A Tolerogenic Artificial APC Durably Ameliorates ... · ORCIDs: 0000-0003-3798-5301 (K.A.S.);...

of July 9, 2018.This information is current as T Cells+ and CD8+CD4

Autoreactive−Modulating Myelin PeptideEncephalomyelitis by Directly and Selectively

Ameliorates Experimental Autoimmune A Tolerogenic Artificial APC Durably

Chuanlai ShenShilong Song, Xiaoxiao Jin, Limin Wang, Chen Zhao and Xin Wan, Weiya Pei, Khawar Ali Shahzad, Lei Zhang,

ol.1800108http://www.jimmunol.org/content/early/2018/07/06/jimmun

published online 9 July 2018J Immunol

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The Journal of Immunology

A Tolerogenic Artificial APC Durably AmelioratesExperimental Autoimmune Encephalomyelitis by Directlyand Selectively Modulating Myelin Peptide–AutoreactiveCD4+ and CD8+ T Cells

Xin Wan, Weiya Pei, Khawar Ali Shahzad, Lei Zhang, Shilong Song, Xiaoxiao Jin,

Limin Wang, Chen Zhao, and Chuanlai Shen

In this study, a tolerogenic artificial APC (TaAPC) was developed to directly and selectively modulatemyelin-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-b1). 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-g/IL-17A secretion and

elevated IL-10/TGF-b1 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. The

Journal of Immunology, 2018, 201: 000–000.

Multiple sclerosis (MS) is an inflammatory disease ofCNS in which immune cells target and destroy myelinsheath on nerve cells, thereby causing autoimmune

demyelination and consequent neurologic dysfunction (1–3).Myelin Ag–autoreactive CD4+ T cells and proinflammatory CD4+

T cells play a pivotal role in MS and its animal model of exper-imental autoimmune encephalomyelitis (EAE) (4, 5), whereasmyelin Ag–autoreactive CD8+ T cells and B cells also makepartial contributions to the progress of MS and EAE (6–8). But theprecise pathogenesis of MS remains unknown because of itsdiverse performances (2, 9). The current therapeutics such asnatalizumab (9, 10), fingolimod, (11) and immunosuppressiveregimens (laquinimod, cladribine, alemtuzumab, caclizumab, andrituximab) are primarily anti-inflammatory and non–Ag-specific

in nature and have severe side effects (12–14). As a result of long-term medication, most of these treatments for MS display sup-pression of overall immune response, which increases the risks ofinfection and cancer (15, 16).Thus, recently, increasing attentions have shifted toward the

autoantigen-specific immunotherapy. Numerous strategies havebeen demonstrated in the treatment of EAE or MS, such as thesoluble myelin peptide immunotherapy including glatiramer ace-tate, mixture of MBP85–99, PLP139–151 and myelin oligodendrocyteglycoprotein (MOG)35–55 (17), and altered peptide ligand (18);soluble autoantigen arrays (SAgAs) like codelivery of PLP139–151and 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

Department of Microbiology and Immunology, Medical School, Southeast Univer-sity, Nanjing, Jiangsu 210009, China

ORCIDs: 0000-0003-3798-5301 (K.A.S.); 0000-0002-3748-3742 (C.S.).

Received for publication January 24, 2018. Accepted for publication June 19, 2018.

This work was supported by grants from the National Natural Science Foundation ofChina (81172823 and 81372448), the Science and Technology Support Program ofJiangsu Province (BE2017714), and the Postgraduate Research and Practice Innova-tion Program of Jiangsu Province (KYCX17_0164). The sponsors had no role instudy design, data collection and analysis, preparation of the manuscript, or decisionto submit the article for publication.

C.S. designed and supervised the research. X.W., W.P., and K.A.S. performed themain experiments of this study. X.W. analyzed and organized the whole data. L.Z.and S.S. assisted in the generation and characterization of polylactic-coglycolic acidmicroparticles and tolerogenic artificial APCs. X.J. and L.W. assisted in the flowcytometry, confocal imaging, and near-infrared imaging and the consequent data

analysis. C.Z. assisted in animal experiments. C.S. and X.W. wrote the manuscriptwith discussions from all authors.

Address correspondence and reprint requests to Prof. Chuanlai Shen, Departmentof Microbiology and Immunology, Medical School of Southeast University, 87Dingjiaqiao Road, Nanjing, Jiangsu 210009, China. E-mail address: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: DC, dendritic cell; EAE, experimental autoimmuneencephalomyelitis; ICG, indocyanine green; LFB, Luxol Fast Blue; LNC, lymphnode cell; MNC, mononuclear cell; MNP, micro- and nanoparticle; MOG, myelinoligodendrocyte glycoprotein; MP, microparticle; MS, multiple sclerosis; NP, influ-enza A virus nucleoprotein; PEI, polyethyleneimine; PLGA, polylactic-coglycolicacid; PLGA-MPTGF-b1, TGF-b1–encapsulated PLGA MP; pMHC, peptide-loadedMHC; SPC, splenocyte; TaAPC, tolerogenic artificial APC; Treg, regulatory T cell.

Copyright� 2018 by TheAmerican Association of Immunologists, Inc. 0022-1767/18/$35.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800108

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encapsulated polystyrene or polylactic-coglycolic acid (PLGA)particles (23–25); and recombinant TCR ligand–like RTL1000,a recombinant fusion protein of HLA-DR2–ɑ1b1–hMOG35–55

used in MS clinical trials (26). However, most of these modu-lators act in a semi or indirect Ag-specific manner to induceautoantigen-specific suppression and tolerance. These treat-ments prevent the development or relapse of EAE or MS in largepart because of their ability to induce tolerogenic APCs, espe-cially DCs and macrophages, by alternative activation afteruptake of myelin Ags or peptides. Then the induced tolerogenicAPCs will secrete inhibitory cytokines (like TGF-b and IL-10)and express regulatory molecules (like FasL and PD-L1), sub-sequently promote regulatory T cell (Treg) production, and in-hibit the function of myelin protein–autoreactive Th1 and Th17cells through various signal pathways. Although these strategiespresent intriguing potential to confer tolerance in EAE or MS,multiple factors can influence the induction of tolerogenic APCsin vivo. These include the diverse types (27, 28), tissue speci-ficities (23, 24, 29, 30), and surface/nuclear receptors (23, 31,32) of APCs. Additionally, these treatments rarely involve theconcomitant modulation of myelin-autoreactive CD8+ T celland CD4+ T cells. By now, few studies focus on the direct de-pletion and modulation of myelin-autoreactive T cells, one ofthe ideal strategies for the treatment of T cell–mediated EAEand 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-loadedMHC (pMHC) multimers and multiple regulatory molecules inthe same spatial and temporal context for the treatment of EAEand MS. For this purpose, a tolerogenic artificial APC (TaAPC)was established by using a polymeric biomimetic microparticle(MP) platform. PLGA, a biocompatible and biodegradablepolymer widely used in drug and vaccine delivery systemsin human (33), was employed to generate cell-sized MPs asa scaffold to cocoupling the target Ags (MOG40–54/H-2D

b-Igdimer, MOG35–55/I-A

b-biotin multimer), regulatory molecules(anti-Fas and PD-L1–Fc), and “self-marker” CD47-Fc ontheir surface and encapsulating inhibitory cytokine (TGF-b1)inside. The multipotent TaAPCs were i.v. administered intoMOG35–55-induced active EAE murine model and followed bythe investigation of therapeutic outcomes, precise mechanism,and side effects. This study provides the first experimentalevidence, to our knowledge, for the capability of TaAPCs todirectly modulate autoreactive T cells by surface presentationof multiple ligands and paracrine release of cytokine, thus sug-gesting a novel Ag-specific immunotherapy, a desirable avenue,for the treatment of EAE and MS.

