In this study, a tolerogenic artificial APC (TaAPC) was developed to directly and selectively modulate myelin-autoreactive CD4+ and CD8+ T cells in the myelin oligodendrocyte glycoprotein (MOG)35–55 peptide–induced experimental autoimmune encephalomyelitis in C57BL/6J mice. Cell-sized polylactic-coglycolic acid microparticles were generated to cocouple target Ags (MOG40–54/H-2Db-Ig dimer, MOG35–55/I-Ab multimer), regulatory molecules (anti-Fas and PD-L1-Fc), and “self-marker” CD47-Fc and encapsulate inhibitory cytokine (TGF-β1). Four infusions of the TaAPCs markedly and durably inhibited the experimental autoimmune encephalomyelitis progression and reduced the local inflammation in CNS tissue. They circulated throughout vasculature into peripheral lymphoid tissues and various organs, but not into brain, with retention of 36 h and exerted direct effects on T cells in vivo and in vitro. Two infusions of the TaAPCs depleted 65–79% of MOG35–55-specific CD4+ and 46–62% of MOG40–54-specific CD8+ T cells in peripheral blood, spleen, and CNS tissues in an Ag-specific manner and regulatory molecule–dependent fashion; induced robust T cell apoptosis; inhibited the activation and proliferation of MOG peptide–reactive T cells; reduced MOG peptide–reactive Th1, Th17, and Tc17 cells; and expanded regulatory T cells. They also inhibited IFN-γ/IL-17A secretion and elevated IL-10/TGF-β1 production in splenocytes but not in CNS tissue. More importantly, the TaAPCs treatment did not obviously suppress the overall immune function of host. To our knowledge, this study provides the first experimental evidence for the capability of TaAPCs to directly modulate autoreactive T cells by surface presentation of multiple ligands and paracrine release of cytokine, thus suggesting a novel Ag-specific immunotherapy for the T cell–mediated autoimmune diseases.

Multiple sclerosis (MS) is an inflammatory disease of CNS in which immune cells target and destroy myelin sheath on nerve cells, thereby causing autoimmune demyelination and consequent neurologic dysfunction (13). Myelin Ag–autoreactive CD4+ T cells and proinflammatory CD4+ T cells play a pivotal role in MS and its animal model of experimental autoimmune encephalomyelitis (EAE) (4, 5), whereas myelin Ag–autoreactive CD8+ T cells and B cells also make partial contributions to the progress of MS and EAE (68). But the precise pathogenesis of MS remains unknown because of its diverse performances (2, 9). The current therapeutics such as natalizumab (9, 10), fingolimod, (11) and immunosuppressive regimens (laquinimod, cladribine, alemtuzumab, caclizumab, and rituximab) are primarily anti-inflammatory and non–Ag-specific in nature and have severe side effects (1214). As a result of long-term medication, most of these treatments for MS display suppression of overall immune response, which increases the risks of infection and cancer (15, 16).

Thus, recently, increasing attentions have shifted toward the autoantigen-specific immunotherapy. Numerous strategies have been demonstrated in the treatment of EAE or MS, such as the soluble myelin peptide immunotherapy including glatiramer acetate, mixture of MBP85–99, PLP139–151 and myelin oligodendrocyte glycoprotein (MOG)35–55 (17), and altered peptide ligand (18); soluble autoantigen arrays (SAgAs) like codelivery of PLP139–151 and regulatory molecules by hyaluronic acid polymer chain (19, 20); myelin Ag–decorated apoptotic dendritic cells (DCs) (21) or spleen cells (22); myelin protein or peptide-decorated or encapsulated polystyrene or polylactic-coglycolic acid (PLGA) particles (2325); and recombinant TCR ligand–like RTL1000, a recombinant fusion protein of HLA-DR2–ɑ1β1–hMOG35–55 used in MS clinical trials (26). However, most of these modulators act in a semi or indirect Ag-specific manner to induce autoantigen-specific suppression and tolerance. These treatments prevent the development or relapse of EAE or MS in large part because of their ability to induce tolerogenic APCs, especially DCs and macrophages, by alternative activation after uptake of myelin Ags or peptides. Then the induced tolerogenic APCs will secrete inhibitory cytokines (like TGF-β and IL-10) and express regulatory molecules (like FasL and PD-L1), subsequently promote regulatory T cell (Treg) production, and inhibit the function of myelin protein–autoreactive Th1 and Th17 cells through various signal pathways. Although these strategies present intriguing potential to confer tolerance in EAE or MS, multiple factors can influence the induction of tolerogenic APCs in vivo. These include the diverse types (27, 28), tissue specificities (23, 24, 29, 30), and surface/nuclear receptors (23, 31, 32) of APCs. Additionally, these treatments rarely involve the concomitant modulation of myelin-autoreactive CD8+ T cell and CD4+ T cells. By now, few studies focus on the direct depletion and modulation of myelin-autoreactive T cells, one of the ideal strategies for the treatment of T cell–mediated EAE and MS.

The present study aims to develop a direct Ag-specific immune modulator targeting the myelin-autoreactive CD4+ and CD8+ T cells by the concomitant delivery of myelin peptide-loaded MHC (pMHC) multimers and multiple regulatory molecules in the same spatial and temporal context for the treatment of EAE and MS. For this purpose, a tolerogenic artificial APC (TaAPC) was established by using a polymeric biomimetic microparticle (MP) platform. PLGA, a biocompatible and biodegradable polymer widely used in drug and vaccine delivery systems in human (33), was employed to generate cell-sized MPs as a scaffold to cocoupling the target Ags (MOG40–54/H-2Db-Ig dimer, MOG35–55/I-Ab-biotin multimer), regulatory molecules (anti-Fas and PD-L1–Fc), and “self-marker” CD47-Fc on their surface and encapsulating inhibitory cytokine (TGF-β1) inside. The multipotent TaAPCs were i.v. administered into MOG35–55-induced active EAE murine model and followed by the investigation of therapeutic outcomes, precise mechanism, and side effects. This study provides the first experimental evidence, to our knowledge, for the capability of TaAPCs to directly modulate autoreactive T cells by surface presentation of multiple ligands and paracrine release of cytokine, thus suggesting a novel Ag-specific immunotherapy, a desirable avenue, for the treatment of EAE and MS.

Female C57BL/6J mice were obtained from the Comparative Medicine Center of Yangzhou University (Yangzhou, China) and maintained in the specific pathogen-free Laboratory Animal Centre of Southeast University (Nanjing, China). Animal welfare and experimental procedures were approved by the Animal Ethics Committee of Southeast University and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (publications no. 8023, revised 1978). Eight approximately 10-wk-old mice were used in the experiments. Yac-1 cell line and B16F10 melanoma cell line were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). MOG35–55 (MEVGWYRSPFSRVVHLYRNGK), MOG40–54 (YRSPFSRVVHLYRNG), and influenza A virus nucleoprotein (NP) 366–374 (ASNENMETM) were synthesized by ChinaPeptides Biotech (Suzhou, China) with a purity >95%.

Polyethyleneimine (PEI)–coated PLGA MPs were fabricated by using double-emulsion water-in-oil-in-water method as we previously described (34), with the minor modifications of cytokine encapsulation. Twenty milligrams of PLGA polymer was dissolved in 1 ml of methylene chloride, and then TGF-β1 (2.5 μg) (PeproTech) dissolved in PBS was added in the suspension. Double-emulsion method was followed to generate the TGF-β1–encapsulated PLGA MPs (PLGA-MPsTGF-β1), followed by preparation of PEI-conjugated PLGA-MPsTGF-β1 by using modified EDC/NHS chemistry. Additionally, the PEI-conjugated PLGA MPs without encapsulation of TGF-β1 were also prepared in the same way. The prepared PLGA MPs were then characterized by using scanning electron microscopy (SEM; ZEISS EVO 18; Oberkochen, Germany). The size was measured by dynamic light scattering (BI-90 Particle Sizer; Brookhaven Instruments, Holtsville, NY), and the ζ potential was detected by PALS ζ instrument (Brookhaven Instruments).

MOG40–54/H-2Db-Ig dimers and NP366–374/H-2Db-Ig dimers were prepared by coincubating H-2Db-Ig Dimer X (BD Biosciences) with MOG40–54 or NP366–374 peptide for 48 h at 4°C according to the manufacturer’s instructions. After that, PEI-coated PLGA-MPsTGF-β1 (1 × 108 beads) were coincubated with MOG40–54/H-2Db-Ig dimers (10 μg), anti-Fas mAb (5 μg; BD Biosciences), PD-L1–Fc (2.5 μg; Sino Biological, Beijing, China), CD47-Fc (0.6 μg; R&D Systems), and streptavidin (7 μg; ProZyme) in sterile PBS overnight at 4°C on rotator. Then, MPs were blocked with 30% BSA in PBS for another 24 h at 4°C on rotator. After centrifugation to move the supernatant, the MPs were collected and further incubated with MOG35–55/I-Ab-biotin monomer (5 μg; MBL, Nagoya, Japan) for 4 h at 4°C on rotator. Finally, the resulting MPs, termed M/M-TaAPCs, were washed with PBS and resuspended in sterile PBS for further use. In parallel, several controls of the M/M-TaAPCs were also prepared by following the similar procedure, such as N/O-TaAPCs (cocoupling NP366–374/H-2Db-Ig dimers, OVA323–339/I-Ab-biotin monomers, and other immune molecules), M/M-MPs (the MPs only cocoupling MOG40–54/H-2Db-Ig dimers and MOG35–55/I-Ab-biotin monomers), and blank MPs (the MPs only blocking with BSA).

To monitor the sustained release of TGF-β1 encapsulated into PLGA MPs, TaAPCs in-house (1 × 107) were resuspended in 1 ml of sterile PBS and incubated on rotator at 37°C. The supernatant was collected at indicated time intervals, and an equal volume of sterile PBS was supplemented. The amount of TGF-β1 released in the supernatant was quantified by using TGF-β1 ELISA kit (Dakewe Biotech, Shenzhen, China) and followed by the calculation of release curve. For phenotypic analyses, TaAPCs were stained with FITC-anti–hamster-IgG (eBioscience), PE–anti-mouse I-Ab (eBioscience), and allophycocyanin–anti-mouse IgG1 (BD Biosciences) or allophycocyanin–anti-human IgG1 (Miltenyi Biotec, Bergisch Gladbach, Germany) for 30 min at 4°C. After washing, the TaAPCs were acquired on a FACSCalibur flow cytometer (BD Biosciences) and observed under confocal laser scanning microscopy (FV1000; Olympus, Tokyo, Japan).

On day 0, active EAE model was induced in female C57BL/6J mice by multipoint s.c. injections with MOG35–55 peptide (ChinaPeptides Biotech, Suzhou, China) emulsified in CFA (Sigma-Aldrich) at a dosage of 300 μg per mouse. Prior to use, CFA was prepared by mixing heat-killed Bacillus Calmette–Guérin (Ruichun Biotech, Shanghai, China) into IFA (Sigma-Aldrich) to achieve a concentration of 10 mg ml−1 of Bacillus Calmette–Guérin. On days 0 and 2, pertussis toxin (Sigma-Aldrich) was administered i.p. into the mice at a dosage of 250 ng per mouse per time point.

Then, mice were randomly assigned to one of five treatment groups (6–8 mice per group) and injected i.v. with PBS, blank MPs, M/M-MPs, N/O-TaAPCs, and M/M-TaAPCs, respectively, on days 8, 18, 28, and 38 after MOG immunization (1 × 107 MPs per mouse per time point). After EAE induction and TaAPCs treatments, mice were monitored daily by two investigators in a blinded manner, and the severity of disease was scored as described (35) with the following criteria: grade 0, normal; grade 1, flaccid tail; grade 2, mild hindlimb weakness (quick righting reflex); grade 3, severe hindlimb weakness (slow righting reflex); grade 4, hindlimb paralysis; grade 5, hindlimb paralysis and partial forelimb weakness or death.

In parallel, i.p. injections and s.c. injections were also carried out on days 8, 18, 28, and 38 after MOG immunization (1 × 107 MPs per mouse per time point) in a similar way. The late therapeutic administrations on days 18, 28, and 38 via tail vein were performed in the EAE mice with the same dosage.

