MOG or scFv GL117:MOG, or left untreated (naive). After 48 h, CD11c+ DCs were isolated from spleens, analyzed by flow cytometry (A), and stimulated with LPS overnight. Cytokines were detected in the supernatants by ELISA (B). (A) Histogram overlays show B7H expressions in dark gray and the respective isotype controls in light gray. Graphs show the means ± SD of three independent experiments. *p < 0.01. (B) Concentrations of IL-10 and TGF-β in supernatants of the unstimulated and LPS-stimulated isolated CD11c+ DCs are shown. Data shown represent the mean ± SD of five independent experiments. *p < 0.01, **p < 0.005.

In addition to expression of suppressive surface molecules, DCs may also be able to suppress immune reactions by secretion of regulatory cytokines such as IL-10, TGF-β, or both. To test whether the release of these cytokines by DCs is stimulated by scFv DEC:MOG, we isolated DCs from mice 2 d after injection of the respective scFv conjugates, and cultured them with and without an activating stimulus provided by LPS. Tissue culture supernatants were tested for IL-10 and TGF-β by ELISA. The results depicted in Fig. 4B show that splenic DCs isolated from scFv DEC:MOG-injected mice produced significantly more IL-10, as well as TGF-β, after stimulation than control cells. Of note, even the spontaneous secretion of TGF-β was elevated in DEC:MOG groups. Thus, these data indicate that targeting of the MOG Ag via scFv DEC:MOG to DCs induces a suppressive phenotype of DCs that expresses B7H1 on the surface and secretes IL-10 and TGF-β.

Injection of scFv DEC:MOG prevents EAE

Because our data so far indicated that injection of scFv DEC:MOG stimulates immunoregulatory mechanisms in vivo, we next tried to inhibit autoimmunity in a murine EAE model by injecting scFv fusion proteins. In a preventive approach, mice were injected i.v. with scFv DEC:MOG or scFv GL117:MOG or left untreated before EAE was induced (Fig. 5A, left panel). The disease of the animals was scored every day according to a standardized scale. Fig. 5A shows that injection of scFv DEC:MOG prevents the development of EAE. In contrast, untreated as well as scFv GL117:MOG injected controls showed severe symptoms of EAE. In these groups, two to three mice (out of six) showed complete paralysis of the hind limbs (score, 7). To further rule out that DEC targeting alone may trigger anti-EAE mechanisms, we also injected mice with anti-DEC scFv Abs carrying the EAE unrelated TRP peptide (scFv DEC:TRP2). Similar to controls (Fig. 5A right panel), these animals had severe EAE symptoms, indicating that targeting of the DEC205 receptor alone was not sufficient to induce tolerance to EAE.

FIGURE 5.
FIGURE 5.

Injection of scFv DEC:MOG prevents EAE. Mice were i.v. injected with 10 μg scFv DEC:MOG, scFv GL117:MOG, or scFv DEC:TRP2, or left untreated (EAE) before EAE was induced. In some experiments, anti–TGF-β Ab (15 mg/kg body weight) was i.p. injected before, during, and after scFv DEC:MOG treatment. (A) Schematic of a preventive EAE experiment. Each graph shows the average scores of one characteristic experiment (n = 6) out of four. (B) Average score of EAE symptoms of a preventive EAE experiment with different injection routes for the scFv DEC-MOG (s.c., i.p., or i.v.). Graph shows the average score of one characteristic experiment (n = 6) out of two. (C) H&E staining of spinal cord sections shows typical inflammatory infiltrates in EAE- and scFv GL117:MOG-treated mice. (D) Pictures of the isolated brains, cerebellum/brainstem sections, and FACS analysis of brain cell suspensions of respective mice stained with CD4. The FL-1 channel was not used. Shown in all cases is one exemplary result, typical for three independent experiments. Scale bars, 100 μm. PT, Pertussis toxin.

Because TGF-β secretion by DCs is enhanced after scFv DEC:MOG targeting (Fig. 4B), we next tested whether it is instrumental for the EAE reduction by scFv DEC:MOG. Therefore, we blocked TGF-β by injections of anti–TGF-β Abs before and during treatment of mice with scFv DEC:MOG (Fig. 5A). In this study, we could completely abrogate the immunosuppressive effects of scFv DEC:MOG, demonstrating that TGF-β plays a substantial role in preventing EAE.