Materials and MethodsMice, cell lines, and peptides

Female C57BL/6J mice were obtained from the Comparative MedicineCenter of Yangzhou University (Yangzhou, China) and maintained in thespecific pathogen-free Laboratory Animal Centre of Southeast University(Nanjing, China). Animal welfare and experimental procedures were ap-proved by the Animal Ethics Committee of Southeast University andperformed in accordance with the Guidelines for the Care and Use ofLaboratoryAnimals (Ministry of Science and Technology of China, 2006) andthe National Institutes of Health Guide for the Care and Use of LaboratoryAnimals (publications no. 8023, revised 1978). Eight approximately10-wk-old mice were used in the experiments. Yac-1 cell line and B16F10melanoma cell line were obtained from the Cell Bank of Type CultureCollection of Chinese Academy of Sciences (Shanghai, China). MOG35–55

(MEVGWYRSPFSRVVHLYRNGK), MOG40–54 (YRSPFSRVVHLYRNG),and influenza A virus nucleoprotein (NP) 366–374 (ASNENMETM) weresynthesized by ChinaPeptides Biotech (Suzhou, China) with a purity.95%.

Fabrication and characterization of polyethyleneimine-coatedand TGF-b1–encapsulated PLGA MPs

Polyethyleneimine (PEI)–coated PLGA MPs were fabricated by usingdouble-emulsion water-in-oil-in-water method as we previously described(34), with the minor modifications of cytokine encapsulation. Twentymilligrams of PLGA polymer was dissolved in 1 ml of methylene chloride,and then TGF-b1 (2.5 mg) (PeproTech) dissolved in PBS was added in thesuspension. Double-emulsion method was followed to generate theTGF-b1–encapsulated PLGA MPs (PLGA-MPsTGF-b1), followed bypreparation of PEI-conjugated PLGA-MPsTGF-b1 by using modified EDC/NHS chemistry. Additionally, the PEI-conjugated PLGA MPs withoutencapsulation of TGF-b1 were also prepared in the same way. The pre-pared PLGA MPs were then characterized by using scanning electronmicroscopy (SEM; ZEISS EVO 18; Oberkochen, Germany). The size wasmeasured by dynamic light scattering (BI-90 Particle Sizer; BrookhavenInstruments, Holtsville, NY), and the z potential was detected by PALS zinstrument (Brookhaven Instruments).

Generation and phenotypic analyses of multivalent TaAPCs

MOG40–54/H-2Db-Ig dimers and NP366–374/H-2D

b-Ig dimers were preparedby coincubating H-2Db-Ig Dimer X (BD Biosciences) with MOG40–54 orNP366–374 peptide for 48 h at 4˚C according to the manufacturer’s instructions.After that, PEI-coated PLGA-MPsTGF-b1 (1 3 108 beads) were coincubated withMOG40–54/H-2D

b-Ig dimers (10 mg), anti-Fas mAb (5 mg; BD Biosciences),PD-L1–Fc (2.5 mg; Sino Biological, Beijing, China), CD47-Fc (0.6 mg; R&DSystems), and streptavidin (7 mg; ProZyme) in sterile PBS overnight at 4˚C onrotator. Then, MPs were blocked with 30% BSA in PBS for another 24 h at4˚C on rotator. After centrifugation to move the supernatant, the MPs werecollected and further incubated with MOG35–55/I-A

b-biotin monomer (5 mg;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 PBSfor further use. In parallel, several controls of the M/M-TaAPCs were alsoprepared by following the similar procedure, such as N/O-TaAPCs (cocouplingNP366–374/H-2D

b-Ig dimers, OVA323–339/I-Ab-biotin monomers, and other im-

mune 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-b1 encapsulated into PLGAMPs,

TaAPCs in-house (1 3 107) were resuspended in 1 ml of sterile PBS andincubated on rotator at 37˚C. The supernatant was collected at indicated timeintervals, and an equal volume of sterile PBS was supplemented. The amountof TGF-b1 released in the supernatant was quantified by using TGF-b1ELISA kit (Dakewe Biotech, Shenzhen, China) and followed by the calcu-lation of release curve. For phenotypic analyses, TaAPCs were stained withFITC-anti–hamster-IgG (eBioscience), PE–anti-mouse I-Ab (eBioscience), andallophycocyanin–anti-mouse IgG1 (BD Biosciences) or allophycocyanin–anti-human IgG1 (Miltenyi Biotec, Bergisch Gladbach, Germany) for 30 min at4˚C. After washing, the TaAPCs were acquired on a FACSCalibur flowcytometer (BD Biosciences) and observed under confocal laser scanning mi-croscopy (FV1000; Olympus, Tokyo, Japan).

Induction and treatment of active EAE

On day 0, active EAE model was induced in female C57BL/6J mice bymultipoint s.c. injections with MOG35–55 peptide (ChinaPeptides Bio-tech, Suzhou, China) emulsified in CFA (Sigma-Aldrich) at a dosage of300 mg per mouse. Prior to use, CFAwas prepared by mixing heat-killedBacillus Calmette–Guerin (Ruichun Biotech, Shanghai, China) into IFA(Sigma-Aldrich) to achieve a concentration of 10 mg ml21 of BacillusCalmette–Guerin. On days 0 and 2, pertussis toxin (Sigma-Aldrich) wasadministered i.p. into the mice at a dosage of 250 ng per mouse per timepoint.

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 38after MOG immunization (1 3 107 MPs per mouse per time point). AfterEAE induction and TaAPCs treatments, mice were monitored daily by twoinvestigators in a blinded manner, and the severity of disease was scored asdescribed (35) with the following criteria: grade 0, normal; grade 1, flaccidtail; grade 2, mild hindlimb weakness (quick righting reflex); grade 3, se-vere 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 days8, 18, 28, and 38 after MOG immunization (1 3 107 MPs per mouse pertime point) in a similar way. The late therapeutic administrations on days18, 28, and 38 via tail vein were performed in the EAE mice with the samedosage.

2 A TOLEROGENIC ARTIFICIAL APC TREATS EAE


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Histopathological analyses

The spinal cord tissues were isolated from the EAEmice after i.v. injectionson day 100 after MOG immunization. All specimens were embedded inparaffin, and sections with a thickness of 5–7 mm were prepared. Thensections were routinely stained with H&E and Luxol Fast Blue (LFB;Boster Biological Technology, Wuhan, China), respectively. Inflammatoryinfiltration and demyelization in the tissues were evaluated under micro-scope (Eclipse 80i; Nikon, Tokyo, Japan). The mean number of inflam-matory cells was obtained by counting five separated fields (1003) byImage-Pro Plus software (Media Cybernetics, Rockville, MD). Quantifi-cation of spinal cord demyelization was assessed by two investigators in ablinded manner as described previously (36): score 0, no demyelination;score 1, mild demyelination; score 2, moderate demyelination, and score3: severe demyelination. The average score from five spinal cord sectionsof each animal was calculated.