The spinal cord tissues were isolated from the EAE mice after i.v. injections on day 100 after MOG immunization. All specimens were embedded in paraffin, and sections with a thickness of 5–7 μm were prepared. Then sections were routinely stained with H&E and Luxol Fast Blue (LFB; Boster Biological Technology, Wuhan, China), respectively. Inflammatory infiltration and demyelization in the tissues were evaluated under microscope (Eclipse 80i; Nikon, Tokyo, Japan). The mean number of inflammatory cells was obtained by counting five separated fields (100×) by Image-Pro Plus software (Media Cybernetics, Rockville, MD). Quantification of spinal cord demyelization was assessed by two investigators in a blinded manner as described previously (36): score 0, no demyelination; score 1, mild demyelination; score 2, moderate demyelination, and score 3: severe demyelination. The average score from five spinal cord sections of each animal was calculated.

To monitor the in vivo trafficking of TaAPCs, indocyanine green (ICG)–inlayed TaAPCs were generated in the same way. Briefly, ICG (Sigma-Aldrich), TGF-β1, and PLGA polymer were dissolved in dichloromethane, and the double-emulsion solvent evaporation method was followed to generate the ICG-inlayed PLGA-MPsTGF-β1. Then the MPs were surface modified with PEI and cocoupled with MOG-specific pMHC multimers, anti-Fas, PD-L1–Fc, and CD47-Fc to prepare the ICG-encapsulated M/M-TaAPCs. Similarly, the ICG-inlayed M/M-TaAPCs (CD47-Fc), N/O-TaAPCs, and blank MPs were generated in parallel. On day 18 after MOG immunization, the EAE mice were randomized into four groups followed by the injections of different ICG MPs, respectively, via the tail vein (1 × 107 MPs per mouse). The mice were then anesthetized by isoflurane and imaged by using Maestro in vivo fluorescence imaging system (CRi, Woburn, MA) at indicated time points. Images were captured at an excitation wavelength of 710–745 nm and an emission wavelength of 780–840 nm. At 4 h after injection, the mice from each group were sacrificed, and organs (liver, spleen, kidneys, lymph nodes, heart, brain, and lungs) were dissected surgically for ex vivo imaging.

PE-labeled TaAPCs were generated by cocoupling PE–streptavidin (BD Biosciences) and other immune molecules onto PEI-conjugated PLGA-MPsTGF-β1. In parallel, PE-labeled M/M-TaAPCs (CD47-Fc), N/O-TaAPCs, and blank MPs were also generated as controls. On day 18 after MOG immunization, the PE-labeled control MPs and TaAPCs were injected, respectively, into the EAE mice (1 × 107 MPs per mouse) via tail vein. Peripheral blood from the orbital venous plexus, spleen, and lymph nodes were collected at 30 min, 4h, and 4 h after injection, respectively, in dark, and processed to single-cell suspensions. Wright’s staining was carried out, and slides were observed under optical microscope (Eclipse 80i; Nikon). The spleen cells and lymph node cells (LNCs) were freshly acquired on a FACSCalibur flow cytometer (BD Biosciences) without any staining.

Additionally, spleens were collected in dark from each group and embedded in freezing medium (O.C.T, Sakura Finetek). Frozen sections with a thickness of 9–10 μm were prepared and stained with FITC–anti-mouse CD4 (GK1.5), FITC–anti-mouse CD8a (53-6.7), FITC–anti-mouse CD11c (N418), FITC–anti-mouse CD19 (MB19-1), or FITC–anti-mouse F4/80 (BM8) (all from eBioscience) for 1 h at room temperature. After washing, the sections were further stained with DAPI (Sigma-Aldrich) for 5 min and finally visualized under confocal laser scanning microscopy (Olympus). In parallel, the isotype controls were also stained using FITC–rat IgG2bκ or FITC–rat IgG2aκ (eBioscience). Meanwhile, the spleens from EAE mice were processed into single-cells suspensions, stained with FITC-labeled mAbs specific for CD4+ T cells, CD8+ T cells, B cells, DCs, and macrophages, respectively, and followed by flow cytometric analyses. The PE+/FITC+ signals (presumably MP–cell conjugates) were quantified with a visible percentage in the spleen cell suspensions.

Peripheral blood, spleens, and CNS tissues (brain and spinal cord) were collected from EAE mice after i.v. administrations on day 20 after MOG immunization and processed to single-cell suspensions. PBMCs or splenocytes (SPCs) were prepared routinely. The mononuclear cells (MNCs) in CNS tissues were further isolated by using discontinuous 70%/30% Percoll gradients (Pharmacia, Stockholm, Sweden) as previously described (37). The cells were then seeded in 24-well cell culture plate (5 × 106 cells well−1) and coincubated with MOG35–55 peptide (20 μg ml−1) plus IL-2 (30 pg ml−1; PeproTech) or MOG40–54 peptide (20 μg ml−1) plus IL-2 (100 pg ml−1) for 6 d in RPMI 1640 medium supplemented with 10% FBS (Life Technologies), at 37°C in 5% CO2 and humidified conditions.

The cells were then harvested and incubated with anti-mouse CD16/CD32 (eBioscience) for 20 min at 4°C to block the Fc receptors. To detect MOG Ag–specific CD4+ T cells, the cells were further incubated with allophycocyanin-labeled MOG35–55/I-Ab tetramer or allophycocyanin-labeled OVA323–339/I-Ab tetramer (10 μl per tube) (MBL) for 30 min in dark at 4°C. FITC–anti-mouse CD4 (GK 1.5) and PE–anti-mouse CD3e (145-2C11) (eBioscience) were added to each tube for another 30 min incubation at 4°C in the dark. For the detection of MOG Ag–specific CD8+ T cells, H-2Db-Ig/peptide dimers (BD Biosciences) were incubated with allophycocyanin-labeled anti-mouse IgG1 (BD Biosciences) for 1 h at 4°C. Then, the cells were stained by the mixture of MOG40–54/H-2Db-Ig dimers or NP366–374/H-2Db-Ig dimers with allophycocyanin-labeled anti-mouse IgG1 for 30 min at 4°C. Finally, the cells were stained with FITC–anti-mouse CD8a (53-6.7; eBioscience) and PE–anti-mouse CD3e for 30 min at 4°C. In parallel, the isotype controls were stained also by using PE–Armenian hamster IgG, FITC–rat IgG2bκ, or FITC–rat IgG2aκ (eBioscience). After washing with PBS, the cells were acquired on a FACSCalibur flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR).

Peripheral blood and spleens were collected from EAE mice after i.v. administrations on day 20 after MOG immunization. The PBMCs and SPCs were prepared and stained with allophycocyanin–anti-mouse CD4 (GK1.5), allophycocyanin–anti-mouse CD8a (53-6.7), or allophycocyanin–anti-mouse CD3e (145-2C11) (eBioscience) for 30 min at 4°C. After washing with PBS, the cells were stained with annexin V and propidium iodide according to the manufacturer’s protocol (eBioscience) and analyzed by flow cytometry. To evaluate T cells activation, the SPCs were freshly stained with FITC–anti-mouse CD3e (145-2C11), allophycocyanin–anti-mouse CD4 (GK1.5) or CD8a (53-6.7), and PE–anti-mouse CD44 (IM7) or CD69 (H1.2F3) (eBioscience) for 30 min at 4°C and followed by flow cytometry. Isotype control staining was also performed as described.

Spleens were separated from the EAE mice after i.v. administrations on day 20 after MOG immunization. SPCs were prepared and incubated with 5 μM of CFSE (Sigma-Aldrich) for 10 min at 37°C, and immediately washed three times with ice-cold RPMI 1640 medium (Life Technologies). Then, the CFSE-labeled SPCs were seeded into round-bottom 96-well plates (1 × 105 cells/well) (BD Falcon) and coincubated with MOG35–55 or MOG40–54 peptides (20 μg ml−1) for 7 d in complete RPMI 1640 medium at 37°C with 5% CO2 and humidified conditions. Cells were harvested, stained with PE–anti-mouse CD3e and allophycocyanin–anti-mouse CD4 or CD8a (eBioscience) for 30 min at 4°C, and analyzed by flow cytometry. Cell divisions were demarcated according to CFSE staining brightness.

For the detection of IFN-γ– or IL-17A–secreting CD4+ T cells and CD8+ T cells, SPCs or LNCs from EAE mice after i.v. administrations were prepared on day 20 and cocultured in 24-well plate (1 × 106 cells well−1) with PMA/ionomycin and BFA/monensin mixture (Multi Sciences, Shanghai, China) for 4 h or stimulated by MOG35–55 or MOG40–54 peptide (20 μg ml−1) for 16 h and followed by addition of protein transport inhibitor BFA for another 5 h at 37°C under 5% CO2 and humidified conditions. Then, the cells were harvested, blocked with anti-mouse CD16/CD32 for 20 min at 4°C, incubated with allophycocyanin–anti-mouse CD8a and FITC–anti-mouse CD4 for 30 min at 4°C, and followed by PE–anti-mouse IFN-γ (XMG1.2) or PE–anti-mouse IL-17A (eBio17B7) (eBioscience) staining for another 30 min at 4°C after fixation/permeabilization. PE–rat IgG1κ (eBRG1) or PE–rat IgG2aκ isotype (eBR2a) staining was carried out in parallel. After washing with PBS, cells were analyzed by flow cytometry. For the detection of Tregs, the Mouse Regulatory T Cell Staining Kit (eBioscience) was used according to the manufacturer’s protocol. Briefly, the fresh SPCs or LNCs were blocked with anti-mouse CD16/CD32, then stained with allophycocyanin–anti-mouse CD25 (PC61.5) and FITC–anti-mouse CD4 (RM4-5). After fixation, the intracellular staining with PE–anti-mouse Foxp3 (FJK-16s) was performed and finally analyzed by flow cytometry.

The supernatants of SPCs were collected from the T cell proliferation assay, and the homogenates of CNS tissues (brain and spinal cord) were prepared. Then, the concentrations of cytokines (IL-17A, IFN-γ, IL-10, and TGF-β1) in these samples were detected by using the mouse cytokine ELISA kit (Dakewe Biotech).

SPCs were prepared from EAE mice after i.v. administrations on day 40 after MOG immunization. A total of 1 × 107 cells were labeled with CFSE as described and then used as effector cells to coculture with target cells (Yac-1 cells, 1 × 105 cells well−1) at indicated ratios of effector to target in round-bottom 96-well plates in complete RPMI 1640 medium for 5 h at 37°C, 5% CO2, and humidified conditions. Cells were then harvested and analyzed by flow cytometry after staining with 7-amino-actinomycin D (7-AAD; eBioscience). The cytotoxic activity of NK cells was calculated as the percentage of 7-AAD–positive cells within the CFSE-negative cell population.

The EAE mice were s.c. injected with B16F10 melanoma cells (1 × 106 cells per mouse) in the right groin on day 3 after MOG immunization. Then, the mice were randomized into three groups (seven mice per group) and administered via tail vein with M/M-TaAPCs, blank MPs, or PBS on days 8, 18, 28, and 38 after MOG immunization (1 × 107 MPs per mouse per time point). The tumor size was measured daily after it became detectable, with a venire caliper, until the size was up to 2500 mm3. The products of perpendicular diameters were determined, and the tumor volume was calculated with following formula: (the shortest diameter)2 × (the longest diameter) × 0.5.

EAE mice were randomized into three groups and administered via tail vein with PBS, blank MPs, or M/M-TaAPCs on days 8, 18, 28, and 38 after MOG immunization. On day 40, the spleen cells were obtained from each EAE mice, labeled with CFSE, and seeded into round-bottom 96-well plates as responder cells (1 × 105 cells well−1), then coincubated with SPCs from BALB/c mice (pretreated with mitomycin C, 1 × 105 cells well−1) or irrelevant peptides (OVA323–339 plus NP366–374, 20 μg ml−1). Cells were cocultured for 7 d in complete RPMI 1640 medium at 37°C, 5% CO2, and humidified conditions, then harvested and stained with allophycocyanin–anti-mouse CD3e for 30 min at 4°C, and finally analyzed by flow cytometry. Cell divisions were demarcated according to CFSE staining brightness.

