Nanoparticle-mediated codelivery of myelin antigenand a tolerogenic small molecule suppressesexperimental autoimmune encephalomyelitisAda Yeste, Meghan Nadeau, Evan J. Burns, Howard L. Weiner, and Francisco J. Quintana1

Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115

Edited by Lawrence Steinman, Beckman Center for Molecular Medicine, Stanford, CA, and accepted by the Editorial Board May 21, 2012 (received for reviewDecember 14, 2011)

The immune response is normally controlled by regulatory T cells(Tregs). However, Treg deficits are found in autoimmune diseases,and therefore the induction of functional Tregs is considereda potential therapeutic approach for autoimmune disorders. Theactivation of the ligand-activated transcription factor aryl hydro-carbon receptor by 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylicacid methyl ester (ITE) or other ligands induces dendritic cells (DCs)that promote FoxP3+ Treg differentiation. Here we report the useof nanoparticles (NPs) to coadminister ITE and a T-cell epitope frommyelin oligodendrocyte glycoprotein (MOG)35–55 to promote thegeneration of Tregs by DCs. NP-treated DCs displayed a tolerogenicphenotype and promoted the differentiation of Tregs in vitro.Moreover, NPs carrying ITE and MOG35–55 expanded the FoxP3+

Treg compartment and suppressed the development of experi-mental autoimmune encephalomyelitis, an experimental modelof multiple sclerosis. Thus, NPs are potential new tools to inducefunctional Tregs in autoimmune disorders.

Multiple sclerosis (MS) is an autoimmune disease driven byan immune response directed against antigens in the CNS

(1). The autoreactive components of the immune system arenormally under the control of specialized regulatory T cells(Tregs); of particular importance are FoxP3+ (2) and IL-10+Tregs (3). Treg deficits have been found in MS and other au-toimmune diseases (4, 5). Conversely, Tregs have been shown toarrest the development of several experimental models of au-toimmune disease (5). Thus, the induction of antigen-specifictolerance is considered a promising approach for the treatmentof MS and other autoimmune disorders (6).As a result of our studies on immunoregulation in the zebra-

fish (7), we found that the ligand-activated transcription factoraryl hydrocarbon receptor (AhR) controls the differentiation ofFoxP3+ and IL-10+ Tregs and Th17 cells in mice and humans(8–12). AhR activation with the nontoxic mucosal ligand 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE)expands Tregs and suppresses EAE (11). We (11) and others(13–15) showed that the generation of Tregs by AhR ligandsinvolves the induction of tolerogenic dendritic cells (DCs). In-deed, the activation of AhR signaling in DCs by ITE or otherligands induces DCs that promote FoxP3+ Treg differentiation(11, 13–15).Nanoparticles (NPs) have unique features that prompted their

use in medicine. For example, NPs have been used for in vivotumor detection and targeting (16) and for the delivery of anti-angiogenic compounds (17). NPs have also been used to inducepathogen-specific immunity in vaccination regimens (18, 19). Inthe context of the therapeutic management of inflammation, NPshave been recently used to deliver siRNAs to silence ccr2 ex-pression and prevent the accumulation of inflammatory mono-cytes at sites of inflammation (20). However, the use of NPs toinduce antigen-specific tolerance and treat autoimmune dis-orders remains largely unexplored.In this work, we report the use of NPs to coadminister ITE and

the T-cell epitope from myelin oligodendrocyte protein locatedbetween residues 35 and 55 to promote the generation of CNS-specific Tregs by DCs. NP-treated DCs displayed a tolerogenic

phenotype and promoted the differentiation of Tregs. Moreover,NPs carrying ITE and a peptide corresponding to residues 35–55of the myelin oligodendrocyte glycoprotein (MOG35–55) expandedthe FoxP3+ Treg compartment and suppressed the developmentof experimental autoimmune encephalomyelitis (EAE), an ex-perimental model of MS. Thus, NPs are potential new tools forthe codelivery of T-cell antigens and tolerogenic small moleculesto induce antigen-specific Tregs in autoimmune disorders.