In vivo and ex vivo near-infrared imaging

To monitor the in vivo trafficking of TaAPCs, indocyanine green (ICG)–inlayed TaAPCs were generated in the same way. Briefly, ICG (Sigma-Aldrich), TGF-b1, and PLGA polymer were dissolved in dichloromethane,and the double-emulsion solvent evaporation method was followed to gen-erate the ICG-inlayed PLGA-MPsTGF-b1. Then the MPs were surface mod-ified 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-Fc2), N/O-TaAPCs, andblank MPs were generated in parallel. On day 18 after MOG immunization,the EAE mice were randomized into four groups followed by the injectionsof different ICG MPs, respectively, via the tail vein (1 3 107 MPs permouse). The mice were then anesthetized by isoflurane and imaged by usingMaestro in vivo fluorescence imaging system (CRi, Woburn, MA) at indi-cated time points. Images were captured at an excitation wavelength of710–745 nm and an emission wavelength of 780–840 nm. At 4 h after in-jection, the mice from each group were sacrificed, and organs (liver, spleen,kidneys, lymph nodes, heart, brain, and lungs) were dissected surgically forex vivo imaging.

Tissue distribution of TaAPCs and colocalizationswith immune cells

PE-labeled TaAPCs were generated by cocoupling PE–streptavidin(BD Biosciences) and other immune molecules onto PEI-conjugatedPLGA-MPsTGF-b1. In parallel, PE-labeled M/M-TaAPCs (CD47-Fc2),N/O-TaAPCs, and blank MPs were also generated as controls. On day 18after MOG immunization, the PE-labeled control MPs and TaAPCs wereinjected, respectively, into the EAE mice (13 107 MPs per mouse) via tailvein. Peripheral blood from the orbital venous plexus, spleen, and lymphnodes were collected at 30 min, 4h, and 4 h after injection, respectively, indark, and processed to single-cell suspensions. Wright’s staining wascarried out, and slides were observed under optical microscope (Eclipse80i; Nikon). The spleen cells and lymph node cells (LNCs) were freshlyacquired on a FACSCalibur flow cytometer (BD Biosciences) without anystaining.

Additionally, spleens were collected in dark from each group and em-bedded in freezing medium (O.C.T, Sakura Finetek). Frozen sections with athickness of 9–10 mm were prepared and stained with FITC–anti-mouseCD4 (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 wash-ing, the sections were further stained with DAPI (Sigma-Aldrich) for 5 minand finally visualized under confocal laser scanning microscopy (Olym-pus). In parallel, the isotype controls were also stained using FITC–ratIgG2bk or FITC–rat IgG2ak (eBioscience). Meanwhile, the spleens fromEAE mice were processed into single-cells suspensions, stained withFITC-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 quantifiedwith a visible percentage in the spleen cell suspensions.

MHC multimer staining and flow cytometry

Peripheral blood, spleens, and CNS tissues (brain and spinal cord) werecollected from EAE mice after i.v. administrations on day 20 after MOGimmunization and processed to single-cell suspensions. PBMCs or splenocytes(SPCs)were prepared routinely. Themononuclear cells (MNCs) inCNS tissueswere further isolated by using discontinuous 70%/30% Percoll gradients(Pharmacia, Stockholm, Sweden) as previously described (37). The cells werethen seeded in 24-well cell culture plate (5 3 106 cells well21)and coincubated with MOG35–55 peptide (20 mg ml21) plus IL-2

(30 pg ml21; PeproTech) or MOG40–54 peptide (20 mg ml21) plus IL-2(100 pg ml21) 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 detectMOG Ag–specific CD4+ T cells, the cells were further incubated withallophycocyanin-labeled MOG35–55/I-A

b tetramer or allophycocyanin-labeled OVA323–339/I-A

b tetramer (10 ml per tube) (MBL) for 30 min indark 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 minincubation 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 withallophycocyanin-labeled anti-mouse IgG1 (BD Biosciences) for 1 h at 4˚C.Then, the cells were stained by the mixture of MOG40–54/H-2D

b-Ig dimers orNP366–374/H-2D

b-Ig dimers with allophycocyanin-labeled anti-mouse IgG1for 30 min at 4˚C. Finally, the cells were stained with FITC–anti-mouseCD8a (53-6.7; eBioscience) and PE–anti-mouse CD3e for 30 minat 4˚C. In parallel, the isotype controls were stained also by usingPE–Armenian hamster IgG, FITC–rat IgG2bk, or FITC–rat IgG2ak(eBioscience). After washing with PBS, the cells were acquired ona FACSCalibur flow cytometer (BD Biosciences) and analyzed withFlowJo software (Tree Star, Ashland, OR).

Analyses of T cell apoptosis and activation

Peripheral blood and spleens were collected from EAE mice after i.v.administrations on day 20 after MOG immunization. The PBMCs and SPCswere 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 wash-ing with PBS, the cells were stained with annexin V and propidium iodideaccording to the manufacturer’s protocol (eBioscience) and analyzed byflow cytometry. To evaluate T cells activation, the SPCs were freshlystained with FITC–anti-mouse CD3e (145-2C11), allophycocyanin–anti-mouse CD4 (GK1.5) or CD8a (53-6.7), and PE–anti-mouse CD44 (IM7) orCD69 (H1.2F3) (eBioscience) for 30 min at 4˚C and followed by flowcytometry. Isotype control staining was also performed as described.

T cell proliferation assay

Spleens were separated from the EAE mice after i.v. administrations on day20 after MOG immunization. SPCs were prepared and incubated with 5 mMof CFSE (Sigma-Aldrich) for 10 min at 37˚C, and immediately washedthree times with ice-cold RPMI 1640 medium (Life Technologies). Then,the CFSE-labeled SPCs were seeded into round-bottom 96-well plates(1 3 105 cells/well) (BD Falcon) and coincubated with MOG35–55 orMOG40–54 peptides (20 mg ml21) for 7 d in complete RPMI 1640 mediumat 37˚C with 5% CO2 and humidified conditions. Cells were harvested,stained with PE–anti-mouse CD3e and allophycocyanin–anti-mouse CD4or CD8a (eBioscience) for 30 min at 4˚C, and analyzed by flow cytometry.Cell divisions were demarcated according to CFSE staining brightness.