Brain and spinal cord were isolated from the EAE mice after i.v. administrations on day 20 after MOG immunization. The MNCs were prepared and then stained with allophycocyanin–anti-mouse CD3e (145-2C11), FITC–anti-mouse CD8a (53-6.7), and PE–anti-mouse CD4 (GK1.5) (eBioscience) for 30 min at 4°C, followed by flow cytometry.

Spleen and lymph nodes were harvested from the treated EAE mice on day 40 after MOG immunization. The SPCs and LNCs were prepared and stained with allophycocyanin–anti-mouse CD3e (145-2C11), FITC–anti-mouse CD8a (53-6.7), PE–anti-mouse CD4 (GK1.5), FITC–anti-mouse CD19 (MB19-1), and FITC–anti-mouse NK1.1 (PK136) (eBioscience), respectively, for 30 min at 4°C. After washing with PBS, cells were analyzed by flow cytometry. In parallel, peripheral blood was collected from orbital venous plexus of EAE mice on day 40. Routine blood tests were performed using automated hematology analyzer (XE-2100; Sysmex, Kobe, Japan).

GraphPad Prism 6.0 (GraphPad, La Jolla, CA) software was used to analyze the data statistically. Wilcoxon signed rank test was used to analyze the clinical score curves of EAE and the tumor sizes. To determine the survival curve of mice, a Kaplan–Meier graph was constructed, and a log-rank comparison of the groups was used to calculate the mice survival curve after tumor cells challenge. For other experiments, a two-tailed unpaired Student t test was used to determine differences across groups. All data were presented as the mean ± SD. A result of p < 0.05 was considered significant.

The PEI-conjugated and PLGA-MPsTGF-β1 were generated in-house using double-emulsion method and displayed a spherical shape with smooth surface as characterized by SEM (Fig. 1A). The average diameter of the MPs was 5.08 ± 1.9 μm, and 77.8% of MPs were 3.27–7.78 μm in diameter (Fig. 1B). The mean ζ potential was 45.3 ± 4.38 mV as detected by the PALS ζ instrument, suggesting a high capacity to covalently couple proteins (Fig. 1C).

FIGURE 1.

Characterization of PLGA MPs and multivalent TaAPCs. (A) Representative SEM image of PLGA MPs. (B) Size distribution and (C) ζ-potential (millivolt) distribution of PLGA MPs. (D) Release curve of TGF-β1 from M/M-TaAPCs and blank MPs over 30 d. (E) Phenotypic analyses of TaAPCs by flow cytometry. Blank MPs, M/M-TaAPCs, M/M-TaAPCs (PD-L1), and M/M-TaAPCs (CD47) were generated in parallel and followed by three-color fluorescence staining with PE-anti–I-Ab, FITC–anti-hamster IgG (binding to anti-Fas), and allophycocyanin–anti-mouse IgG1 (binding to H-2Db) or allophycocyanin–anti-human IgG1 (binding to both CD47-Fc and PD-L1–Fc). (F) Phenotypic analyses of TaAPCs using confocal laser scanning microscope after three-color staining (original magnification ×400). (G) Flow cytometric dot plots of TaAPCs were presented in a two-color manner with the percentage of double-positive TaAPCs in the top right quadrant.

FIGURE 1.

Characterization of PLGA MPs and multivalent TaAPCs. (A) Representative SEM image of PLGA MPs. (B) Size distribution and (C) ζ-potential (millivolt) distribution of PLGA MPs. (D) Release curve of TGF-β1 from M/M-TaAPCs and blank MPs over 30 d. (E) Phenotypic analyses of TaAPCs by flow cytometry. Blank MPs, M/M-TaAPCs, M/M-TaAPCs (PD-L1), and M/M-TaAPCs (CD47) were generated in parallel and followed by three-color fluorescence staining with PE-anti–I-Ab, FITC–anti-hamster IgG (binding to anti-Fas), and allophycocyanin–anti-mouse IgG1 (binding to H-2Db) or allophycocyanin–anti-human IgG1 (binding to both CD47-Fc and PD-L1–Fc). (F) Phenotypic analyses of TaAPCs using confocal laser scanning microscope after three-color staining (original magnification ×400). (G) Flow cytometric dot plots of TaAPCs were presented in a two-color manner with the percentage of double-positive TaAPCs in the top right quadrant.

Close modal

To fabricate the multivalent TaAPCs, PLGA-MPsTGF-β1 were further cocoupled with the target Ags (MOG40–54/H-2Db-Ig dimer, MOG35–55/I-Ab multimer), regulatory molecules (anti-Fas, PD-L1–Fc), and self-marker CD47-Fc onto their surface, termed M/M-TaAPCs. As shown in Fig. 1D, the cumulative release efficiency of TGF-β1 was ∼75.2% as measured by ELISA. The total cumulative TGF-β1 released from 1 × 107 beads of M/M-TaAPCs was nearly 188.1 ng over 30 d in a sustained manner, with the rapid release during the first 2 d. To confirm the immobilization of multiple molecules on the surface of TaAPCs, control MPs (M/M-MPs, N/O-TaAPCs, PD-L1 M/M-TaAPCs, and CD47 M/M-TaAPCs) were generated in parallel and followed by three-color staining. The histograms (Fig. 1E) showed that each kind of surface molecule was effectively coupled onto TaAPCs with the strong fluorescence signals. Furthermore, confocal images also confirmed the copresence of the five kinds of surface molecules onto TaAPCs (Fig. 1F). The flow cytometric dot plots (Fig. 1G) revealed that nearly 60–80% of TaAPCs codisplayed the five kinds of surface molecules as determined by double-positive staining in each two-color dot plots. Each batch of TaAPCs was routinely evaluated in this manner prior to use.

Active EAE was induced in female C57BL/6 mice by MOG35–55 peptide immunization as described. The mice were then randomized into five groups and followed by i.v. injections with PBS, blank MPs, M/M-MPs, N/O-TaAPCs, or M/M-TaAPCs on days 8, 18, 28, and 38 after MOG immunization. The clinical manifestation (clinical scores) was recorded daily for 100 d. According to the clinical score curves and pathological analyses, active EAE induced in this study always presented a classic clinical course containing acute onset (near day 10), peak stage (near day 18), and later on stable chronic remission. For each treatment group, the mean clinical score (Fig. 2A), peak clinical score (Fig. 2B), clinical score on day 100 (Fig. 2C), and cumulative clinical score (Fig. 2D) were presented. Mice in the PBS, blank MP, and M/M-MP treatment groups displayed apparent motor dysfunction, without significant difference across groups. Expectedly, the M/M-TaAPC treatment group presented markedly and durably lower clinical scores than the N/O-TaAPC group and other control groups from acute onset to stable chronic remission stage, as analyzed by Wilcoxon signed rank test and paired two-tailed Student t test. Meanwhile, as a noncognate Ag control group, EAE mice treated by N/O-TaAPCs only displayed a transient and slight relief of motor dysfunction at peak stage with no significant difference from other control groups.

FIGURE 2.

TaAPCs markedly and durably ameliorate EAE and reduce local autoimmune response. MOG35–55-induced EAE mice were randomized into five groups and followed by four-time i.v. injections of M/M-TaAPCs or control MPs. (A) The mean clinical scores, (B) peak clinical scores, (C) clinical scores on day 100, and (D) cumulative clinical scores of each treatment group were presented. The clinical effects of TaAPCs were replicated in three independent experiments with 6–8 mice per group in each independent experiment, so the total number in each group was 20–22 mice as shown in the scatter plots of (B) and (C). The data have been analyzed using the uniform average of three independent experiments. Brain and spinal cord tissues were isolated from each group on day 100. (E) Frequencies and numbers of CD3+, CD4+, and CD8+ T cells infiltrated into CNS tissues were determined by flow cytometry. Representative dot plots from M/M-TaAPC and blank MP groups are presented. (F) Inflammatory cells in the spinal cord were detected by H&E staining. The representative staining images from M/M-TaAPC and blank MP groups are presented. White arrows indicate inflammatory cells. The numbers of inflammatory cells are displayed in histograms for each group. (G) Demyelination in the spinal cord was determined by LFB staining. Representative images from M/M-TaAPC and blank MP groups and the demyelination scores in each group are displayed respectively. White arrows point at the demyelination area (n = 6 to 8 mice in each group). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

TaAPCs markedly and durably ameliorate EAE and reduce local autoimmune response. MOG35–55-induced EAE mice were randomized into five groups and followed by four-time i.v. injections of M/M-TaAPCs or control MPs. (A) The mean clinical scores, (B) peak clinical scores, (C) clinical scores on day 100, and (D) cumulative clinical scores of each treatment group were presented. The clinical effects of TaAPCs were replicated in three independent experiments with 6–8 mice per group in each independent experiment, so the total number in each group was 20–22 mice as shown in the scatter plots of (B) and (C). The data have been analyzed using the uniform average of three independent experiments. Brain and spinal cord tissues were isolated from each group on day 100. (E) Frequencies and numbers of CD3+, CD4+, and CD8+ T cells infiltrated into CNS tissues were determined by flow cytometry. Representative dot plots from M/M-TaAPC and blank MP groups are presented. (F) Inflammatory cells in the spinal cord were detected by H&E staining. The representative staining images from M/M-TaAPC and blank MP groups are presented. White arrows indicate inflammatory cells. The numbers of inflammatory cells are displayed in histograms for each group. (G) Demyelination in the spinal cord was determined by LFB staining. Representative images from M/M-TaAPC and blank MP groups and the demyelination scores in each group are displayed respectively. White arrows point at the demyelination area (n = 6 to 8 mice in each group). *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

In parallel, the EAE mice were also administered by i.p. injections or i.v. injections using the same timeline and dosage. As compared with the control groups, i.p. injections of M/M-TaAPCs caused only partial inhibitory effects on EAE progress (Supplemental Fig. 1A), whereas s.c. injections did not present any protective effects (Supplemental Fig. 1B). In addition, the late therapeutic administrations of TaAPCs were carried out in the EAE mice. As shown in Supplemental Fig. 1C, three i.v. injections of M/M-TaAPCs on days 18, 28, and 38 significantly inhibited the progression of EAE as compared with control groups, but the clinical severity was not ameliorated as well as the early administrations on days 8, 18, 28, and 38. Thus, in the later experiments, the immune responses after i.p., i.v., or late on day 18 injections were not further investigated.

To evaluate the local infiltration of T cells after TaAPCs treatment, brain and spinal cord were collected from the EAE mice after i.v. administrations at peak stage (day 20, 2 d after second treatment) and processed to single-cell suspensions followed by fluorescence staining and flow cytometry. The frequencies and numbers of CD3+ cells, CD4+ T cells, and CD8+ T cells in CNS were obviously and concurrently reduced in the M/M-TaAPC–treated mice, as compared with the N/O-TaAPC or blank MP treatment group (Fig. 2E).

Moreover, the local inflammations and myelin loss in spinal cord were investigated with H&E and LFB staining at a long time point (on day 100 after MOG immunization). The numbers of inflammatory cells (Fig. 2F) and the scores of demyelination (Fig. 2G) in CNS tissues were significantly decreased by the i.v. administrations of M/M-TaAPCs relative to the N/O-TaAPC group and other control groups. Both therapeutic outcomes and pathological analyses demonstrated that four injections of M/M-TaAPCs obviously inhibited the progression of EAE with a sustained inhibitory effect over the clinical course of 100 d and reduced local autoimmune response of CNS. As compared with the regulatory molecule–negative control group (M/M-MPs) and noncognate Ag control group (N/O-TaAPCs), the multipotent M/M-TaAPCs worked in an Ag-specific manner and regulatory molecule–dependent fashion in vivo.

EAE mice at peak stage (day 18 after MOG immunization) were injected via tail vein with PE-labeled TaAPCs or PBS. Then the PE-TaAPCs were observed in peripheral blood at 30 min, in spleen and lymph nodes at 4 h after injection by Wright’s staining, and were absent in the cell suspensions from PBS group, a negative control (Fig. 3A). Also, the spleen and LNC suspensions were harvested at 4 h and freshly detected by flow cytometry without any fluorescence staining. Fig. 3B displayed the PE-TaAPCs in these cell suspensions, which may be free or bound to cells. This experiment aims to confirm the distribution of TaAPCs in secondary lymphatic tissues with a visible percentage after i.v. injection.