ResultsConstruction of NPs Containing MOG35–55 and ITE. MS and EAE arecaused by an autoimmune response directed against the CNS (1).To induce CNS-specific Tregs, we constructed NPs containing theAhR ligand ITE and MOG35–55, which contains a CD4+ T-cellepitope targeted by effector and Tregs during the course of EAE(21). By using gold particles (60 nm in diameter), we constructedfour types of NPs that were stabilized with a layer of thiol-poly-ethylene glycol (PEG) (16): (i) unloaded NPs, (ii) NPs loadedwith ITE (NPITE), (iii) NPs loaded with MOG35–55 (NPMOG), and(iv) NPs loaded with ITE and MOG35–55 (NPITE+MOG; Fig. 1A).As a first step toward the characterization of the NPs we stud-

ied their UV–visible absorption spectra. We detected a prominentabsorption at 530 nm in unloaded gold NPs, which results fromthe excitation of surface plasmon vibrations in the gold NPs (22)(Fig. 1B). Loading of ITE, MOG35–55, or MOG35–55 and ITEshifted the absorption peak to 560 nm, reflecting the binding ofITE and/or MOG35–55 to the NPs (Fig. 1B). MOG35–55 was de-tected in NPMOG and NPITE+MOG, but not in NP or NPITE, bygel electrophoresis followed by silver staining (Fig. 1C). Furtheranalysis by transmission EM showed that the NPs had a roundmorphology and a diameter of ∼60 nm (Fig. 1D).We analyzed the ability of NP-delivered ITE to activate AhR

by using a mammalian cell line stably transfected with a constructcarrying the luciferase gene under the control of an AhR-re-sponsive promoter. We found that treatment of the reporter cellline with ITE-containing NPs (NPITE and NPITE+MOG) led to thesignificant activation of the AhR-responsive promoter (Fig. 1E).The AhR-responsive promoter, however, was not activated byNPs that did not carry ITE (NP and NPMOG; Fig. 1E). Thus, ITEloaded into NPs can be released to trigger AhR-dependentsignaling.Compounds loaded onto NPs are protected from enzymatic

degradation (23), so we investigated the effect that the in-corporation of ITE into NPs might have on its degradation byliver enzymes. Free ITE and NPITE were preincubated witha preparation of hepatic microsomes (24), and their ability to

Author contributions: A.Y., H.L.W., and F.J.Q. designed research; A.Y., M.N., and E.J.B.performed research; A.Y., M.N., E.J.B., H.L.W., and F.J.Q. analyzed data; and A.Y. and F.J.Q.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. L.S. is a guest editor invited by the Editorial Board.1To whom correspondence should be addressed. E-mail: fquintana@rics.bwh.harvard.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1120611109/-/DCSupplemental.

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activate an AhR-responsive promoter was analyzed in aliquotstaken at different time points. Incubation with hepatic microsomesfor 1 h decreased the ability of free ITE to activate AhR to ∼50%of its initial value, whereas ITE in NPs maintained its ability toactivate AhR under the same experimental conditions (Fig. 1D).To investigate whether the stabilization of ITE by NPs might

have any effect on its in vivo activity, we compared the suppres-sive activity of free and NP-bound ITE on the experimental au-toimmune disease EAE. In agreement with our previous results,daily administration of ITE (100 μg per mouse, i.p.) led to a sig-nificant suppression of EAE, but no significant suppression wasobserved when ITE was administered once per week (Fig. S1).However, the weekly administration of NP-bound ITE (i.p.) ledto a significant suppression of EAE development (Fig. S1). Takentogether, these data suggest that NPs protect ITE from enzymaticdegradation and boost its immunoregulatory activity in vivo.