Intracellular cytokine staining

For the detection of IFN-g– or IL-17A–secreting CD4+ T cells and CD8+

T cells, SPCs or LNCs from EAE mice after i.v. administrations wereprepared on day 20 and cocultured in 24-well plate (1 3 106 cells well21)with PMA/ionomycin and BFA/monensin mixture (Multi Sciences, Shang-hai, China) for 4 h or stimulated by MOG35–55 or MOG40–54 peptide(20 mg ml21) for 16 h and followed by addition of protein transportinhibitor BFA for another 5 h at 37˚C under 5% CO2 and humidifiedconditions. Then, the cells were harvested, blocked with anti-mouseCD16/CD32 for 20 min at 4˚C, incubated with allophycocyanin–anti-mouseCD8a and FITC–anti-mouse CD4 for 30 min at 4˚C, and followedby PE–anti-mouse IFN-g (XMG1.2) or PE–anti-mouse IL-17A (eBio17B7)(eBioscience) staining for another 30 min at 4˚C after fixation/permeabilization. PE–rat IgG1k (eBRG1) or PE–rat IgG2ak isotype(eBR2a) staining was carried out in parallel. After washing with PBS, cellswere analyzed by flow cytometry. For the detection of Tregs, the MouseRegulatory T Cell Staining Kit (eBioscience) was used according to themanufacturer’s protocol. Briefly, the fresh SPCs or LNCs were blockedwith anti-mouse CD16/CD32, then stained with allophycocyanin–anti-mouse CD25 (PC61.5) and FITC–anti-mouse CD4 (RM4-5). After fixa-tion, the intracellular staining with PE–anti-mouse Foxp3 (FJK-16s) wasperformed and finally analyzed by flow cytometry.

Quantification of cytokines by ELISA

The supernatants of SPCs were collected from the T cell proliferation assay,and the homogenates of CNS tissues (brain and spinal cord) were prepared.

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Then, the concentrations of cytokines (IL-17A, IFN-g, IL-10, and TGF-b1)in these samples were detected by using the mouse cytokine ELISA kit(Dakewe Biotech).

Cytotoxicity assay of NK cells

SPCs were prepared from EAE mice after i.v. administrations on day 40 afterMOG immunization. A total of 1 3 107 cells were labeled with CFSE asdescribed and then used as effector cells to coculture with target cells (Yac-1cells, 1 3 105 cells well21) 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 analyzedby flow cytometry after staining with 7-amino-actinomycin D (7-AAD;eBioscience). The cytotoxic activity of NK cells was calculated as the per-centage of 7-AAD–positive cells within the CFSE-negative cell population.

Tumor cells challenge

The EAE mice were s.c. injected with B16F10 melanoma cells (1 3 106

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 ondays 8, 18, 28, and 38 after MOG immunization (1 3 107 MPs per mouseper time point). The tumor size was measured daily after it became de-tectable, with a venire caliper, until the size was up to 2500 mm3. Theproducts of perpendicular diameters were determined, and the tumorvolume was calculated with following formula: (the shortest diameter)2

3 (the longest diameter) 3 0.5.

T cell proliferation in response to allogenic cells and irrelativeantigenic peptides

EAE mice were randomized into three groups and administered via tail veinwith PBS, blank MPs, or M/M-TaAPCs on days 8, 18, 28, and 38 after MOGimmunization. On day 40, the spleen cells were obtained from each EAEmice, labeled with CFSE, and seeded into round-bottom 96-well plates asresponder cells (1 3 105 cells well21), then coincubated with SPCs fromBALB/c mice (pretreated with mitomycin C, 1 3 105 cells well21) or ir-relevant peptides (OVA323–339 plus NP366–374, 20 mg ml21). Cells werecocultured for 7 d in complete RPMI 1640 medium at 37˚C, 5% CO2, andhumidified 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.

Enumeration of various immune cells in CNS tissue, spleen,lymph nodes, and peripheral blood

Brain and spinal cord were isolated from the EAE mice after i.v. admin-istrations on day 20 after MOG immunization. The MNCs were preparedand 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 EAEmice on day40 after MOG immunization. The SPCs and LNCs were prepared and stainedwith allophycocyanin–anti-mouse CD3e (145-2C11), FITC–anti-mouseCD8a (53-6.7), PE–anti-mouse CD4 (GK1.5), FITC–anti-mouse CD19(MB19-1), and FITC–anti-mouse NK1.1 (PK136) (eBioscience), respec-tively, for 30 min at 4˚C. After washing with PBS, cells were analyzed byflow cytometry. In parallel, peripheral blood was collected from orbital ve-nous plexus of EAE mice on day 40. Routine blood tests were performedusing automated hematology analyzer (XE-2100; Sysmex, Kobe, Japan).

Statistical analyses

GraphPad Prism 6.0 (GraphPad, La Jolla, CA) software was used to analyzethe data statistically.Wilcoxon signed rank test was used to analyze the clinicalscore curves of EAE and the tumor sizes. To determine the survival curve ofmice, a Kaplan–Meier graph was constructed, and a log-rank comparison ofthe groups was used to calculate the mice survival curve after tumor cellschallenge. For other experiments, a two-tailed unpaired Student t test wasused to determine differences across groups. All data were presented as themean 6 SD. A result of p , 0.05 was considered significant.

ResultsGeneration and characterization of PLGA MPs andmultivalent TaAPCs

The PEI-conjugated and PLGA-MPsTGF-b1 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 6 1.9 mm, and 77.8%of MPs were 3.27–7.78 mm in diameter (Fig. 1B). The mean z

potential was 45.3 6 4.38 mV as detected by the PALS z instru-ment, suggesting a high capacity to covalently couple proteins(Fig. 1C).To fabricate the multivalent TaAPCs, PLGA-MPsTGF-b1 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 efficiencyof TGF-b1 was ∼75.2% as measured by ELISA. The total cumu-lative TGF-b1 released from 1 3 107 beads of M/M-TaAPCs wasnearly 188.1 ng over 30 d in a sustained manner, with the rapidrelease during the first 2 d. To confirm the immobilization ofmultiple molecules on the surface of TaAPCs, control MPs(M/M-MPs, N/O-TaAPCs, PD-L12 M/M-TaAPCs, and CD472

M/M-TaAPCs) were generated in parallel and followed by three-color staining. The histograms (Fig. 1E) showed that each kind ofsurface molecule was effectively coupled onto TaAPCs with thestrong fluorescence signals. Furthermore, confocal images alsoconfirmed the copresence of the five kinds of surface moleculesonto TaAPCs (Fig. 1F). The flow cytometric dot plots (Fig. 1G)revealed that nearly 60–80% of TaAPCs codisplayed the five kindsof surface molecules as determined by double-positive staining ineach two-color dot plots. Each batch of TaAPCs was routinelyevaluated in this manner prior to use.

TaAPCs markedly and durably ameliorate EAE and reducelocal autoimmune response after i.v. injections

Active EAE was induced in female C57BL/6 mice by MOG35–55

peptide immunization as described. The mice were then ran-domized 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 manifes-tation (clinical scores) was recorded daily for 100 d. According tothe clinical score curves and pathological analyses, active EAEinduced in this study always presented a classic clinical coursecontaining acute onset (near day 10), peak stage (near day 18), andlater on stable chronic remission. For each treatment group, themean 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, andM/M-MP treatment groups displayed apparent motor dysfunction,without significant difference across groups. Expectedly, theM/M-TaAPC treatment group presented markedly and durablylower clinical scores than the N/O-TaAPC group and other controlgroups from acute onset to stable chronic remission stage, asanalyzed by Wilcoxon signed rank test and paired two-tailedStudent t test. Meanwhile, as a noncognate Ag control group,EAE mice treated by N/O-TaAPCs only displayed a transient andslight relief of motor dysfunction at peak stage with no significantdifference from other control groups.In parallel, the EAEmicewere also administered by i.p. injections

or i.v. injections using the same timeline and dosage. As comparedwith the control groups, i.p. injections of M/M-TaAPCs caused onlypartial 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 adminis-trations of TaAPCs were carried out in the EAE mice. As shown inSupplemental Fig. 1C, three i.v. injections of M/M-TaAPCs on days18, 28, and 38 significantly inhibited the progression of EAE ascompared with control groups, but the clinical severity was notameliorated as well as the early administrations on days 8, 18, 28,