FIGURE 3.

Tissue distribution and in vivo trafficking of TaAPCs in EAE mice. (A) Wright’s staining for peripheral blood cells, spleen cells, and LNCs from EAE mice at different time points after i.v. injection of TaAPCs or PBS. White arrows indicate the TaAPCs observed under light microscope at original magnification ×1000. (B) Flow cytometric analyses without any fluorescence staining for the fresh spleen cells and LNCs from EAE mice at 4 h after i.v. injection of PE-labeled TaAPCs or PBS. The upper panel is the control SSC/FL-2 flow cytometric dot plots and FL-2 histograms running only the TaAPCs or PE-TaAPCs without any cells. (C) Whole-body near-infrared imaging at indicated time points and ex vivo near-infrared imaging for organs dissected surgically from EAE mice at 4 h time point after i.v. injection of ICG-inlayed TaAPCs or control MPs. (D) The mean intensities of fluorescence in whole body and dissected organs after ICG-inlayed TaAPCs or control MPs injection (n = 3 mice per time point per group). *p < 0.05, **p < 0.01.

FIGURE 3.

Tissue distribution and in vivo trafficking of TaAPCs in EAE mice. (A) Wright’s staining for peripheral blood cells, spleen cells, and LNCs from EAE mice at different time points after i.v. injection of TaAPCs or PBS. White arrows indicate the TaAPCs observed under light microscope at original magnification ×1000. (B) Flow cytometric analyses without any fluorescence staining for the fresh spleen cells and LNCs from EAE mice at 4 h after i.v. injection of PE-labeled TaAPCs or PBS. The upper panel is the control SSC/FL-2 flow cytometric dot plots and FL-2 histograms running only the TaAPCs or PE-TaAPCs without any cells. (C) Whole-body near-infrared imaging at indicated time points and ex vivo near-infrared imaging for organs dissected surgically from EAE mice at 4 h time point after i.v. injection of ICG-inlayed TaAPCs or control MPs. (D) The mean intensities of fluorescence in whole body and dissected organs after ICG-inlayed TaAPCs or control MPs injection (n = 3 mice per time point per group). *p < 0.05, **p < 0.01.

Close modal

To define the fate of TaAPCs in vivo and find out the effects of targeting Ags and self-marker CD47 molecules onto TaAPCs, ICG-inlayed M/M-TaAPCs, M/M-TaAPCs (CD47), N/O-TaAPCs, or blank MPs were injected via tail vein into the EAE mice at peak stage. As revealed by the whole-body fluorescence images at various time points (Fig. 3C, left panel), the fluorescent intensity in mice was strongest during 6 h after ICG–M/M-TaAPCs injection and then decreased slowly with a retention time of more than 36 h. As controls, ICG–M/M-TaAPCs (CD47), ICG–N/O-TaAPCs (noncognate), and ICG–blank MPs showed the in vivo trackings similar to ICG–M/M-TaAPCs, but a much shorter retention time with very weak fluorescent intensity at 36 h time point (Fig. 3D, left panel). The ex vivo imaging of dissected organs displayed that at 4 h after injection, M/M-TaAPCs and the control MPs appeared in liver, spleen, kidney, and lungs with visible fluorescence, but M/M-TaAPCs displayed significantly more accumulation in liver and spleen with a higher mean fluorescence intensity than the control MPs (Fig. 3C, right panel; Fig. 3D, right panel). These differences may imply that target Ags and CD47 molecules enable the TaAPCs to target MOG Ag–specific T cells and resist phagocytosis, and thus make the in vivo trafficking distinct to the N/O-TaAPCs, CD47 M/M-TaAPCs, and blank MPs. Notably, no visible fluorescent signal of M/M-TaAPCs or control MPs was observed in the head of EAE mice over 36 h, suggesting that most of the cell-sized TaAPCs or control MPs could not go into the CNS through vascular circulation.

To find the evidence that multipotent TaAPCs can directly contact or interplay with target T cells in vivo, PE-labeled M/M-TaAPCs or control MPs were i.v. injected into the EAE mice at peak stage. Four hours later, spleens were collected from each group. Frozen sections were prepared and followed by immune fluorescence staining. As shown by confocal micrographs (Fig. 4A), M/M-TaAPCs mainly distributed in the red pulp and marginal zone and displayed many colocalizations with CD4+ T cells and CD8+ T cells but few colocalizations with B cells, macrophages, and DCs. CD47-negative M/M-TaAPCs showed fewer or similar colocalizations with CD4+ and CD8+ T cells relative to M/M-TaAPCs but many contacts with B cells, macrophages, and DCs. Blank MPs and N/O-TaAPCs were found having few contacts with T cells and many colocalizations with others. In parallel, the spleen single-cell suspensions were prepared and followed by immune fluorescence staining. As shown by flow cytometry (Fig. 4B), the PE+/FITC+ signals (presumably MP–cell conjugates) were quantified with a visible percentage in the spleen cell suspensions. When compared with the PE–blank MP or PE–N/O-TaAPC group, PE–M/M-TaAPCs displayed the significantly higher percentages of TaAPC–CD4+ T cell conjugates (PE+/CD4-FITC+) and TaAPC–CD8+ T cell conjugates (PE+/CD8a-FITC+), but significantly lower percentages of TaAPC–B cell conjugates (PE+/CD19-FITC+) and TaAPC–macrophage conjugates (PE+/F4/80-FITC+), than the PE–M/M-TaAPCs(CD47) injection group.

FIGURE 4.

TaAPCs prefer to contact with CD4+ T cells and CD8+ T cells in vivo and in vitro. (A) Spleens were collected from EAE mice at 4 h after i.v. injection of PE-labeled TaAPCs or control MPs. Frozen sections were prepared. Then, CD8+ T cells, CD4+ T cells, B cells, macrophages, and DCs were stained with FITC-labeled mAbs, respectively, and observed by confocal imaging in the spleen section at original magnification ×400. White arrows point at the colocalizations of TaAPCs with stained cells. (B) Spleen single-cell suspensions were prepared from EAE mice at 4 h after i.v. injection of PE-labeled TaAPCs or control MPs and followed by staining with FITC-labeled mAbs above and flow cytometry. PE+/FITC+ signals were quantified in each cell suspension (n = 3). Furthermore, SPCs from the EAE mice without PE-TaAPCs injection were cocultured with PE-labeled blank MPs, N/O-TaAPCs, M/M-TaAPCs (CD47), or M/M-TaAPCs for 4 h. (C) The mixed suspensions of cells and MPs were processed to cell smears on slide and stained with FITC-labeled mAbs as above and visualized by laser confocal microscope. White arrows point at the colocalizations of TaAPCs or MPs with immune cells. The percentage of colocalized cells with TaAPCs or MPs was calculated by counting the confocal cells among 100 FITC-positive cells. (D) The mixed suspensions of cells and MPs were stained with FITC-labeled mAbs as above and analyzed by flow cytometry to detect the percentages of cell–MP conjugates (PE+/FITC+). Three to five replicate wells for each coculture group. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4.

TaAPCs prefer to contact with CD4+ T cells and CD8+ T cells in vivo and in vitro. (A) Spleens were collected from EAE mice at 4 h after i.v. injection of PE-labeled TaAPCs or control MPs. Frozen sections were prepared. Then, CD8+ T cells, CD4+ T cells, B cells, macrophages, and DCs were stained with FITC-labeled mAbs, respectively, and observed by confocal imaging in the spleen section at original magnification ×400. White arrows point at the colocalizations of TaAPCs with stained cells. (B) Spleen single-cell suspensions were prepared from EAE mice at 4 h after i.v. injection of PE-labeled TaAPCs or control MPs and followed by staining with FITC-labeled mAbs above and flow cytometry. PE+/FITC+ signals were quantified in each cell suspension (n = 3). Furthermore, SPCs from the EAE mice without PE-TaAPCs injection were cocultured with PE-labeled blank MPs, N/O-TaAPCs, M/M-TaAPCs (CD47), or M/M-TaAPCs for 4 h. (C) The mixed suspensions of cells and MPs were processed to cell smears on slide and stained with FITC-labeled mAbs as above and visualized by laser confocal microscope. White arrows point at the colocalizations of TaAPCs or MPs with immune cells. The percentage of colocalized cells with TaAPCs or MPs was calculated by counting the confocal cells among 100 FITC-positive cells. (D) The mixed suspensions of cells and MPs were stained with FITC-labeled mAbs as above and analyzed by flow cytometry to detect the percentages of cell–MP conjugates (PE+/FITC+). Three to five replicate wells for each coculture group. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

Spleens were collected from the EAE mice on day 18 without injection with PE-labeled TaAPCs or control MPs and processed to SPC suspensions. Then, the cells were cocultured (1 × 105 beads well−1) with PE–blank MPs, PE–N/O-TaAPCs, PE–M/M-TaAPCs (CD47), and PE-M/M-TaAPCs (1 × 105 beads well−1), respectively, in 96-well cell culture plate. After 4 h coculture, the mixed suspensions of cells and MPs were harvested and dropped on slides to prepare cell smears. After being naturally dried, the cell smears were stained by FITC-labeled mAbs specific for CD4, CD8a, CD19, CD11c, and F4/80, respectively, and finally visualized under laser confocal scanning microscopy (Fig. 4C). In parallel, the mixed suspensions of cells and MPs were harvested and stained with the FITC-labeled mAbs as above and detected by flow cytometry to quantify the MP cell conjugates (Fig. 4D).

As shown in Fig. 4C, PE–M/M-TaAPCs displayed many more contacts with CD4+ T cells and CD8+ T cells than PE–blank MPs or PE–N/O-TaAPCs, but significantly fewer colocalizations with B cells, DCs, and macrophages than PE–M/M-TaAPCs(CD47). Fig. 4D showed that PE–M/M-TaAPCs showed significantly higher percentages of TaAPC–CD4+ T cell conjugates (PE+/CD4-FITC+) and TaAPC–CD8+ T cell conjugates (PE+/CD8a-FITC+) than the PE–blank MP or PE–N/O-TaAPC group but significantly lower percentages of TaAPC–B cell conjugates (PE+/CD19-FITC+), TaAPC–DC conjugates (PE+/CD11c-FITC+), and TaAPC–macrophage conjugates (PE+/F4/80-FITC+) than PE–M/M-TaAPCs (CD47).

These results from in vitro experiments were consistent with the observations in vivo and further demonstrate that TaAPCs can directly contact with T cells in secondary lymphatic tissues by autoantigen targeting, and CD47-Fc can minimize the engulfment of TaAPCs by phagocytes in vivo.

PBMCs, SPCs, and MNCs in CNS tissues were collected from the EAE mice in each treatment group on day 20 (2 d after the second injection of TaAPCs) and followed by the 6-d ex vivo incubation with MOG35–55 or MOG40–54 peptide. Then, MOG35–55-specific CD4+ T cells and MOG40–54-specific CD8+ T cells were enumerated by MOG35–55/I-Ab tetramer and MOG40–54/H-2Db-Ig dimer staining, respectively. When compared with the blank MP group, two infusions of M/M-TaAPCs reduced the frequencies of MOG35–55-specific CD4+ T cells by 65.3% in peripheral blood, 79.5% in spleen, and 66.7% in CNS, whereas the noncognate N/O-TaAPCs treatment did not lead to significant reduction (Fig. 5A). Similarly, the frequencies of MOG40–54-specific CD8+ T cells were reduced by 46.4% in peripheral blood, 56.4% in spleen, and 62.4% in CNS after two injections of M/M-TaAPCs, whereas no obvious reduction was displayed after N/O-TaAPCs treatment (Fig. 5B). As the negative controls of pMHC multimer staining, the noncognate OVA323–339/I-Ab tetramer and NP366–374/H-2Db-Ig dimer staining were also carried out in parallel for each sample. Both OVA323–339-specific CD4+ T cells and NP366–374-specific CD8+ T cells showed very low frequencies in the PBMCs and SPCs, with no significant difference across groups (Supplemental Fig. 2).

FIGURE 5.