NPITE+MOG Induces Tolerogenic DC. DCs play a central role in theactivation and polarization of T cells in vivo (25), so we in-vestigated the effect of NPs on DC function. Splenic DCs wereisolated from naive mice and incubated with NPs, and NP uptakewas analyzed by transmission EM. NPs could be detected insideDCs 1 h after their addition to the cells, and were still detectableinside DCs 24 h later (Fig. 2A). To investigate whether the up-take of NPs carrying ITE activates AhR signaling in DCs, weanalyzed the expression of cyp1a1, a gene that is directly trans-activated by AhR (26). We found that cyp1a1 expression was up-regulated in DCs treated with NPITE and NPITE+MOG, but not byNP and NPMOG (Fig. 2B). Thus, ITE-containing NPs are up-taken by DCs and activate AhR signaling.We (11) and others (13–15) have shown that AhR activation

induces tolerogenic DCs that have a decreased ability to polarizenaive T cells into effector Th1 or Th17 cells and promote thedifferentiation of Tregs. Thus, we studied the effects of NPs onthe response of DCs to stimulation with the Toll-like receptoragonist Escherichia coli lipopolysaccharide (LPS). Splenic DCsisolated from naive mice were incubated in vitro with NPs and

activated with LPS. Incubation with empty NPs did not modifythe expression of the class II MHC or costimulatory molecules inDCs, and did not affect their ability to activate naive T cells (Fig.S2 A and B). However, we found that NPITE+MOG-treated DCsshowed a significant decrease in the expression of MHC-II,CD40, and CD86, but no significant change was seen in CD80(Fig. S2C). Moreover, incubation with NPITE or NPITE+MOG ledto a significant reduction in the production of the Th1 and Th17polarizing cytokines il12 and il6, respectively (Fig. 2C).To directly investigate the effects of NPs on the activation and

polarization of T cells by DCs, NP-treated DCs were activated withLPS and used to stimulate CD4+ 2D2+ T cells, which expressa transgenic T-cell receptor that recognizes MOG35–55 (27). Wefound that incubation of naive CD4+ 2D2+ T cells with DCs andNPMOG resulted in the proliferation of the 2D2+ T cells in theabsence of exogenous MOG35–55, demonstrating that NP-deliveredMOG35–55 is presented by the DCs (Fig. 2D). Activation of 2D2+ Tcells with DCs and NPITE+MOG, however, triggered a significantlyreduced proliferative T-cell response (Fig. 2D), which resembles thereduced response of T cells incubated with ITE-treated DCs (11).We also observed a significant decrease in the proliferative responseof 2D2+T cells activated withDCs andNPITE+MOG orNPITE in thepresence of exogenously added MOG35–55 (Fig. S2D).The analysis of cytokine secretion showed that 2D2+ T-cell ac-

tivation with DCs and NPMOG triggered the production of signifi-cant amounts of IFN-γ and IL-17, indicative of the polarization ofTh1 and Th17 cells (Fig. 2E). The production of IFN-γ and IL-17,however, was significantly reduced when 2D2+ T cells were acti-vated with DCs and NPITE+MOG (Fig. 2E). Conversely, DCs in-cubated with NPITE+MOG showed an increased ability to promotethe differentiation of FoxP3+ Tregs (Fig. 2F). Taken together,these results are in agreement with the reported effects of AhRactivation on the ability of DCs to activate an polarize effector andTregs (11, 13–15), and demonstrate that NPITE+MOG induces tol-erogenic DCs that favor the generation of FoxP3+ Tregs.

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Fig. 1. Characterization of NPs containing ITE andMOG35–55. (A) Schematic representation of NPITE+MOG.(B) Optical absorption obtained from NPs. (C) Gelelectrophoresis and silver staining of NPs. (D) Trans-mission EM analysis of pegylated NPs. (E) HEK293 cellstransfected with a reporter construct coding for lucif-erase under the control of an AhR-responsive promoterwere incubated with NPs and luciferase activity wasmeasured after 24 h. Cotransfection with a TK-Renillaconstruct was used for normalization purposes. (F) AhRactivation detected after incubation of free ITE or NPITEwith a preparation of hepatic microsomes for 0, 10, 30,or 60 min (*P < 0.05, **P < 0.01, and ***P < 0.001 vs.NPITE).