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and 38. Thus, in the later experiments, the immune responses afteri.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) andprocessed to single-cell suspensions followed by fluorescencestaining and flow cytometry. The frequencies and numbers of CD3+

cells, CD4+ T cells, and CD8+ T cells in CNS were obviously andconcurrently reduced in the M/M-TaAPC–treated mice, as com-pared 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 inflam-matory cells (Fig. 2F) and the scores of demyelination (Fig. 2G) inCNS tissues were significantly decreased by the i.v. administrationsof M/M-TaAPCs relative to the N/O-TaAPC group and other control

groups. Both therapeutic outcomes and pathological analyses dem-onstrated that four injections of M/M-TaAPCs obviously inhibitedthe progression of EAE with a sustained inhibitory effect over theclinical course of 100 d and reduced local autoimmune response ofCNS. As compared with the regulatory molecule–negative controlgroup (M/M-MPs) and noncognate Ag control group (N/O-TaAPCs), the multipotent M/M-TaAPCs worked in an Ag-specificmanner and regulatory molecule–dependent fashion in vivo.

Tissue distribution and in vivo trafficking of TaAPCsin EAE mice

EAE mice at peak stage (day 18 after MOG immunization) wereinjected via tail vein with PE-labeled TaAPCs or PBS. Then the PE-TaAPCs were observed in peripheral blood at 30 min, in spleen andlymph nodes at 4 h after injection by Wright’s staining, and wereabsent in the cell suspensions from PBS group, a negative control

FIGURE 1. Characterization of PLGA MPs and multivalent TaAPCs. (A) Representative SEM image of PLGA MPs. (B) Size distribution and (C)

z-potential (millivolt) distribution of PLGA MPs. (D) Release curve of TGF-b1 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-L12), and M/M-TaAPCs (CD472) 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 3400). (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.



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(Fig. 3A). Also, the spleen and LNC suspensions were harvestedat 4 h and freshly detected by flow cytometry without any fluo-rescence staining. Fig. 3B displayed the PE-TaAPCs in these cellsuspensions, which may be free or bound to cells. This experimentaims to confirm the distribution of TaAPCs in secondary lym-phatic tissues with a visible percentage after i.v. injection.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, orblank MPs were injected via tail vein into the EAE mice at peakstage. As revealed by the whole-body fluorescence images atvarious time points (Fig. 3C, left panel), the fluorescent intensityin mice was strongest during 6 h after ICG–M/M-TaAPCs injec-tion and then decreased slowly with a retention time of morethan 36 h. As controls, ICG–M/M-TaAPCs (CD472), ICG–N/O-TaAPCs (noncognate), and ICG–blank MPs showed the in vivo

trackings similar to ICG–M/M-TaAPCs, but a much shorter re-tention time with very weak fluorescent intensity at 36 h timepoint (Fig. 3D, left panel). The ex vivo imaging of dissected or-gans displayed that at 4 h after injection, M/M-TaAPCs and thecontrol MPs appeared in liver, spleen, kidney, and lungs withvisible fluorescence, but M/M-TaAPCs displayed significantlymore accumulation in liver and spleen with a higher mean fluo-rescence intensity than the control MPs (Fig. 3C, right panel;Fig. 3D, right panel). These differences may imply that target Agsand CD47 molecules enable the TaAPCs to target MOG Ag–specific T cells and resist phagocytosis, and thus make the in vivotrafficking distinct to the N/O-TaAPCs, CD472 M/M-TaAPCs, andblank MPs. Notably, no visible fluorescent signal of M/M-TaAPCsor control MPs was observed in the head of EAE mice over 36 h,suggesting that most of the cell-sized TaAPCs or control MPs couldnot go into the CNS through vascular circulation.

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 de-

myelination area (n = 6 to 8 mice in each group). *p , 0.05, **p , 0.01, ***p , 0.001.



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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 31000. (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 (Figure legend continues)



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TaAPCs prefer to colocalize with CD4+ T cells andCD8+ T cells in EAE mice

To find the evidence that multipotent TaAPCs can directly contactor interplay with target T cells in vivo, PE-labeled M/M-TaAPCsor control MPs were i.v. injected into the EAE mice at peak stage.Four hours later, spleens were collected from each group. Frozensections were prepared and followed by immune fluorescencestaining. As shown by confocal micrographs (Fig. 4A), M/M-TaAPCs mainly distributed in the red pulp and marginal zoneand displayed many colocalizations with CD4+ T cells and CD8+

T cells but few colocalizations with B cells, macrophages, andDCs. CD47-negative M/M-TaAPCs showed fewer or similarcolocalizations 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 contactswith T cells and many colocalizations with others. In parallel,the spleen single-cell suspensions were prepared and followedby immune fluorescence staining. As shown by flow cytometry(Fig. 4B), the PE+/FITC+ signals (presumably MP–cell conju-gates) were quantified with a visible percentage in the spleen cellsuspensions. When compared with the PE–blank MP or PE–N/O-TaAPC group, PE–M/M-TaAPCs displayed the significantlyhigher 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 cellconjugates (PE+/CD19-FITC+) and TaAPC–macrophage conju-gates (PE+/F4/80-FITC+), than the PE–M/M-TaAPCs(CD472)injection group.

TaAPCs prefer to contact with CD4+ T cells and CD8+ T cellsin cocultures

Spleens were collected from the EAE mice on day 18 withoutinjection with PE-labeled TaAPCs or control MPs and processed toSPC suspensions. Then, the cells were cocultured (1 3 105 beadswell21) with PE–blank MPs, PE–N/O-TaAPCs, PE–M/M-TaAPCs(CD472), and PE-M/M-TaAPCs (1 3 105 beads well21), re-spectively, in 96-well cell culture plate. After 4 h coculture, themixed suspensions of cells and MPs were harvested and droppedon slides to prepare cell smears. After being naturally dried, thecell smears were stained by FITC-labeled mAbs specific for CD4,CD8a, CD19, CD11c, and F4/80, respectively, and finally visu-alized under laser confocal scanning microscopy (Fig. 4C). Inparallel, the mixed suspensions of cells and MPs were harvestedand stained with the FITC-labeled mAbs as above and detected byflow 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 orPE–N/O-TaAPCs, but significantly fewer colocalizations with B cells,DCs, and macrophages than PE–M/M-TaAPCs(CD472). Fig. 4Dshowed that PE–M/M-TaAPCs showed significantly higher per-centages of TaAPC–CD4+ T cell conjugates (PE+/CD4-FITC+)and TaAPC–CD8+ T cell conjugates (PE+/CD8a-FITC+) than thePE–blank MP or PE–N/O-TaAPC group but significantly lowerpercentages 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 (CD472).

These results from in vitro experiments were consistent with theobservations in vivo and further demonstrate that TaAPCs candirectly contact with T cells in secondary lymphatic tissues byautoantigen targeting, and CD47-Fc can minimize the engulfmentof TaAPCs by phagocytes in vivo.