TaAPCs markedly eliminate MOG Ag–specific CD4+ and CD8+ T cells in EAE mice. PBMCs, SPCs, and MNCs in CNS were isolated from EAE mice at 2 d after the second i.v. injection of M/M-TaAPCs or control MPs and incubated with the MOG35–55 peptide or MOG40–54 peptide for 6 d. Then, MOG Ag–specific T cells were enumerated. (A) Frequencies of MOG35–55-specific CD4+ T cells as determined by MOG35–55/I-Ab tetramer staining. (B) Frequencies of MOG40–54-specific CD8+ T cells as determined by MOG40–54/ H-2Db-Ig dimer staining. Percentage displayed in the top quadrant of representative dot plots represents the average value of three mice in each group and was also shown in the corresponding histogram as mean ± SD. *p < 0.05, **p < 0.01.

FIGURE 5.

TaAPCs markedly eliminate MOG Ag–specific CD4+ and CD8+ T cells in EAE mice. PBMCs, SPCs, and MNCs in CNS were isolated from EAE mice at 2 d after the second i.v. injection of M/M-TaAPCs or control MPs and incubated with the MOG35–55 peptide or MOG40–54 peptide for 6 d. Then, MOG Ag–specific T cells were enumerated. (A) Frequencies of MOG35–55-specific CD4+ T cells as determined by MOG35–55/I-Ab tetramer staining. (B) Frequencies of MOG40–54-specific CD8+ T cells as determined by MOG40–54/ H-2Db-Ig dimer staining. Percentage displayed in the top quadrant of representative dot plots represents the average value of three mice in each group and was also shown in the corresponding histogram as mean ± SD. *p < 0.05, **p < 0.01.

Close modal

To find out the mechanisms by which M/M-TaAPCs reduce the MOG Ag–specific T cells, the apoptosis of fresh CD4+ T cells and CD8+ T cells in peripheral blood and spleen was analyzed first. Two injections of M/M-TaAPCs led to the mean percentage of total apoptotic CD4+ T cells ∼3.1-fold higher in PBMCs and 1.5 folds higher in SPCs than the blank MP group; similarly, two injections of M/M-TaAPCs increased the number of apoptotic CD4+ T cells by 189% in PBMCs and 57% in SPCs compared with the blank MP group (Fig. 6A). As shown in Fig. 6B, the mean percentage of total apoptotic CD8+ T cells increased ∼1.5-fold more in SPCs than the blank MP group, whereas the number of apoptotic CD8+ T cells increased by 88% in SPCs and 21% in PBMCs after M/M-TaAPCs treatment. In contrast, M/M-MP (no regulatory molecules) and N/O-TaAPC (irrelative Ags) treatment caused no significant increase of apoptotic CD4+ T cells and CD8+ T cells as compared with blank MP group. These data suggested that the M/M-TaAPCs depleted the myelin peptide–autoreactive T cells and induced T cells apoptosis in EAE mice in an Ag-specific and regulatory molecule–dependent manner.

FIGURE 6.

TaAPCs markedly induce T cells apoptosis and inhibit the activation and proliferation of CD4+ and CD8+ T cells. PBMCs and SPCs were isolated from EAE mice at 2 d after the second i.v. injection of M/M-TaAPCs or control MPs, and apoptotic T cells were detected in the fresh PBMCs and SPCs. (A) Frequencies of apoptotic CD4+ T cells. (B) Frequencies of apoptotic CD8+ T cells. Furthermore, SPCs were separated from the EAE mice at 2 d after the second administration of M/M-TaAPCs or control MPs and freshly stained with PE–anti-mouse CD44 or CD69. The frequencies of CD4+/CD44+ or CD4+/CD69+ T cells (C) and CD8+/CD44+ or CD8+/CD69+ T cells (D) were presented. In addition, the SPCs were labeled with CFSE and cocultured with MOG35–55 or MOG40–54 peptide for 7 d. Then, the proliferation percentages of CD4+ T cells and CD8+ T cells (E) were determined by flow cytometry (n = 3–5 mice in each group). *p < 0.05, **p < 0.01.

FIGURE 6.

TaAPCs markedly induce T cells apoptosis and inhibit the activation and proliferation of CD4+ and CD8+ T cells. PBMCs and SPCs were isolated from EAE mice at 2 d after the second i.v. injection of M/M-TaAPCs or control MPs, and apoptotic T cells were detected in the fresh PBMCs and SPCs. (A) Frequencies of apoptotic CD4+ T cells. (B) Frequencies of apoptotic CD8+ T cells. Furthermore, SPCs were separated from the EAE mice at 2 d after the second administration of M/M-TaAPCs or control MPs and freshly stained with PE–anti-mouse CD44 or CD69. The frequencies of CD4+/CD44+ or CD4+/CD69+ T cells (C) and CD8+/CD44+ or CD8+/CD69+ T cells (D) were presented. In addition, the SPCs were labeled with CFSE and cocultured with MOG35–55 or MOG40–54 peptide for 7 d. Then, the proliferation percentages of CD4+ T cells and CD8+ T cells (E) were determined by flow cytometry (n = 3–5 mice in each group). *p < 0.05, **p < 0.01.

Close modal

In addition to the apoptosis induction, TaAPCs may also exert inhibitory effects on MOG Ag–specific T cells by the surface presentation of PD-L1 and paracrine release of TGF-β1. On day 20 (2 d after the second administration of TaAPCs), SPCs were separated from the EAE mice in each treatment group and freshly stained with PE–anti-mouse CD44 or CD69 for monitoring the activation of T cells. The frequencies of CD4+/CD44+ or CD4+/CD69+ T cells (Fig. 6C) and CD8+/CD44+ or CD8+/CD69+ T cells (Fig. 6D) were obviously decreased in M/M-TaAPCs treatment group, whereas there was no significant inhibition in N/O-TaAPC and M/M-MP groups, when compared with the blank MP group. Furthermore, the fresh SPCs were also coincubated with MOG35–55 or MOG40–54 peptide for 7 d. The proliferation of CD4+ and CD8+ T cells was determined by CFSE staining and flow cytometry analyses. After the MOG peptide stimulation, the proliferation of both CD4+ T cells and CD8+ T cells was reduced by 59.1 and 52.7%, respectively, in the M/M-TaAPC group as compared with blank MP group, whereas there was no significant reduction in N/O-TaAPC group and M/M-MP group (Fig. 6E).

To evaluate the effects of TaAPCs in polarizing T cells in vivo, SPCs and LNCs were separated from the EAE mice in each treatment group on day 20 (2 d after the second injection of TaAPCs) and followed by Treg detection. As compared with the blank MP group, the frequencies and numbers of CD4+/CD25+/Foxp3+ T cells in the M/M-TaAPC–treated mice were significantly increased in SPCs (Fig. 7A) but not in LNCs (Supplemental Fig. 3A). Meanwhile, no difference was found across the control groups. Furthermore, the fresh SPCs and LNCs were coincubated with MOG35–55 or MOG40–54 peptides for 16 h or with PMA for 4 h followed by the detection of IFN-γ– or IL-17A–secreting CD4+ T cells and CD8+ T cells. After MOG35–55 peptide stimulation, the frequencies of IFN-γ+/CD4+ T cells and IL-17A+/CD4+ T cells (presumably MOG35–55-reactive Th1 and Th17 cells) decreased, respectively, by 60.1 and 55.7% in the SPCs, and 40.5 and 41.6% in the LNCs from M/M-TaAPC–treated mice, when compared with the blank MP group. The numbers of IFN-γ+/CD4+ T cells and IL-17A+/CD4+ T cells also displayed the trend similar to the frequencies across groups. Meanwhile, M/M-MPs led to no significant decrease of Th1 and Th17 either in SPCs or LNCs, but N/O-TaAPCs also caused the significant reduction of Th1 and Th17 cells in SPCs relative to the blank MP group (Fig. 7B, 7C). In addition, after PMA stimulation, the frequencies of IFN-γ+/CD4+ T and IL-17A+/CD4+ T cells (presumably general Th1 and Th17 cells) did not show any difference across groups (Supplemental Fig. 3B, 3C). After MOG40–54 peptide stimulation, the percentages of IL-17A+/CD8+ T cells (presumably MOG40–54-reactive Tc17 cells) also decreased, respectively, by 50.1% in the SPCs and 53.7% in the LNCs from M/M-TaAPC–treated mice in comparison with mice of control groups (Fig. 7D), but no significant decrease of IFN-γ+/ /CD8+ T cells (presumably MOG40–54-reactive Tc1 cells) was found either in SPCs or LNCs (Supplemental Fig. 3D).

FIGURE 7.

TaAPCs induce Tregs and inhibit MOG Ag–reactive Th1, Th17, and Tc17 cells. Spleen, lymph nodes, brain, and spinal cords were collected from EAE mice at 2 d after the second i.v. administration of M/M-TaAPCs or control MPs and processed into single-cell suspensions. (A) Frequencies of CD4+/CD25+/Foxp3+ T cells in the CD4+ T cell populations from fresh SPCs and their numbers in spleen. (B) Frequencies and numbers of IFN-γ+/CD4+ T cells (MOG Ag–reactive Th1 cells) in SPCs and LNCs after 16 h incubation with MOG35–55 peptide. (C) Frequencies and numbers of IL-17A+/CD4+ T cells (MOG Ag–reactive Th17 cells) in SPCs and LNCs after 16 h incubation with MOG35–55 peptide. (D) Frequencies and numbers of IL-17A+/CD8+ T cells (MOG Ag–reactive Tc17 cells) in SPCs and LNCs after 16 h incubation with MOG40–54 peptide. (E) Concentrations of IFN-γ, IL-17A, IL-10, and TGF-β1 in the supernatants of SPCs after 7-d incubation with MOG35–55 peptide (n = 3 mice in each group). *p < 0.05, **p < 0.01.

FIGURE 7.

TaAPCs induce Tregs and inhibit MOG Ag–reactive Th1, Th17, and Tc17 cells. Spleen, lymph nodes, brain, and spinal cords were collected from EAE mice at 2 d after the second i.v. administration of M/M-TaAPCs or control MPs and processed into single-cell suspensions. (A) Frequencies of CD4+/CD25+/Foxp3+ T cells in the CD4+ T cell populations from fresh SPCs and their numbers in spleen. (B) Frequencies and numbers of IFN-γ+/CD4+ T cells (MOG Ag–reactive Th1 cells) in SPCs and LNCs after 16 h incubation with MOG35–55 peptide. (C) Frequencies and numbers of IL-17A+/CD4+ T cells (MOG Ag–reactive Th17 cells) in SPCs and LNCs after 16 h incubation with MOG35–55 peptide. (D) Frequencies and numbers of IL-17A+/CD8+ T cells (MOG Ag–reactive Tc17 cells) in SPCs and LNCs after 16 h incubation with MOG40–54 peptide. (E) Concentrations of IFN-γ, IL-17A, IL-10, and TGF-β1 in the supernatants of SPCs after 7-d incubation with MOG35–55 peptide (n = 3 mice in each group). *p < 0.05, **p < 0.01.

Close modal

Moreover, proinflammatory cytokines (IFN-γ, IL-17A) and inhibitory cytokines (IL-10, TGF-β1) were quantified in the culture supernatants of SPCs after 7-d incubation with MOG35–55 peptide and in the CNS tissue homogenates collected on day 20. As shown in Fig. 7E, the concentrations of IFN-γ and IL-17A decreased by 45.7 and 75.3%, respectively, in the cell culture supernatants of SPCs from M/M-TaAPC–treated mice, whereas the levels of IL-10 and TGF-β1 elevated by 56.6 and 33.3%, respectively, as compared with the blank MP group. Also, the SPCs from N/O-TaAPC–treated EAE mice secreted much less IL-17A than that from other control groups, but without significant difference (Fig. 7E). However, the M/M-TaAPC treatment did not change these cytokines profiles in CNS tissue homogenates (Supplemental Fig. 3E).