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NPITE+MOG Administration Suppresses EAE. We then studied theeffects of NPs in vivo. Following parenteral administration,pegylated gold NPs have been reported to home to the spleen(16). DCs control T-cell activation and polarization in vivo (25),so we studied the effect of NP administration on splenic DCs.NPs were parenterally administered to naive mice, and, 6 h later,AhR activation in splenic DCs was analyzed by quantitativePCR. We found a significant up-regulation of cyp1a1 expressionin DCs isolated from NPITE- or NPITE+MOG-treated mice, butnot in those isolated from NP- or NPMOG-treated mice (Fig. 3A).We also found a significant activation of AhR, as indicated bycyp1a1 expression, in splenic macrophages and B cells isolatedfrom NPITE+MOG-treated mice (Fig. S3A).Given the central role of DCs in priming encephalitogenic Th1

and Th17 cells in vivo (25), we analyzed the effect of NP ad-ministration on the production of Th1 and Th17 polarizingcytokines. DCs from NPITE+MOG-treated mice showed a signifi-cant decrease in the production of IL-6 and IL-12 in response toactivation with LPS ex vivo (Fig. S3 B and C). Taken together,these data demonstrate that ITE-containing NPs activate AhRsignaling in splenic DCs in vivo and decrease the production ofTh1 and Th17 polarizing cytokines.Based on the effects of NPITE+MOG on the activation and

antigen presenting cell (APC) function of DCs in vitro andin vivo, and the role played by DCs in the differentiation ofencephalitogenic and Tregs (25), we studied the effects of NPson EAE. EAE was induced by immunization with MOG35–55 innaive B6 mice, NPs were administered weekly (6 μg per mouse)starting on the day of EAE induction, and the animals weremonitored daily for the development of the disease. NPITE+MOGadministration resulted in a significant suppression of EAE de-velopment (Fig. 3B). Treatment with NPITE also led to a significant

amelioration in EAE symptoms, but the protective effects of NPITEwere not as strong as those observed with NPITE+MOG, and thisdifference in the protection achieved by NPITE or NPITE+MOGtreatment was found to be significant (Fig. 3B). Of note, the co-administration of free MOG35–55 and ITE had no significant effecton the development of EAE (Fig. S4).EAE in B6 mice is driven by MOG35–55–specific Th1 and Th17

cells (21). Thus, to study the suppression of EAE by NPITE+MOG,we analyzed the effect of NP administration on the T-cell recallresponse to MOG35–55. Treatment with NPITE and NPITE+MOGresulted in a significant decrease in the proliferation and the pro-duction of IFN-γ and IL-17 triggered by MOG35–55 in recall exper-iments; this decrease was significantly stronger in the NPITE+MOGgroup (Fig. 3 C and D). No significant effect was detected whenthe response to anti-CD3 stimulation was investigated (Fig. S5A and B), suggesting that NP-treated mice are not systemicallyimmune-suppressed. Thus, NPITE+MOG suppress the encephalito-genic Th1 and Th17 T-cell response and the development of EAE.To investigate the potential therapeutic value of the co-

administration of antigen and ITE using NPs, we used the SJLmodel of EAE induced by immunization with the 139 to 151region of the proteolipid protein (PLP). In this model, thechronic phase of EAE is characterized by the spreading of theT-cell response to the PLP epitope placed between residues 178and 191 (PLP178–191) (28). Thus, we tested the therapeutic effecton SJL EAE of treatment with NPITE, NPs loaded with ITE andPLP139–151 (NPITE+139), and ITE and PLP178–191 (NPITE+178). Inaddition, an experimental group was treated with both NPITE+139and NPITE+178; empty NPs were used as controls. EAE was in-duced by immunization with PLP139–151 in naive SJL mice, and,on day 17 after disease induction, the mice were assigned to the