In a further set of experiments, we tested whether the injection route of scFv DEC:MOG affects the potency of the EAE suppression. Therefore, we injected groups of mice with scFv DEC:MOG i.v., i.p., and s.c., respectively (Fig. 5B), and according to the protocol depicted in Fig. 5A, EAE was induced 3 d after the last injection. In these experiments, the i.v. injection gave the most striking results, with protecting >90% of the mice from EAE. However, s.c. and i.p. injection were also able to alleviate EAE as compared with untreated controls, but the potency of these injection routes to suppress EAE was significantly reduced as compared with i.v. injection. Thus, in further experiments, we used i.v. application.

The macroscopic signs of protection from EAE by scFv DEC:MOG injection were also corroborated by other immunologic parameters. For instance, H&E staining of spinal cord sections revealed infiltrating leukocytes (Fig. 5C). When whole brains were prepared from differently treated mice (Fig. 5D), it became apparent that untreated and scFv GL117:MOG-treated brains were swollen and displayed increased vascularization. Microscopically increased numbers of infiltrating leukocytes at the cerebellum–brainstem interface were detectable by HE staining. Immunohistology of brain sections, as well as FACS analysis, revealed massive infiltration by CD4+ T cells. Collectively, these parameters are all signs of brain inflammation in untreated and in scFv GL117:MOG-injected mice. In contrast, scFv DEC:MOG-treated mice showed no signs of inflammation and remained healthy throughout the experiments.

Injection of scFv DEC:MOG reduces Th1/Th17 cells and induces a suppressive phenotype of DCs

Because EAE is a Th1/Th17-driven disease, we next investigated the cytokine profile of CD4+ T cells, isolated from differently treated groups and different organs. Intracellular cytokines were quantified after overnight stimulation using standard protocols. Fig. 6A shows exemplary results of a FACS staining and a summary of all data obtained. In this study, it became apparent that massive induction of IL-17, as well as IFN-γ, occurs in CD4+ T cells in lymphoid organs and in brains of untreated and scFv GL117:MOG-injected mice (Fig. 6A). Moreover, when isolating total brain cells and cultivating them overnight without any further activating stimuli, ELISA revealed significantly elevated levels of IL-17 and IFN-γ in untreated and in scFv GL117:MOG-treated animals (Fig. 6B). In contrast, all cytokine levels remained normal in scFv DEC:MOG-injected mice as compared with healthy controls.

FIGURE 6.
FIGURE 6.

Injection of scFv DEC:MOG reduces Th1/Th17 cells and induces a suppressive phenotype of DCs. Mice were i.v. injected twice with 10 μg scFv DEC:MOG or scFv GL117:MOG, or left untreated (EAE) before EAE was induced. (A) Intracellular detection of IL-17 and IFN-γ in CD4+ T cells using flow cytometry. Dot plots show one exemplary FACS result from the spleens. Graphs show the mean percentages ± SD of IL-17– or IFN-γ–producing CD4+ T cells in LN, spleen, and brain of three independent experiments, each with five mice per group. (B) Concentrations of IL-17 and IFN-γ in supernatants of isolated brain cells. Data shown represent the mean ± SD of three independent experiments. (C and D) CD11c+ DCs were isolated from spleens on day 15 after EAE induction, and (C) were stimulated with LPS for 24 h to measure the cytokines in the supernatants or (D) were cocultured with allogeneic CD4+ T cells to detect the stimulatory capacity. Data show the mean values ± SD of three independent experiments. *p < 0.05, **p < 0.001, indicate significant differences between the mice with severe EAE symptoms (EAE) and the scFv DEC:MOG-treated group.

Because EAE is induced by injection of MOG peptide emulsified in CFA, which contains DC-activating bacteria, we next wanted to investigate whether the less stimulatory phenotype of CD11c+ DCs induced by DEC targeting (Fig. 4) is maintained during the course of EAE. We therefore analyzed DC functions after induction of EAE. Fig. 6C shows that CD11c+ DCs derived from scFv DEC:MOG-injected animals, as compared with controls, still secreted significantly more TGF-β and IL-10 even 15 d after EAE induction.