To obtain more convincing evidence for the direct effects of TaAPCs on T cells and the role of each kind of regulatory molecule, the cocultures of TaAPCs or control MPs with purified T cells were carried out. The control MPs cocarrying autoantigens (MOG40–54/H-2Db multimer, MOG35–55/I-Ab multimer) and single kinds of regulatory molecules (anti-Fas, PD-L1-Fc or TGF-β1) were newly prepared and termed M/M/anti-Fas–MPs, M/M/PD-L1–MPs, and M/M/TGF-β1–MPs, respectively. Then, spleens were collected from the EAE mice without treatment of TaAPCs on day 18 after MOG immunization and processed to single-cell suspensions. The T cells were purified from the spleen cells by using a mouse CD3 T cell negative magnetic sorting kit (Stemcell Technologies, Vancouver, Canada), then seeded in 96-well cell culture plate (1 × 105 cells well−1), and coincubated with PBS, blank MPs, M/M/anti-Fas–MPs, M/M/PD-L1–MPs, M/M/TGF-β1–MPs, N/O-TaAPCs, or M/M-TaAPCs (1 × 105 beads well−1) plus IL-2 (30 pg ml−1) in RPMI 1640 medium supplemented with 10% FBS at 37°C in 5% CO2 and humidified conditions. After 24 h, the cells were harvested and followed by the detections of apoptotic CD4+ and CD8+ T cells, Tregs, and activated CD4+ and CD8+ T cells.

As shown in Fig. 8A and 8B, compared with the PBS and blank MP group, the number and frequencies of apoptotic CD4+ and CD8+ T cells increased obviously in M/M-TaAPC group and M/M/anti-Fas group, but not in other control groups. These results show that TaAPCs can directly induce T cells apoptosis without the requirement of other cells, and anti-Fas onto TaAPCs exerts pivotal effects on the induction of apoptosis. In parallel, the frequencies of CD44+/CD4+ T cells (Fig. 8C) and CD44+/CD8+ T cells (Fig. 8D) were decreased only in M/M-TaAPC group, but not in other control groups, as compared with blank MP group. As shown in Fig. 8E, the frequency of Tregs was enhanced significantly in M/M-TaAPCs and M/M/TGF-β1 groups but not in other control groups when compared with PBS control group. These data imply that TaAPCs can directly inhibit T cells activation and polarize T cells to Tregs without the requirement of other cells. To a certain extent, these results also imply that TGF-β1 released from TaAPCs may be the key molecule for inducing Tregs. Although PD-L1 is thought to inhibit T cell proliferation, inhibiting T cell activation requires the combined effects of anti-Fas, PD-L1, and TGF-β1 molecules.

FIGURE 8.

TaAPCs induce T cells apoptosis, polarize Tregs, and inhibit T cell activation in the cocultures with purified T cells. T cells were purified from the spleen cells of EAE mice on day 18 after MOG immunization and coincubated with PBS, control MPs, or TaAPCs for 24 h, followed by the detection of apoptotic CD4+ and CD8+ T cells, Tregs, and activated CD4+ and CD8+ T cells using fluorescence Ab staining and flow cytometry. (A) Frequencies of apoptotic CD4+ T cells in CD4+ T cell populations and their numbers in spleen. (B) Frequencies of apoptotic CD8+ T cells in CD8+ T cell populations and their numbers in spleen. (C) Frequencies of CD44+/CD4+ T cells in CD4+ T cell populations. (D) Frequencies of CD44+/CD8+ T cells in CD8+ T cell populations. (E) Frequencies of CD4+/CD25+/Foxp3+ T cells in CD4+ T cell populations and their numbers in spleen (n = 3 mice in each group). Statistical data are also shown in the corresponding histogram as mean ± SD. Three to five replicate wells for each coculture group. The data were from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 8.

TaAPCs induce T cells apoptosis, polarize Tregs, and inhibit T cell activation in the cocultures with purified T cells. T cells were purified from the spleen cells of EAE mice on day 18 after MOG immunization and coincubated with PBS, control MPs, or TaAPCs for 24 h, followed by the detection of apoptotic CD4+ and CD8+ T cells, Tregs, and activated CD4+ and CD8+ T cells using fluorescence Ab staining and flow cytometry. (A) Frequencies of apoptotic CD4+ T cells in CD4+ T cell populations and their numbers in spleen. (B) Frequencies of apoptotic CD8+ T cells in CD8+ T cell populations and their numbers in spleen. (C) Frequencies of CD44+/CD4+ T cells in CD4+ T cell populations. (D) Frequencies of CD44+/CD8+ T cells in CD8+ T cell populations. (E) Frequencies of CD4+/CD25+/Foxp3+ T cells in CD4+ T cell populations and their numbers in spleen (n = 3 mice in each group). Statistical data are also shown in the corresponding histogram as mean ± SD. Three to five replicate wells for each coculture group. The data were from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

The side effects of TaAPCs treatment were preliminarily investigated. EAE mice were randomized into three groups and administered via tail vein with PBS, blank MPs, or M/M-TaAPCs on days 8, 18, 28, and 38 after MOG immunization. On day 40 (2 d after the final injection of TaAPCs), the numbers of lymphocytes, monocytes, and neutrophils in peripheral blood (Supplemental Fig. 4A) were detected by routine blood tests. The percentages of T cells (Supplemental Fig. 4B), B cells, NK cells (Supplemental Fig. 4C), and apoptotic T cells (Supplemental Fig. 4D) in SPCs or LNCs were detected by flow cytometry. No obvious difference was found across groups.

Furthermore, the cytotoxicity of NK cells in each group was measured without significant difference across groups (Supplemental Fig. 4E). The tumor model was established to evaluate the antitumor ability of EAE mice after TaAPCs treatment. EAE mice were challenged with B16 melanoma cells on day 3 after MOG immunization, followed by i.v. administrations of M/M-TaAPCs or control MPs on days 8, 18, 28, and 38. As shown, the tumor growth curves (Supplemental Fig. 4F) and survival rates (Supplemental Fig. 4G) of tumor-bearing EAE mice displayed no significant difference across treatment groups. Finally, the proliferation of host T cells in response to alloantigen and irrelative antigenic peptides was further investigated in cocultures. On day 40, spleen cells were prepared from EAE mice as above, labeled with CFSE, and coincubated with SPCs from BALB/c mice (pretreated with mitomycin C) or irrelevant peptides (OVA323–339 plus NP366–374) for 7 d. Then, the cells were stained with allophycocyanin–anti-mouse CD3e and finally analyzed by flow cytometry. As shown in Supplemental Fig. 4H, the proliferation of T cells from the EAE mice treated with TaAPCs or control MPs was quantified, and no obvious difference was found across groups.

These preliminary data suggested that TaAPCs treatment did not lead to the visible nonspecific killing of various immune cells or the impairment of overall immune functions. The TaAPCs strategy achieved the desirable outcomes by directly and selectively depleting or modulating myelin Ag–specific T cells in the EAE mice.

Epitope spreading is an important issue to evaluating the translation potential of TaAPCs. In this study, the MOG119–132-induced (38), PLP178–191-induced (39), and MOG35–55-induced EAE models have been established in B6 mice as described and administered i.v. with M/M-TaAPCs, N/O-TaAPCs, and blank MPs, respectively, on day 25 after myelin peptide immunization. Then, the clinical manifestation (clinical scores) has been recorded daily for 40 d to clarify whether epitope spreading can be observed in the B6/MOG and B6/PLP models. As shown in Supplemental Fig. 1, one infusion of M/M-TaAPCs on day 25 significantly decreased the mean clinical scores of chronic remission phase in both MOG35–55-induced EAE (Supplemental Fig. 1D) and MOG119–132-induced EAE (Supplemental Fig. 1E) but not in PLP178–191-induced EAE (Supplemental Fig. 1F). Of note is that the M/M-TaAPCs codisplaying MOG35–55/I-Ab multimer and MOG40–54/H-2Db-Ig dimer are designed to directly modulate MOG35–55-specific CD4+ T cells and MOG40–54-specific CD8+ T cells in MOG35–55-induced EAE. These new data indicated, to some extent, the epitope spreading of TaAPC treatment in MOG peptide–induced EAE, but not in PLP peptide–induced EAE, in B6 mice.

Numerous researchers around the world have reported the biomimetic micro- and nanoparticles (MNPs) carrying myelin peptides or proteins along with toxin or regulatory molecules for the treatment of EAE or MS (30, 40, 41). s.c. prophylactic and therapeutic vaccination with PLGA NPs encapsulating MOG35–55 peptide and IL-10 ameliorated the MOG-induced EAE in mice (42). Similarly, i.v. infusions of PLGA MPs or polystyrene NP– bearing PLP139–151 peptide could prevent the onset of the PLP-induced relapsing EAE and induce long-term T cell tolerance (23, 24). Gold NPs cocoupling MOG35–55 peptide and aryl hydrocarbon receptor (31), or PLG-NPs coencapsulating myelin protein or peptide and rapamycin (30), also induced durable and Ag-specific immune tolerance and suppressed EAE after i.v. administration. Although these MNPs were decorated or encapsulated with myelin peptides or protein, most of the therapeutics underlie the Ag presentation by cellular uptake of MNPs and the following induction of tolerogenic APCs and Tregs, in which T cell tolerance was indirectly induced. Therefore, these strategies may be called an indirect Ag-specific immunotherapy.

In this study, four points are different from the previous research. First, the MOG peptide–loaded pMHC multimers were coupled onto PLGA MPs as target Ags which can directly bind with MOG peptide–specific TCRs on the autoreactive T cells without the requirement of presentation by APCs, thus tailoring the direct modulation on MOG-reactive T cells. To minimize the cellular uptake of autoantigens, cell-sized PLGA MPs were used as carriers instead of NPs. As known, the size greatly affects the ability of MNPs to pass through biological barriers, be engulfed by phagocytes, and interplay with target cells via surface presentation of ligands (43). The closer the particle is to cellular size, the more potent the effect on the target cell (44, 45). Cell-sized MPs present a reduced risk of engulfment by phagocytes relative to NPs (43, 46). Only phagocytic cells can take up MPs (especially 0.5–10 μm), whereas most of the cell types in vivo may be able to ingest nanoscale particles by pinocytosis. Moreover, CD47-Fc molecules were cocoupled onto the PLAG MPs as a self-marker (47, 48) because CD47 can interact with signal regulatory protein-α (SIRPα) on phagocytes to prevent phagocytosis (49) and has been used to construct the “stealth particles” in NP-mediated drug delivery systems for increasing the circulation time of NP vehicles in human (50). In this study, the cocultures of TaAPCs with purified T cells confirmed the direct effects of TaAPCs on T cells; many colocalizations or conjugates of TaAPCs with CD4+ and CD8+ T cells were observed in spleen sections and cocultures. These in vivo and in vitro data suggest the direct and selective modulations of TaAPCs on T cells, but we cannot eliminate the possible phagocytosis occurring in vivo, especially at the later stage during the retention time of 36 h. Therefore, it is reasonable to conclude that the TaAPCs modulate the autoreactive T cells in vivo mainly in a direct contact way, but an indirect modulation mediated by tolerogenic APCs and Tregs may also be involved in the immunotherapy.

Second, the PLGA MPs were cocoupled with pMHC class II (MOG35–55/I-Ab multimer) and class I (MOG40–54/H-2Db-Ig dimer) multimers, and were thus enabled to target and modulate both CD4+ T cells and CD8+ T cells specific for MOG Ags. It is well documented that myelin Ag–reactive CD4+ T cells, such as Th17, Th1, and Tregs, work as crucial drivers or regulators in autoimmune demyelinating diseases (4, 5, 51). The corresponding proinflammatory cytokines (IL-17A and IFN-γ) and inhibitory cytokines (IL-10 and TGF-β1) produced by Th17, Th1, and Tregs, respectively, are also demonstrated to be important in EAE and MS. Defects in Th17 and Th1 cells help to prevent EAE, whereas Tregs participate in the maintenance of self-tolerance and the regulation of inflammatory autoimmune system by various mechanisms (5254). Therefore, most Ag-specific strategies focus on the autoreactive CD4+ T cells. However, increasing studies have also demonstrated the secondary contributions of myelin Ag–specific CD8+ T cells in MS and EAE progress (68). Tc17 cells, which are newly defined by producing IL-17, are thought to be primarily proinflammatory cells and induce many of the same effects as Th1 and Th17 cells in several autoimmune diseases, including MS and EAE (55). Therefore, the multivalent PLGA MPs were generated in our study to enact the combination therapy targeting both pathogenic CD4+ and CD8+ T cell subsets.