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Fig. 2. NPITE+MOG induces tolerogenic DCs. (A)Transmission EM analysis of uptake of NPITE+MOG byDCs in culture. (B) Analysis of cyp1a1 expression byDCs coincubated with NPs 24 h after initiation ofcell cultures. (C) Quantitative PCR analysis of il6 andil12 expression in DCs incubated in vitro with NPsand activated with LPS for 12 h; results presentedrelative to gapdh mRNA. (D–F) DCs were coincu-bated in vitro with NPs, activated with LPS, andused to stimulate naive 2D2+ CD4+ T cells. Pro-liferation (D) and cytokine secretion (E) to thesupernatants were analyzed at 72 and 48 h, re-spectively. (F) The frequency of CD4+ FoxP3+ cellswas analyzed by FACS at 72 h. Representative dataof one of three experiments that produced similarresults (*P < 0.05, **P < 0.01, and ***P < 0.001).

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different experimental groups and treated with NPs (6 μg permouse); the animals were treated weekly during the duration ofthe experiment. We observed that treatment with NPITE had nosignificant effect in the development of EAE (Fig. 3E). We alsofound that the administration of NPITE+139 had a transientbeneficial effect on the development of EAE; however, the ad-ministration of NPITE+139 and NPITE+178 led to a significantreduction in the EAE score, maximal EAE score, and thenumber of relapses after the initiation of NP treatment (Fig. 3Eand Table S1). Treatment with NPITE+139 alone had no signifi-cant effect on the chronic phase of EAE. Taken together, thesedata suggest that a combination of NPs targeting several relevantT-cell reactivities can be engineered to control epitope spreadingand chronic inflammation in established CNS autoimmunity.

FoxP3+ Tregs Mediate Suppression of EAE by NPITE+MOG. We (9–11)and others (13, 14, 29–31) have shown that AhR activation canexpand the FoxP3+ Treg compartment, so we investigated theeffect of ITE on FoxP3+ Treg. We found that the suppression ofEAE development by AhR activation with NPITE+MOG was as-sociated with a significant increase in the frequency of CD4+

Foxp3+ Tregs in the spleen (Fig. 4B) and the blood (Fig. 4B).Thus, NPITE+MOG administration suppresses the encephalito-genic Th1/Th17 (Fig. 3D) whereas it promotes the generation ofFoxP3+ Tregs.We then performed transfer experiments to study the role of

CD4+ Foxp3+ Tregs in the suppression of EAE triggered byNPITE+MOG. EAE was induced in Foxp3gpf mice carrying a GFPreporter in the foxp3 gene (32), the mice were treated with NP orNPITE+MOG, and CD4+ T cells were isolated and transferred intonaive mice. We found that naive recipients could be protected

from the development of EAE by the transfer of 5 × 106 CD4+ Tcells isolated from NPITE+MOG-treated mice, but not with cellsisolated from NP-treated mice (Fig. 4C). Removal of theCD4+FoxP3:GFP+ Treg fraction abrogated the protective effectof the transferred cells (Fig. 4C), suggesting that NPITE+MOG-induced CD4+FoxP3:GFP+ Tregs are responsible for the controlof the encephalitogenic T-cell response.

DiscussionNPs are being actively studied as tools for the modulation of theimmune response. Most of these studies, however, are focused onthe induction of pathogen-specific effector immunity in the contextof vaccine development (18, 19). Although the generation of anti-gen-specific Treg is considered a promising approach for thetreatment of autoimmune disorders (6), the use of NPs to induceantigen-specific tolerance and treat autoimmune disorders remainslargely unexplored. Here we describe NPs designed to coadministera tissue specific antigen (i.e., MOG35–55) and an AhR ligand (i.e.,ITE) to induce tolerogenic APCs that promote the differentiationof CNS-specific Tregs and suppress the development of EAE.Methods based on DNA vaccination (33, 34), oral (35), nasal (36),and transdermal (37) tolerization, or administration of antigencoupled to red blood cells (38) have also been developed to expandantigen-specific Tregs, and their translational relevance is now be-ing investigated in clinical trials. However, compared with othermethods for antigen delivery, an advantage of NPs is their ability tocodeliver target antigens in combination with well-defined tolero-genic small molecules to control APC activity.Tsai et al. reported the use of NPs containing recombinant