TaAPCs markedly eliminate MOG Ag–specific T cells andinduce apoptosis of T cells in EAE mice

PBMCs, SPCs, and MNCs in CNS tissues were collected from theEAE mice in each treatment group on day 20 (2 d after the secondinjection of TaAPCs) and followed by the 6-d ex vivo incubationwithMOG35–55 or MOG40–54 peptide. Then, MOG35–55-specific CD4+

T cells and MOG40–54-specific CD8+ T cells were enumerated byMOG35–55/I-A

b tetramer and MOG40–54/H-2Db-Ig dimer staining,

respectively. When compared with the blank MP group, two infu-sions of M/M-TaAPCs reduced the frequencies of MOG35–55-specific CD4+ T cells by 65.3% in peripheral blood, 79.5% inspleen, and 66.7% in CNS, whereas the noncognate N/O-TaAPCstreatment did not lead to significant reduction (Fig. 5A). Similarly,the frequencies of MOG40–54-specific CD8+ T cells were reducedby 46.4% in peripheral blood, 56.4% in spleen, and 62.4% inCNS after two injections of M/M-TaAPCs, whereas no obviousreduction was displayed after N/O-TaAPCs treatment (Fig. 5B).As the negative controls of pMHC multimer staining, the noncognateOVA323–339/I-A

b tetramer and NP366–374/H-2Db-Ig dimer staining were

also carried out in parallel for each sample. Both OVA323–339-specificCD4+ T cells and NP366–374-specific CD8+ T cells showed very lowfrequencies in the PBMCs and SPCs, with no significant differenceacross groups (Supplemental Fig. 2).To find out the mechanisms by which M/M-TaAPCs reduce the

MOG Ag–specific T cells, the apoptosis of fresh CD4+ T cells andCD8+ T cells in peripheral blood and spleen was analyzed first.Two injections of M/M-TaAPCs led to the mean percentage oftotal apoptotic CD4+ T cells ∼3.1-fold higher in PBMCs and 1.5folds higher in SPCs than the blank MP group; similarly, twoinjections of M/M-TaAPCs increased the number of apoptoticCD4+ T cells by 189% in PBMCs and 57% in SPCs comparedwith the blank MP group (Fig. 6A). As shown in Fig. 6B, the meanpercentage of total apoptotic CD8+ T cells increased ∼1.5-foldmore in SPCs than the blank MP group, whereas the number ofapoptotic CD8+ T cells increased by 88% in SPCs and 21% inPBMCs after M/M-TaAPCs treatment. In contrast, M/M-MP (noregulatory molecules) and N/O-TaAPC (irrelative Ags) treatmentcaused no significant increase of apoptotic CD4+ T cells and CD8+

T cells as compared with blank MP group. These data suggestedthat the M/M-TaAPCs depleted the myelin peptide–autoreactiveT cells and induced T cells apoptosis in EAE mice in an Ag-specific and regulatory molecule–dependent manner.

TaAPCs inhibit the activation and proliferation of MOGAg–reactive T cells in EAE mice

In addition to the apoptosis induction, TaAPCs may also exert in-hibitory effects on MOG Ag–specific T cells by the surface presen-tation of PD-L1 and paracrine release of TGF-b1. On day 20(2 d after the second administration of TaAPCs), SPCs were sepa-rated from the EAE mice in each treatment group and freshly stainedwith PE–anti-mouse CD44 or CD69 for monitoring the activation ofT cells. The frequencies of CD4+/CD44+ or CD4+/CD69+ T cells

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.



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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 3400.

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 (CD472), 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.



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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 6 SD. *p , 0.05, **p , 0.01.



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(Fig. 6C) and CD8+/CD44+ or CD8+/CD69+ T cells (Fig. 6D) wereobviously decreased in M/M-TaAPCs treatment group, whereasthere was no significant inhibition in N/O-TaAPC and M/M-MPgroups, 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 wasdetermined by CFSE staining and flow cytometry analyses. Afterthe MOG peptide stimulation, the proliferation of both CD4+ T cellsand CD8+ T cells was reduced by 59.1 and 52.7%, respectively, inthe M/M-TaAPC group as compared with blank MP group, whereasthere was no significant reduction in N/O-TaAPC group and M/M-MP group (Fig. 6E).

TaAPCs induce Tregs and inhibit MOG Ag-reactive Th1, Th17,and Tc17 cells in EAE mice

To evaluate the effects of TaAPCs in polarizing T cells in vivo, SPCsandLNCswere separated from the EAEmice in each treatment groupon day 20 (2 d after the second injection of TaAPCs) and followed byTreg detection. As compared with the blank MP group, the fre-quencies 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 differencewas found across the control groups. Furthermore, the fresh SPCsand LNCs were coincubated with MOG35–55 or MOG40–54 peptidesfor 16 h or with PMA for 4 h followed by the detection of IFN-g– orIL-17A–secreting CD4+ T cells and CD8+ T cells. After MOG35–55

peptide stimulation, the frequencies of IFN-g+/CD4+ T cells and IL-17A+/CD4+ T cells (presumably MOG35–55-reactive Th1 and Th17cells) decreased, respectively, by 60.1 and 55.7% in the SPCs, and40.5 and 41.6% in the LNCs from M/M-TaAPC–treated mice, whencompared with the blank MP group. The numbers of IFN-g+/CD4+

T cells and IL-17A+/CD4+ T cells also displayed the trend similar tothe frequencies across groups. Meanwhile, M/M-MPs led to nosignificant decrease of Th1 and Th17 either in SPCs or LNCs, butN/O-TaAPCs also caused the significant reduction of Th1 and Th17cells in SPCs relative to the blank MP group (Fig. 7B, 7C). In ad-dition, after PMA stimulation, the frequencies of IFN-g+/CD4+ Tand 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) alsodecreased, respectively, by 50.1% in the SPCs and 53.7% in theLNCs from M/M-TaAPC–treated mice in comparison with mice ofcontrol groups (Fig. 7D), but no significant decrease of IFN-g+//CD8+ T cells (presumably MOG40–54-reactive Tc1 cells) was foundeither in SPCs or LNCs (Supplemental Fig. 3D).Moreover, proinflammatory cytokines (IFN-g, IL-17A) and in-

hibitory cytokines (IL-10, TGF-b1) were quantified in the culturesupernatants of SPCs after 7-d incubation with MOG35–55 peptideand in the CNS tissue homogenates collected on day 20. As shownin Fig. 7E, the concentrations of IFN-g and IL-17A decreased by45.7 and 75.3%, respectively, in the cell culture supernatants ofSPCs from M/M-TaAPC–treated mice, whereas the levels of IL-10and TGF-b1 elevated by 56.6 and 33.3%, respectively, as comparedwith the blank MP group. Also, the SPCs from N/O-TaAPC–treatedEAE mice secreted much less IL-17A than that from other controlgroups, but without significant difference (Fig. 7E). However, theM/M-TaAPC treatment did not change these cytokines profiles inCNS tissue homogenates (Supplemental Fig. 3E).