Third, multiple regulatory molecules were copresented with pMHC target Ags by the PLGA MPs in the same spatial and temporal context. The combined uses of anti-Fas, PD-L1, and TGF-β1 provide several signal pathways to powerfully modulate the autoreactive T cells like the tolerogenic DCs, which induce apoptosis, inhibit activation and proliferation, and skew Th cells to Tregs. In this study, the roles of each kind of regulatory molecule were confirmed in the cocultures of TaAPCs with purified T cells. The anti-Fas–dependent deletion of Ag-specific T cells was a key component in the first inhibitory effects on the development of EAE, but the effects on inducing T cells apoptosis exerted by anti-Fas onto PLGA MPs can only persist for 4 d or less in mice, as demonstrated by our previous works (56). Thus, the significant increase of Tregs and obvious inhibition of Ag-specific T cell activation and proliferation mediated by TGF-β and PD-L1 should be closely associated with the long-term (100 d) maintenance of EAE amelioration. Therefore, the TaAPCs delivering three kinds of regulatory molecules may obtain a combined or synergistic inhibitory effect in EAE mice, including Ag-specific T cells deletion and active T cell tolerance induction by multiple signal pathways, although we cannot appreciate the accurate contribution in vivo of each molecule.

Finally, the PLGA MPs highly simulate the natural tolerogenic APCs to modulate T cells by surface presentation of ligands (pMHC multimers, anti-Fas, PD-L1, and CD47) and paracrine release of inhibitory cytokine (TGF-β1), so were termed TaAPCs for the first time, to our knowledge. Another advantage is that the cell-sized TaAPCs cannot circulate into brain, as our data displayed, and thus may evoke much less concern regarding biosafety for the putative clinical use.

A similar but distinct concept, killer artificial APCs, was reported by Schütz et al. (57) in 2008. The CMV or Mart-1 peptide-loaded HLA-A2-Ig dimers and anti-Fas mAb were cocoupled onto cell-sized polymer beads to specifically induce the apoptosis of Ag-specific T cells in static 96-well plates. After that, peptide/H-2Kb-Ig dimer and anti-Fas mAb were covalently cocoupled onto the degradable cell-sized PLGA MPs to selectively deplete OVA257–264-specific CD8+ T cells in vitro (56) and kill the H-2Kb-alloreactive CD8+ T cells in a murine model for prolonging the alloskin graft survival in our recent works (58). In this study, the killer artificial APC platform was upgraded to multipotent TaAPCs and was applied in the treatment of T cell–medicated autoimmune disease for the first time, to our knowledge.

Of note, the route, timeline, and dosage of TaAPC administration should be further optimized in translational studies along with the long-time investigation of biosafety and toxicity issue. For the biomimetic particles carrying peptides, Ags, or pMHC multimers in the treatment of autoimmune diseases, many studies show that i.v. administration leads to much more effective immune tolerance and clinical outcomes than the i.p. or s.c. routes (23, 24, 31). In this study, the results of i.p. or s.c. injections were consistent with the previous research. The in vivo mechanism remains unclear. The safety of i.v. injection has also been demonstrated in the treatment of murine EAE without obvious allergic reactions (23, 24). Our recent works also confirmed the long-time safety of the cell-sized PLGA MPs carrying targeting Ag (H-2Kb alloantigen) and anti-Fas mAb after i.v. administration in the mice of alloskin transplantation. Obvious impairment of the host overall immune function and visible organ toxicity were not found at long-time points (58). In addition, the i.v. administrations of TaAPCs late at peak stage led to much weaker inhibitory effects on EAE progress than the i.v. injections early at onset phase. This difference may be contributed to the dynamic changes in the frequencies and reactivity of MOG-reactive T cells during the EAE course (59) and the difficult reparation of demyelination and neurologic dysfunction after acute onset stage in EAE.

In conclusion, a TaAPC was developed in this study by cocoupling MOG peptide–loaded pMHC multimers, anti-Fas, PD-L1, and CD47 on the surface of cell-sized PLGA MPs and encapsulating TGF-β1 inside for the treatment of MOG-induced EAE. The encouraging results suggest a novel Ag-specific immunotherapy for the T cell–mediated autoimmune diseases by directly modulating autoreactive T cells through multiple signal pathways. The in vivo mechanism, tissue distribution, and effects on overall immune functions were also defined initially, and thus may facilitate the translational studies.

This work was supported by grants from the National Natural Science Foundation of China (81172823 and 81372448), the Science and Technology Support Program of Jiangsu Province (BE2017714), and the Postgraduate Research and Practice Innovation Program of Jiangsu Province (KYCX17_0164). The sponsors had no role in study design, data collection and analysis, preparation of the manuscript, or decision to submit the article for publication.

The online version of this article contains supplemental material.

Abbreviations used in this article:

DC

dendritic cell

EAE

experimental autoimmune encephalomyelitis

ICG

indocyanine green

LFB

Luxol Fast Blue

LNC

lymph node cell

MNC

mononuclear cell

MNP

micro- and nanoparticle

MOG

myelin oligodendrocyte glycoprotein

MP

microparticle

MS

multiple sclerosis

NP

influenza A virus nucleoprotein

PEI

polyethyleneimine

PLGA

polylactic-coglycolic acid

PLGA-MPTGF-β1

TGF-β1–encapsulated PLGA MP

pMHC

peptide-loaded MHC

SPC

splenocyte

TaAPC

tolerogenic artificial APC

Treg

regulatory T cell.