MHC molecules loaded with β-cell epitopes to reestablish im-mune tolerance in nonobese diabetic mice (39). These peptide/

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Fig. 3. NPITE+MOG suppresses EAE. (A)cyp1a1 expression in splenic DCs fromNP-treated mice. (B) EAE was inducedby immunization of naive B6 micewith MOG35–55, and NPs were admin-istered i.p. weekly from the day ofimmunization until the termination ofthe experiment. The course of EAE isshown as the mean EAE score ± SEM(n = 10 mice per group) and also asthe linear regression curves of thedisease for each group (Right). (C)Proliferative response to MOG35–55 ofsplenocytes taken from NP-treatedanimals immunized with MOG35–55 inCFA. Cell proliferation is indicated asthe mean cpm ± SEM in three to fivemice per group. (D) Cytokine secretiontriggered by MOG35–55 in splenocytestaken from NP-treated animals im-munized with MOG35–55 in CFA. (E)EAE was induced by immunization ofnaive SJL mice with PLP131–159, andNPs were administered i.p. weeklyfrom day 17 until the termination ofthe experiment. The course of EAE isshown as the mean EAE score ± SEM(n = 5 mice per group) for the wholeobservation period, and also as thelinear regression curves of the diseasefor each group from day 30 until thetermination of the experiment (Right).Representative data of one of at leastthree experiments that produced sim-ilar results (*P < 0.05, **P < 0.01, and***P < 0.001 vs. NP-treated mice; aP <0.05 vs. NPITE-treated mice).

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MHC NPs induced CD8+ Tregs that suppress the diabetogenicT-cell response, restoring normoglycemia in WT and humanizednonobese diabetic mice (39). Although the work by Tsai et al.showed for the first time the feasibility of using NPs to reestablishimmune tolerance, the use of these NPs in humans is limited by itsreliance on recombinant HLA/peptide complexes. However, it isconceivable that the use of peptide/MHC NPs in combinationwith the NPs described in the present study will promote the gen-eration of both CD4+ and CD8+ Tregs, resulting in more robustimmune regulation and, consequently, improved clinical efficacy.The NPs described in the present study target AhR to promote

the development of tolerogenic APCs. AhR has been shown tocontrol the development of tolerogenic DCs that promote thedifferentiation of Tregs in a retinoic acid and an indoleamine 2,3-dioxygenase–dependent manner (11, 13–15). However, NPs tar-geting other tolerogenic pathways or different APCs could alsobe capable of inducing antigen-specific tolerance. Thus, futurestudies should examine the therapeutic potential of targeting al-ternative tolerogenic pathways and specific APCs with NPs.In the context of neuroinflammation, nanomaterials are being

actively investigated for molecular MRI of the CNS (40), andalso as therapeutic tools. Nanoliposomes have been used todeliver CNS antigens (41) or to deplete macrophages (42) andcontrol disease progression in different EAE models. More re-cently, poly(D,L-lactic-coglycolic acid) NPs were used to delivera peptide designed to interfere with the activation of PLP139–151–specficic T cells (43). Although these poly(D,L-lactic-coglycolicacid) NPs prevented EAE development when tested in a pre-ventive paradigm, this approach failed to treat established dis-ease in SJL mice, reflecting the need to control the spreading ofthe autoimmune T-cell response to other epitopes. These resultsare in agreement with those of Robinson et al., who demon-strated that the successful treatment of ongoing EAE in the SJLmodel using tolerogenic DNA vaccines requires targeting othermyelin antigens in addition to PLP139–151 (44). Taken togetherwith our own data on the effect of tolerogenic NPs on SJL EAE,these results highlight the need to characterize the heteroge-neous immune response directed against multiple CNS targets inMS. In combination with methods for the high-throughputcharacterization of the autoimmune response (33, 45–47), NPsmight provide a new tool to codeliver tissue-specific antigens andtolerogenic small molecules to generate antigen-specific Tregssuited to the individual needs of patients with MS.