To test the function of the DCs, we subjected them to allogeneic MLRs, and a significant difference in their stimulatory capacity was apparent (Fig. 6D). That is, DCs derived from animals that were treated with scFv DEC:MOG before induction of EAE were inferior to induce T cell proliferation as compared with respective controls. Thus, DCs acquire and maintain a nonimmunogenic phenotype after scFv DEC:MOG targeting in the course of EAE.

scFv DEC:MOG does cure early EAE symptoms

Although the “tolerization” approach was clearly effective in protecting the mice from EAE, in a clinical setting it would be beneficial to treat already established EAE. Therefore, we tested whether scFv DEC:MOG are also able to block existing EAE. EAE was induced first, and after all mice showed early symptoms (score 1), animals were treated twice with 10 μg scFv DEC:MOG or 10 μg scFv GL117:MOG, respectively. Control groups were left untreated (Fig. 7). This procedure resulted in abrogation of EAE in scFv DEC:MOG-injected animals. In contrast, mice treated with scFv GL117:MOG experienced a severe EAE similar to untreated controls. Thus, these data indicate that “tolerization” with scFv DEC:MOG potently prevents EAE and, moreover, scFv DEC:MOG is even able to cure already existing EAE symptoms in a therapeutic EAE model.

FIGURE 7.
FIGURE 7.

scFv DEC:MOG does cure early EAE symptoms. Schematic of a therapeutic EAE experiment. Graph shows the average score of one characteristic experiment (n = 6) depicting results typical for three independent experiments. The three groups were either only induced with EAE (EAE) or injected with scFv GL117:MOG or scFv DEC:MOG twice after the first symptoms of EAE were observed.

Discussion

DCs are known to be the key inducers of immunity by taking up Ags in the periphery of the body and presenting them to T cells in secondary lymphoid organs. During infection and inflammation, DCs mature into potent T cell stimulatory cells that express several T cell coactivating molecules, such as CD80 and CD86, as well as proinflammatory cytokines (i.e., IL-12, IL-6) (14). In this situation, endocytosis of Ags by DCs takes place under inflammatory conditions, whereby TLRs and pattern recognition receptors will be triggered in DCs, leading ultimately to an activated phenotype of DC. In contrast, when Ag targeting by Abs is used, DCs take up the respective Ags under noninflammatory conditions and activation of the DC is negligible. Thus, a “steady-state” phenotype of DC results, which does not trigger activation of T cells (3).

In our experiments, we show that steady-state DCs in secondary lymphoid organs can be loaded with Ags using scFv DEC:Ag conjugates, and that these steady-state DCs induce Tregs rather than effector T cells. The DEC205 molecule is an ideal candidate for targeting Ags to DCs in vivo, because this molecule mediates constitutive uptake of Ag and presentation (11). Other surface molecules such as DC-SIGN, DCIR, Langerin, and Clec9A, which are related to the DEC205-like lectin receptors, have been shown to be capable of carrying Ags to DC and to improve vaccination against respective Ags as well (15–17). However, DEC205 contains unique intracellular domains, which route ligands directly to MHC class II+ compartments and mediate repetitive recycling of DEC205 to the cell membrane (11). Thus, Ags taken up by DEC205 are effectively degraded to immunostimulatory peptides and are presented to T cells for up to 3 d, as compared with other Ag receptors (5).

It has been proved repeatedly that DEC205 can be used for the induction of immunity. For instance, targeting of tumor Ags such as TRP2 and survivin to DCs in vivo by anti-DEC205 Ab conjugates has generated long-lasting antitumor immunity, and targeting of DEC205 was even successful to battle infections (10, 18–22). These vaccination approaches were carried out by injecting DEC205:Ag conjugates concomitantly with DC-activating reagents such as CpG, LPS, and polyinosinic-polycytidylic acid, and, therefore, immunity ensued. When Ags are given via DEC205 without any further DC-activating stimuli, tolerogenic effects were observed in experimental settings using OVA and hemagglutinin transgenic animals (7, 23). As basic mechanisms of tolerance induction by DEC205 targeting, apoptosis of effector T cells and induction of Tregs have been shown (6, 7, 24).