1
Lublin
,
F. D.
,
S. C.
Reingold
,
J. A.
Cohen
,
G. R.
Cutter
,
P. S.
Sørensen
,
A. J.
Thompson
,
J. S.
Wolinsky
,
L. J.
Balcer
,
B.
Banwell
,
F.
Barkhof
, et al
.
2014
.
Defining the clinical course of multiple sclerosis: the 2013 revisions.
Neurology
83
:
278
286
.
2
Dendrou
,
C. A.
,
L.
Fugger
,
M. A.
Friese
.
2015
.
Immunopathology of multiple sclerosis.
Nat. Rev. Immunol.
15
:
545
558
.
3
Nylander
,
A.
,
D. A.
Hafler
.
2012
.
Multiple sclerosis.
J. Clin. Invest.
122
:
1180
1188
.
4
Weiner
,
H. L.
2004
.
Multiple sclerosis is an inflammatory T-cell-mediated autoimmune disease.
Arch. Neurol.
61
:
1613
1615
.
5
Bettelli
,
E.
,
M.
Pagany
,
H. L.
Weiner
,
C.
Linington
,
R. A.
Sobel
,
V. K.
Kuchroo
.
2003
.
Myelin oligodendrocyte glycoprotein-specific T cell receptor transgenic mice develop spontaneous autoimmune optic neuritis.
J. Exp. Med.
197
:
1073
1081
.
6
Huseby
,
E. S.
,
D.
Liggitt
,
T.
Brabb
,
B.
Schnabel
,
C.
Ohlén
,
J.
Goverman
.
2001
.
A pathogenic role for myelin-specific CD8(+) T cells in a model for multiple sclerosis.
J. Exp. Med.
194
:
669
676
.
7
Fischer
,
H. J.
,
J.
van den Brandt
,
T.
Lingner
,
F.
Odoardi
,
A.
Flügel
,
A.
Weishaupt
,
H. M.
Reichardt
.
2016
.
Modulation of CNS autoimmune responses by CD8(+) T cells coincides with their oligoclonal expansion.
J. Neuroimmunol.
290
:
26
32
.
8
Leuenberger
,
T.
,
M.
Paterka
,
E.
Reuter
,
J.
Herz
,
R. A.
Niesner
,
H.
Radbruch
,
T.
Bopp
,
F.
Zipp
,
V.
Siffrin
.
2013
.
The role of CD8+ T cells and their local interaction with CD4+ T cells in myelin oligodendrocyte glycoprotein35-55-induced experimental autoimmune encephalomyelitis.
J. Immunol.
191
:
4960
4968
.
9
Goodin
,
D. S.
,
B. A.
Cohen
,
P.
O’Connor
,
L.
Kappos
,
J. C.
Stevens
;
Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology
.
2008
.
Assessment: the use of natalizumab (Tysabri) for the treatment of multiple sclerosis (an evidence-based review): report of the therapeutics and technology assessment subcommittee of the American academy of neurology.
Neurology
71
:
766
773
.
10
Maker-Clark
,
G.
,
S.
Patel
.
2013
.
Integrative therapies for multiple sclerosis.
Dis. Mon.
59
:
290
301
.
11
Gasperini
,
C.
,
S.
Ruggieri
.
2012
.
Development of oral agent in the treatment of multiple sclerosis: how the first available oral therapy, fingolimod will change therapeutic paradigm approach.
Drug Des. Devel. Ther.
6
:
175
186
.
12
Feinstein
,
A.
,
J.
Freeman
,
A. C.
Lo
.
2015
.
Treatment of progressive multiple sclerosis: what works, what does not, and what is needed.
Lancet Neurol.
14
:
194
207
.
13
Hilas
,
O.
,
P. N.
Patel
,
S.
Lam
.
2010
.
Disease modifying agents for multiple sclerosis.
Open Neurol. J.
4
:
15
24
.
14
Wingerchuk
,
D. M.
,
J. L.
Carter
.
2014
.
Multiple sclerosis: current and emerging disease-modifying therapies and treatment strategies.
Mayo Clin. Proc.
89
:
225
240
.
15
Yong
,
H.
,
G.
Chartier
,
J.
Quandt
.
2018
.
Modulating inflammation and neuroprotection in multiple sclerosis.
J. Neurosci. Res.
96
:
927
950
.
16
Buzzard
,
K.
,
W. H.
Chan
,
T.
Kilpatrick
,
S.
Murray
.
2017
.
Multiple sclerosis: basic and clinical.
Adv. Neurobiol.
15
:
211
252
.
17
Walczak
,
A.
,
M.
Siger
,
A.
Ciach
,
M.
Szczepanik
,
K.
Selmaj
.
2013
.
Transdermal application of myelin peptides in multiple sclerosis treatment.
JAMA Neurol.
70
:
1105
1109
.
18
Deraos
,
G.
,
M.
Rodi
,
H.
Kalbacher
,
K.
Chatzantoni
,
F.
Karagiannis
,
L.
Synodinos
,
P.
Plotas
,
A.
Papalois
,
N.
Dimisianos
,
P.
Papathanasopoulos
, et al
.
2015
.
Properties of myelin altered peptide ligand cyclo(87-99)(Ala91,Ala96)MBP87-99 render it a promising drug lead for immunotherapy of multiple sclerosis.
Eur. J. Med. Chem.
101
:
13
23
.
19
Hartwell
,
B. L.
,
A.
Smalter Hall
,
D.
Swafford
,
B. P.
Sullivan
,
A.
Garza
,
J. O.
Sestak
,
L.
Northrup
,
C.
Berkland
.
2016
.
Molecular dynamics of multivalent soluble antigen arrays support a two-signal co-delivery mechanism in the treatment of experimental autoimmune encephalomyelitis.
Mol. Pharm.
13
:
330
343
.
20
Northrup
,
L.
,
J. O.
Sestak
,
B. P.
Sullivan
,
S.
Thati
,
B. L.
Hartwell
,
T. J.
Siahaan
,
C. M.
Vines
,
C.
Berkland
.
2014
.
Co-delivery of autoantigen and b7 pathway modulators suppresses experimental autoimmune encephalomyelitis.
AAPS J.
16
:
1204
1213
.
21
Van Brussel
,
I.
,
W. P.
Lee
,
M.
Rombouts
,
A. H.
Nuyts
,
M.
Heylen
,
B. Y.
De Winter
,
N.
Cools
,
D. M.
Schrijvers
.
2014
.
Tolerogenic dendritic cell vaccines to treat autoimmune diseases: can the unattainable dream turn into reality?
Autoimmun. Rev.
13
:
138
150
.
22
Kleist
,
C.
,
E.
Mohr
,
S.
Gaikwad
,
L.
Dittmar
,
S.
Kuerten
,
M.
Platten
,
W.
Mier
,
M.
Schmitt
,
G.
Opelz
,
P.
Terness
.
2016
.
Autoantigen-specific immunosuppression with tolerogenic peripheral blood cells prevents relapses in a mouse model of relapsing-remitting multiple sclerosis.
J. Transl. Med.
14
:
99
.
23
Getts
,
D. R.
,
A. J.
Martin
,
D. P.
McCarthy
,
R. L.
Terry
,
Z. N.
Hunter
,
W. T.
Yap
,
M. T.
Getts
,
M.
Pleiss
,
X.
Luo
,
N. J.
King
, et al
.
2012
.
Microparticles bearing encephalitogenic peptides induce T-cell tolerance and ameliorate experimental autoimmune encephalomyelitis.
Nat. Biotechnol.
30
:
1217
1224
.
24
Hunter
,
Z.
,
D. P.
McCarthy
,
W. T.
Yap
,
C. T.
Harp
,
D. R.
Getts
,
L. D.
Shea
,
S. D.
Miller
.
2014
.
A biodegradable nanoparticle platform for the induction of antigen-specific immune tolerance for treatment of autoimmune disease.
ACS Nano
8
:
2148
2160
.
25
Getts
,
D. R.
,
L. D.
Shea
,
S. D.
Miller
,
N. J.
King
.
2015
.
Harnessing nanoparticles for immune modulation. [Published erratum appears in 2016 Trends Immunol. 37: 715.]
Trends Immunol.
36
:
419
427
.
26
Benedek
,
G.
,
A. A.
Vandenbark
,
N. J.
Alkayed
,
H.
Offner
.
2017
.
Partial MHC class II constructs as novel immunomodulatory therapy for stroke.
Neurochem. Int.
107
:
138
147
.
27
Tel
,
J.
,
S. P.
Sittig
,
R. A.
Blom
,
L. J.
Cruz
,
G.
Schreibelt
,
C. G.
Figdor
,
I. J.
de Vries
.
2013
.
Targeting uptake receptors on human plasmacytoid dendritic cells triggers antigen cross-presentation and robust type I IFN secretion.
J. Immunol.
191
:
5005
5012
.
28
Reddy
,
S. T.
,
A.
Rehor
,
H. G.
Schmoekel
,
J. A.
Hubbell
,
M. A.
Swartz
.
2006
.
In vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles.
J. Control. Release
112
:
26
34
.
29
Getts
,
D. R.
,
D. M.
Turley
,
C. E.
Smith
,
C. T.
Harp
,
D.
McCarthy
,
E. M.
Feeney
,
M. T.
Getts
,
A. J.
Martin
,
X.
Luo
,
R. L.
Terry
, et al
.
2011
.
Tolerance induced by apoptotic antigen-coupled leukocytes is induced by PD-L1+ and IL-10-producing splenic macrophages and maintained by T regulatory cells.
J. Immunol.
187
:
2405
2417
.
30
Maldonado
,
R. A.
,
R. A.
LaMothe
,
J. D.
Ferrari
,
A. H.
Zhang
,
R. J.
Rossi
,
P. N.
Kolte
,
A. P.
Griset
,
C.
O’Neil
,
D. H.
Altreuter
,
E.
Browning
, et al
.
2015
.
Polymeric synthetic nanoparticles for the induction of antigen-specific immunological tolerance.
Proc. Natl. Acad. Sci. USA
112
:
E156
E165
.
31
Yeste
,
A.
,
M.
Nadeau
,
E. J.
Burns
,
H. L.
Weiner
,
F. J.
Quintana
.
2012
.
Nanoparticle-mediated codelivery of myelin antigen and a tolerogenic small molecule suppresses experimental autoimmune encephalomyelitis.
Proc. Natl. Acad. Sci. USA
109
:
11270
11275
.
32
Cruz
,
L. J.
,
R. A.
Rosalia
,
J. W.
Kleinovink
,
F.
Rueda
,
C. W.
Löwik
,
F.
Ossendorp
.
2014
.
Targeting nanoparticles to CD40, DEC-205 or CD11c molecules on dendritic cells for efficient CD8(+) T cell response: a comparative study.
J. Control. Release
192
:
209
218
.
33
Kapoor
,
D. N.
,
A.
Bhatia
,
R.
Kaur
,
R.
Sharma
,
G.
Kaur
,
S.
Dhawan
.
2015
.
PLGA: a unique polymer for drug delivery.
Ther. Deliv.
6
:
41
58
.
34
Iqbal
,
M.
,
N.
Zafar
,
H.
Fessi
,
A.
Elaissari
.
2015
.
Double emulsion solvent evaporation techniques used for drug encapsulation.
Int. J. Pharm.
496
:
173
190
.
35
Berard
,
J. L.
,
K.
Wolak
,
S.
Fournier
,
S.
David
.
2010
.
Characterization of relapsing-remitting and chronic forms of experimental autoimmune encephalomyelitis in C57BL/6 mice.
Glia
58
:
434
445
.
36
Wen
,
J.
,
R.
Ribeiro
,
M.
Tanaka
,
Y.
Zhang
.
2015
.
Activation of CB2 receptor is required for the therapeutic effect of ABHD6 inhibition in experimental autoimmune encephalomyelitis.
Neuropharmacology
99
:
196
209
.
37
Pino
,
P. A.
,
A. E.
Cardona
.
2011
.
Isolation of brain and spinal cord mononuclear cells using percoll gradients.
J. Vis. Exp.
DOI: 10.3791/2348.
38
Shetty
,
A.
,
S. G.
Gupta
,
M.
Varrin-Doyer
,
M. S.
Weber
,
T.
Prod’homme
,
N.
Molnarfi
,
N.
Ji
,
P. A.
Nelson
,
J. C.
Patarroyo
,
U.
Schulze-Topphoff
, et al
.
2014
.
Immunodominant T-cell epitopes of MOG reside in its transmembrane and cytoplasmic domains in EAE.
Neurol. Neuroimmunol. Neuroinflamm.
1
:
e22
.
39
Kuerten
,
S.
,
T. L.
Gruppe
,
L. M.
Laurentius
,
C.
Kirch
,
M.
Tary-Lehmann
,
P. V.
Lehmann
,
K.
Addicks
.
2011
.
Differential patterns of spinal cord pathology induced by MP4, MOG peptide 35-55, and PLP peptide 178-191 in C57BL/6 mice.
APMIS
119
:
336
346
.
40
Sestak
,
J. O.
,
A.
Fakhari
,
A. H.
Badawi
,
T. J.
Siahaan
,
C.
Berkland
.
2014
.
Structure, size, and solubility of antigen arrays determines efficacy in experimental autoimmune encephalomyelitis.
AAPS J.
16
:
1185
1193
.
41
Capurso
,
N. A.
,
M.
Look
,
L.
Jeanbart
,
H.
Nowyhed
,
C.
Abraham
,
J.
Craft
,
T. M.
Fahmy
.
2010
.
Development of a nanoparticulate formulation of retinoic acid that suppresses Th17 cells and upregulates regulatory T cells.
Self Nonself
1
:
335
340
.
42
Cappellano
,
G.
,
A. D.
Woldetsadik
,
E.
Orilieri
,
Y.
Shivakumar
,
M.
Rizzi
,
F.
Carniato
,
C. L.
Gigliotti
,
E.
Boggio
,
N.
Clemente
,
C.
Comi
, et al
.
2014
.
Subcutaneous inverse vaccination with PLGA particles loaded with a MOG peptide and IL-10 decreases the severity of experimental autoimmune encephalomyelitis.
Vaccine
32
:
5681
5689
.
43
Balmert
,
S. C.
,
S. R.
Little
.
2012
.
Biomimetic delivery with micro- and nanoparticles.
Adv. Mater.
24
:
3757
3778
.
44
Mescher
,
M. F.
1992
.
Surface contact requirements for activation of cytotoxic T lymphocytes.
J. Immunol.
149
:
2402
2405
.
45
Meyer
,
R. A.
,
J. C.
Sunshine
,
J. J.
Green
.
2015
.
Biomimetic particles as therapeutics.
Trends Biotechnol.
33
:
514
524
.
46
Champion
,
J. A.
,
A.
Walker
,
S.
Mitragotri
.
2008
.
Role of particle size in phagocytosis of polymeric microspheres.
Pharm. Res.
25
:
1815
1821
.
47
Oldenborg
,
P. A.
,
A.
Zheleznyak
,
Y. F.
Fang
,
C. F.
Lagenaur
,
H. D.
Gresham
,
F. P.
Lindberg
.
2000
.
Role of CD47 as a marker of self on red blood cells.
Science
288
:
2051
2054
.
48
Poon
,
I. K.
,
C. D.
Lucas
,
A. G.
Rossi
,
K. S.
Ravichandran
.
2014
.
Apoptotic cell clearance: basic biology and therapeutic potential.
Nat. Rev. Immunol.
14
:
166
180
.
49
Tsai
,
R. K.
,
P. L.
Rodriguez
,
D. E.
Discher
.
2010
.
Self inhibition of phagocytosis: the affinity of ‘marker of self’ CD47 for SIRPalpha dictates potency of inhibition but only at low expression levels.
Blood Cells Mol. Dis.
45
:
67
74
.
50
Massarelli
,
E.
,
V.
Papadimitrakopoulou
,
J.
Welsh
,
C.
Tang
,
A. S.
Tsao
.
2014
.
Immunotherapy in lung cancer.
Transl. Lung Cancer Res.
3
:
53
63
.
51
Bielekova
,
B.
,
M. H.
Sung
,
N.
Kadom
,
R.
Simon
,
H.
McFarland
,
R.
Martin
.
2004
.
Expansion and functional relevance of high-avidity myelin-specific CD4+ T cells in multiple sclerosis.
J. Immunol.
172
:
3893
3904
.
52
Volpe
,
E.
,
L.
Battistini
,
G.
Borsellino
.
2015
.
Advances in T helper 17 cell biology: pathogenic role and potential therapy in multiple sclerosis.
Mediators Inflamm.
2015
:
475158
.
53
Sun
,
Y.
,
T.
Tian
,
J.
Gao
,
X.
Liu
,
H.
Hou
,
R.
Cao
,
B.
Li
,
M.
Quan
,
L.
Guo
.
2016
.
Metformin ameliorates the development of experimental autoimmune encephalomyelitis by regulating T helper 17 and regulatory T cells in mice.
J. Neuroimmunol.
292
:
58
67
.
54
Barsheshet
,
Y.
,
G.
Wildbaum
,
E.
Levy
,
A.
Vitenshtein
,
C.
Akinseye
,
J.
Griggs
,
S. A.
Lira
,
N.
Karin
.
2017
.
CCR8+FOXp3+ Treg cells as master drivers of immune regulation.
Proc. Natl. Acad. Sci. USA
114
:
6086
6091
.
55
Gravano
,
D. M.
,
K. K.
Hoyer
.
2013
.
Promotion and prevention of autoimmune disease by CD8+ T cells.
J. Autoimmun.
45
:
68
79
.
56
Wang
,
W.
,
K.
Fang
,
M. C.
Li
,
D.
Chang
,
K. A.
Shahzad
,
T.
Xu
,
L.
Zhang
,
N.
Gu
,
C. L.
Shen
.
2016
.
A biodegradable killer microparticle to selectively deplete antigen-specific T cells in vitro and in vivo.
Oncotarget
7
:
12176
12190
.
57
Schütz
,
C.
,
M.
Fleck
,
A.
Mackensen
,
A.
Zoso
,
D.
Halbritter
,
J. P.
Schneck
,
M.
Oelke
.
2008
.
Killer artificial antigen-presenting cells: a novel strategy to delete specific T cells.
Blood
111
:
3546
3552
.
58
Wang
,
W.
,
K. A.
Shahzad
,
M.
Li
,
A.
Zhang
,
L.
Zhang
,
T.
Xu
,
X.
Wan
,
C.
Shen
.
2017
.
An antigen-presenting and apoptosis-inducing polymer microparticle prolongs alloskin graft survival by selectively and markedly depleting alloreactive CD8+ T cells.
Front. Immunol.
8
:
657
.
59
Wan
,
X.
,
W.
Pei
,
Y.
Zhang
,
L.
Zhang
,
K. A.
Shahzad
,
T.
Xu
,
C.
Shen
.
2018
.
Inconsistence between number and function of autoreactive T cells in the course of experimental autoimmune encephalomyelitis.
Immunol. Invest.
47
:
1
17
.

The authors have no financial conflicts of interest.

Supplementary data