TaAPCs induce T cells apoptosis, polarize Tregs, and inhibitT cell activation in the cocultures with purified T cells

To obtain more convincing evidence for the direct effects ofTaAPCs on T cells and the role of each kind of regulatorymolecule, the

cocultures of TaAPCs or control MPs with purified T cells were carriedout. The control MPs cocarrying autoantigens (MOG40–54/H-2D

b

multimer, MOG35–55/I-Ab multimer) and single kinds of regula-

tory molecules (anti-Fas, PD-L1-Fc or TGF-b1) were newlyprepared and termed M/M/anti-Fas–MPs, M/M/PD-L1–MPs, andM/M/TGF-b1–MPs, respectively. Then, spleens were collectedfrom the EAE mice without treatment of TaAPCs on day 18 afterMOG immunization and processed to single-cell suspensions. TheT cells were purified from the spleen cells by using a mouse CD3T cell negative magnetic sorting kit (Stemcell Technologies,Vancouver, Canada), then seeded in 96-well cell culture plate(1 3 105 cells well21), and coincubated with PBS, blank MPs,M/M/anti-Fas–MPs, M/M/PD-L1–MPs, M/M/TGF-b1–MPs,N/O-TaAPCs, or M/M-TaAPCs (1 3 105 beads well21) plus IL-2(30 pg ml21) in RPMI 1640 medium supplemented with 10% FBSat 37˚C in 5% CO2 and humidified conditions. After 24 h, the cellswere harvested and followed by the detections of apoptotic CD4+ andCD8+ 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+ andCD8+ T cells increased obviously in M/M-TaAPC group andM/M/anti-Fas group, but not in other control groups. These resultsshow that TaAPCs can directly induce T cells apoptosis withoutthe requirement of other cells, and anti-Fas onto TaAPCs exertspivotal effects on the induction of apoptosis. In parallel, the fre-quencies of CD44+/CD4+ T cells (Fig. 8C) and CD44+/CD8+

T cells (Fig. 8D) were decreased only in M/M-TaAPC group, butnot in other control groups, as compared with blank MP group. Asshown in Fig. 8E, the frequency of Tregs was enhanced signifi-cantly in M/M-TaAPCs and M/M/TGF-b1 groups but not in othercontrol groups when compared with PBS control group. Thesedata imply that TaAPCs can directly inhibit T cells activation andpolarize T cells to Tregs without the requirement of other cells. Toa certain extent, these results also imply that TGF-b1 releasedfrom TaAPCs may be the key molecule for inducing Tregs. Al-though PD-L1 is thought to inhibit T cell proliferation, inhibitingT cell activation requires the combined effects of anti-Fas, PD-L1,and TGF-b1 molecules.

TaAPCs treatment does not lead to apparent side effects onimmune cells and overall immune function

The side effects of TaAPCs treatment were preliminarily investigated.EAE mice were randomized into three groups and administered viatail 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 finalinjection of TaAPCs), the numbers of lymphocytes, monocytes, andneutrophils in peripheral blood (Supplemental Fig. 4A) were detectedby routine blood tests. The percentages of T cells (SupplementalFig. 4B), B cells, NK cells (Supplemental Fig. 4C), and apoptoticT cells (Supplemental Fig. 4D) in SPCs or LNCs were detected byflow cytometry. No obvious difference was found across groups.Furthermore, the cytotoxicity of NK cells in each group was

measured without significant difference across groups (SupplementalFig. 4E). The tumor model was established to evaluate the antitu-mor ability of EAE mice after TaAPCs treatment. EAE mice werechallenged with B16 melanoma cells on day 3 after MOG immu-nization, followed by i.v. administrations of M/M-TaAPCs orcontrol MPs on days 8, 18, 28, and 38. As shown, the tumor growthcurves (Supplemental Fig. 4F) and survival rates (SupplementalFig. 4G) of tumor-bearing EAE mice displayed no significant dif-ference across treatment groups. Finally, the proliferation of hostT cells in response to alloantigen and irrelative antigenic peptideswas further investigated in cocultures. On day 40, spleen cells wereprepared from EAE mice as above, labeled with CFSE, and



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coincubated with SPCs from BALB/c mice (pretreated with mito-mycin C) or irrelevant peptides (OVA323–339 plus NP366–374) for 7 d.Then, the cells were stained with allophycocyanin–anti-mouseCD3e and finally analyzed by flow cytometry. As shown inSupplemental Fig. 4H, the proliferation of T cells from the EAEmice treated with TaAPCs or control MPs was quantified, and noobvious 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 TaAPCsstrategy achieved the desirable outcomes by directly and selec-tively depleting or modulating myelin Ag–specific T cells in theEAE mice.

Epitope spreading in the TaAPC treatment for the EAE in B6mice

Epitope spreading is an important issue to evaluating the translationpotential of TaAPCs. In this study, the MOG119–132-induced (38),

FIGURE 6. TaAPCs markedly induce T cells

apoptosis and inhibit the activation and prolifera-

tion 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 apo-

ptotic CD4+ T cells. (B) Frequencies of apoptotic

CD8+ T cells. Furthermore, SPCs were separated

from the EAE mice at 2 d after the second ad-

ministration 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.



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PLP178–191-induced (39), and MOG35–55-induced EAE modelshave been established in B6 mice as described and administeredi.v. with M/M-TaAPCs, N/O-TaAPCs, and blank MPs, respectively,

on day 25 after myelin peptide immunization. Then, the clinicalmanifestation (clinical scores) has been recorded daily for 40 dto clarify whether epitope spreading can be observed in the

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-g+/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-g, IL-17A, IL-10, and TGF-b1 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.



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B6/MOG and B6/PLP models. As shown in Supplemental Fig. 1,one infusion of M/M-TaAPCs on day 25 significantly decreased themean clinical scores of chronic remission phase in both MOG35–55-induced EAE (Supplemental Fig. 1D) and MOG119–132-inducedEAE (Supplemental Fig. 1E) but not in PLP178–191-induced EAE(Supplemental Fig. 1F). Of note is that the M/M-TaAPCs codis-playing MOG35–55/I-A

b multimer and MOG40–54/H-2Db-Ig dimer

are designed to directly modulate MOG35–55-specific CD4+ T cellsand MOG40–54-specific CD8+ T cells in MOG35–55-induced EAE.

These new data indicated, to some extent, the epitope spreading ofTaAPC treatment in MOG peptide–induced EAE, but not in PLPpeptide–induced EAE, in B6 mice.

DiscussionNumerous researchers around the world have reported the bio-mimetic micro- and nanoparticles (MNPs) carrying myelin pep-tides or proteins along with toxin or regulatory molecules forthe treatment of EAE or MS (30, 40, 41). s.c. prophylactic and

FIGURE 8. TaAPCs induce T cells apoptosis,

polarize Tregs, and inhibit T cell activation in the

cocultures with purified T cells. T cells were pu-

rified 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 mean6 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.