In our study, we recorded enhanced secretion of TGF-β by DCs after targeting with scFv DEC:MOG, which is one key factor for the de novo induction of Tregs (25). These observations are in line with results obtained in gene chip arrays of different DC subpopulations. The DEC205+ DCs indeed displayed increased levels of molecules involved in TGF-β signaling pathways, and DEC205+ DCs produced a higher amount of TGF-β as compared with DEC205− counterparts (26).

In addition to increased TGF-β levels, DCs also produced more IL-10 after scFv DEC:MOG targeting. IL-10 can contribute to Treg conversion, too, (27) and is involved in directly suppressing EAE (28, 29). Moreover, IL-10 may also act in an autocrine fashion on DCs by preventing upregulation of T cell costimulatory molecules and keeping DCs immature (30, 31). Therefore, the DC-derived IL-10 may not only have direct effects on Treg generation and EAE suppression, but may be also involved in maintenance of steady-state DCs by an autocrine feedback loop. However, our data indicate that even after applying “general” DC activation stimuli, that is, after the induction of EAE by injection of CFA-MOG, scFv DEC:MOG targeting was able to induce anti-inflammatory mechanisms. This may be due to the fact that DEC205+ DCs, apart from the steady-state, are also intrinsically prone to act tolerogenic. For example, the expression of genes coding for immunosuppressive molecules may be constitutively upregulated in DEC205+ DCs, which was shown for genes of the TGF-β pathway (26). Moreover, a division of functions may exist among DC subpopulations in vivo, whereby the initial induction of EAE is driven by a proinflammatory DEC205−CD8− DC subset, but later scFv DEC:MOG targeting specifically stimulates tolerogenic, DEC205+CD8+ DCs. Thus, the DEC205− proinflammatory DC subset cannot maintain the activation of effector T cells because it is devoid of the MOG Ag. Instead, the MOG-loaded tolerogenic DEC205+CD8+ DCs take over.

Further evidence for tolerogenic functions of DEC205+ DCs derives from observations in spleen, as in this study the DEC205+ subset is specialized to take up and to present apoptotic debris, resulting in apoptosis of allogeneic T cells. Moreover, DEC205+ cells in the liver have been described to possess immunomodulatory functions as compared with their respective DEC205− counterparts (32, 33). Thus, these data are in line with the hypothesis that defines DEC205+ cells as immunosuppressive “veto” cells (34).

Targeting of DEC205 also upregulated expression of B7H1 molecules. Although somewhat debated, B7H1 molecules mainly convey immunosuppressive signals to leukocytes (35). B7H molecules have been shown to downregulate proliferation of T cells and cytokine expression thereof (36–39), and in particular for EAE it has been shown that expression of B7H1 ameliorates the course of the disease (38). In addition to these direct suppressive effects of B7H1, it may also be involved in the interplay with Tregs. For instance, during melanoma growth, it has been shown that Tregs stimulate the expression of B7H1 on myeloid suppressor cells (40). However, similar effects may also be in place in DCs, whereby initial anti-DEC targeting is enabling steady-state DCs to induce Tregs, which, in turn, augments surface expression of immunosuppressive B7H1 molecules.

In summary, these data show that Ag loading of DCs via DEC:Ag conjugates leads to activation of several immunosuppressive mechanisms, including engagement of Tregs, the expression of immunosuppressive B7H1 molecules, and the secretion of TGF-β. Altogether, these mechanisms are able to cure autoimmunity in a murine EAE model. Because human DCs express a homologous DEC205 molecule on their surface (41), future investigation may be initiated to elucidate the potential benefits of DEC targeting in human autoimmune diseases.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Marianne Thome for excellent technical help. We thank Dr. Roland Kontermann (University of Stuttgart) for providing tools for scFv production.

Footnotes

  • This work was supported by Deutsche Forschungsgemeinschaft Grant MA 1924-7/1 (to K.M.).

  • Abbreviations used in this article:

    BMDC
    bone marrow–derived dendritic cell
    DEC205
    dendritic and epithelial cell receptor with an m.w. of 205 kDa
    DC
    dendritic cell
    EAE
    experimental allergic encephalomyelitis
    LN
    lymph node
    MOG
    myelin oligodendrocyte glycoprotein
    scFv
    single-chain fragment variable
    Treg
    regulatory T cell
    TRP2
    tyrosinase-related protein 2.

  • Received September 14, 2012.
  • Accepted July 8, 2013.

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