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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 polystyreneNP– bearing PLP139–151 peptide could prevent the onset of thePLP-induced relapsing EAE and induce long-term T cell tolerance(23, 24). Gold NPs cocoupling MOG35–55 peptide and aryl hy-drocarbon receptor (31), or PLG-NPs coencapsulating myelinprotein or peptide and rapamycin (30), also induced durable andAg-specific immune tolerance and suppressed EAE after i.v. ad-ministration. Although these MNPs were decorated or encapsu-lated with myelin peptides or protein, most of the therapeuticsunderlie the Ag presentation by cellular uptake of MNPs and thefollowing induction of tolerogenic APCs and Tregs, in whichT cell tolerance was indirectly induced. Therefore, these strategiesmay 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 coupledonto PLGA MPs as target Ags which can directly bind with MOGpeptide–specific TCRs on the autoreactive T cells without therequirement of presentation by APCs, thus tailoring the directmodulation on MOG-reactive T cells. To minimize the cellularuptake of autoantigens, cell-sized PLGA MPs were used as car-riers instead of NPs. As known, the size greatly affects the abilityof MNPs to pass through biological barriers, be engulfed byphagocytes, and interplay with target cells via surface presentationof ligands (43). The closer the particle is to cellular size, the morepotent the effect on the target cell (44, 45). Cell-sized MPs presenta reduced risk of engulfment by phagocytes relative to NPs(43, 46). Only phagocytic cells can take up MPs (especially0.5–10 mm), whereas most of the cell types in vivo may be able toingest nanoscale particles by pinocytosis. Moreover, CD47-Fcmolecules were cocoupled onto the PLAG MPs as a self-marker(47, 48) because CD47 can interact with signal regulatory protein-a (SIRPa) on phagocytes to prevent phagocytosis (49) and hasbeen used to construct the “stealth particles” in NP-mediated drugdelivery systems for increasing the circulation time of NP vehiclesin human (50). In this study, the cocultures of TaAPCs with pu-rified T cells confirmed the direct effects of TaAPCs on T cells;many colocalizations or conjugates of TaAPCs with CD4+ andCD8+ T cells were observed in spleen sections and cocultures.These in vivo and in vitro data suggest the direct and selectivemodulations of TaAPCs on T cells, but we cannot eliminate thepossible phagocytosis occurring in vivo, especially at the laterstage during the retention time of 36 h. Therefore, it is reasonableto conclude that the TaAPCs modulate the autoreactive T cellsin vivo mainly in a direct contact way, but an indirect modula-tion mediated by tolerogenic APCs and Tregs may also be in-volved in the immunotherapy.Second, the PLGA MPs were cocoupled with pMHC class II

(MOG35–55/I-Ab multimer) and class I (MOG40–54/H-2D

b-Ig di-mer) multimers, and were thus enabled to target and modulateboth CD4+ T cells and CD8+ T cells specific for MOG Ags. It iswell documented that myelin Ag–reactive CD4+ T cells, such asTh17, Th1, and Tregs, work as crucial drivers or regulators inautoimmune demyelinating diseases (4, 5, 51). The correspondingproinflammatory cytokines (IL-17A and IFN-g) and inhibitorycytokines (IL-10 and TGF-b1) produced by Th17, Th1, and Tregs,respectively, are also demonstrated to be important in EAE andMS. Defects in Th17 and Th1 cells help to prevent EAE, whereasTregs participate in the maintenance of self-tolerance and theregulation of inflammatory autoimmune system by variousmechanisms (52–54). Therefore, most Ag-specific strategies focuson the autoreactive CD4+ T cells. However, increasing studieshave also demonstrated the secondary contributions of myelin

Ag–specific CD8+ T cells in MS and EAE progress (6–8). Tc17cells, which are newly defined by producing IL-17, are thought tobe primarily proinflammatory cells and induce many of the sameeffects as Th1 and Th17 cells in several autoimmune diseases,including MS and EAE (55). Therefore, the multivalent PLGAMPs were generated in our study to enact the combination therapytargeting 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 andtemporal context. The combined uses of anti-Fas, PD-L1, and TGF-b1 provide several signal pathways to powerfully modulate theautoreactive T cells like the tolerogenic DCs, which induce apo-ptosis, inhibit activation and proliferation, and skew Th cells toTregs. In this study, the roles of each kind of regulatory moleculewere confirmed in the cocultures of TaAPCs with purified T cells.The anti-Fas–dependent deletion of Ag-specific T cells was a keycomponent in the first inhibitory effects on the development ofEAE, 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, asdemonstrated by our previous works (56). Thus, the significantincrease of Tregs and obvious inhibition of Ag-specific T cellactivation and proliferation mediated by TGF-b and PD-L1should be closely associated with the long-term (100 d) mainte-nance of EAE amelioration. Therefore, the TaAPCs deliveringthree kinds of regulatory molecules may obtain a combined orsynergistic inhibitory effect in EAE mice, including Ag-specificT cells deletion and active T cell tolerance induction by multiplesignal pathways, although we cannot appreciate the accuratecontribution 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 paracrinerelease of inhibitory cytokine (TGF-b1), so were termed TaAPCsfor the first time, to our knowledge. Another advantage is thatthe cell-sized TaAPCs cannot circulate into brain, as our datadisplayed, and thus may evoke much less concern regarding bio-safety for the putative clinical use.A similar but distinct concept, killer artificial APCs, was reported

by Schutz et al. (57) in 2008. The CMV or Mart-1 peptide-loadedHLA-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 thedegradable cell-sized PLGAMPs to selectively deplete OVA257–264-specific CD8+ T cells in vitro (56) and kill the H-2Kb-alloreactiveCD8+ T cells in a murine model for prolonging the alloskin graftsurvival in our recent works (58). In this study, the killer artificialAPC platform was upgraded to multipotent TaAPCs and was ap-plied in the treatment of T cell–medicated autoimmune disease forthe first time, to our knowledge.Of note, the route, timeline, and dosage of TaAPC administration

should be further optimized in translational studies along with thelong-time investigation of biosafety and toxicity issue. For thebiomimetic particles carrying peptides, Ags, or pMHC multimersin the treatment of autoimmune diseases, many studies show thati.v. administration leads to much more effective immune toleranceand clinical outcomes than the i.p. or s.c. routes (23, 24, 31). In thisstudy, the results of i.p. or s.c. injections were consistent with theprevious research. The in vivo mechanism remains unclear. Thesafety of i.v. injection has also been demonstrated in the treatmentof murine EAE without obvious allergic reactions (23, 24). Ourrecent works also confirmed the long-time safety of the cell-sized PLGA MPs carrying targeting Ag (H-2Kb alloantigen) andanti-Fas mAb after i.v. administration in the mice of alloskin



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transplantation. Obvious impairment of the host overall immunefunction and visible organ toxicity were not found at long-timepoints (58). In addition, the i.v. administrations of TaAPCs late atpeak stage led to much weaker inhibitory effects on EAE progressthan the i.v. injections early at onset phase. This difference may becontributed to the dynamic changes in the frequencies and reac-tivity of MOG-reactive T cells during the EAE course (59) and thedifficult reparation of demyelination and neurologic dysfunctionafter 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 en-capsulating TGF-b1 inside for the treatment of MOG-inducedEAE. The encouraging results suggest a novel Ag-specific im-munotherapy for the T cell–mediated autoimmune diseases bydirectly modulating autoreactive T cells through multiple signalpathways. The in vivo mechanism, tissue distribution, and effectson overall immune functions were also defined initially, and thusmay facilitate the translational studies.

DisclosuresThe authors have no financial conflicts of interest.

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