MethodsMice and Reagents. C57BL/6 mice were purchased from Jackson Laboratoriesand kept, together with 2D2+ (27) and FoxP3gfp (32) mice, in a pathogen-freefacility at the Harvard Institutes of Medicine. All experiments were carriedout in accordance with the guidelines of the standing committee of animalsat Harvard Medical School. ITE was purchased from Sigma-Aldrich and fromTocris Bioscience.

NP Preparation. NPs were produced by using PBS solution, 60 nm tannic acid-stabilized gold particles at a concentration of 2.6 × 1010 particles per milliliter(Ted Pella), methoxy-PEG-SH (molecular weight, ∼5 kDa; Nektar Therapeu-tics), ITE (Tocris Bioscience), and MOG35–55 (MEVGWYRSPFSRVVHLYRNGK).

Freshly prepared solutions of ITE (3.5 mM), MOG35–55 (600 μg/mL), or ITE andMOG35–55 (3.5 mM and 600 μg/mL, respectively) were added drop by drop toa rapidly mixing gold colloid at a 1:6 ITE solution:colloid volume ratio, whichfacilitates even distributions of the molecules on the gold particle surface(16). After 30 min incubation at room temperature with shaking, methoxy-PEG-SH (10 mM) was added drop by drop to the colloids. This surface cov-erage has been shown to result in a complete PEG monolayer on the goldparticle surface, and stabilizes gold colloids against aggregation under var-ious conditions (16). Moreover, it has been reported that the addition of 10-to 20-fold excess PEG-SH does not result in any changes in colloid stability orin the thickness of the polymer coating layer (16). After an additional 30 minincubation at room temperature, the colloids were pelleted by centrifuga-tion, resuspended in PBS solution, and characterized by UV–visible spec-troscopy and transmission EM.

Transmission EM. DC-incubated NPs were fixed in the dish for at least 1 h atroom temperature with 2.5% (vol/vol) glutaraldehyde, 1.25% (vol/vol)paraformaldehyde, and 0.03% picric acid in 0.1M sodium cacodylate buffer(pH 7.4). The cells were then postfixed for 30 min in 1% OsO4/1.5% (wt/vol)KFeCN6, washed in water three times, and incubated in 1% aqueous uranylacetate for 30 min followed by two washes in water and subsequent de-hydration in grades of alcohol [5 min each; 50%, 70%, 95% (vol/vol), twice at100%]. Cells were removed from the dish in propylene oxide, pelleted at1,000 × g for 3 min, and infiltrated for 2 h in a 1:1 mixture of propyleneoxide and TAAB Epon (Marivac). The samples were then embedded in TAABEpon and polymerized at 60 °C for 48 h.

Ultrathin sections (approximately 60 nm) were cut on a Reichert Ultracut-Smicrotome, picked up onto copper grids stained with lead citrate, and ex-amined in TecnaiG2 Spirit BioTWIN, and images were recorded with an AMT2k CCD camera.

Gel Electrophoresis. NPs (3.5 μg per lane) and MOG35–55 (1, 0.1, and 0.01 μg)were run by using NuPAGE 4% to 12% 1.0-mm Bis-Tris gels (Invitrogen), andMOG35–55 was visualized by silver staining (SilverQuest staining kit; Invi-trogen) and Coomassie brilliant blue staining.

Reporter Assays. HEK293 cells were transfected by using FuGENE HD (Roche),and the cells were analyzed after 24 h with the dual luciferase assay kit (NewEngland Biolabs). Tk-Renilla was used for standardization.

Microsomal Degradation. ITE or NPITE was incubated with mouse hepaticmicrosomes (2 mg/mL; Sigma-Aldrich) in a reaction buffer containing NAPDH(1 mM), MgSO4 (8 mM), KCl (45 mM), and 3.3 glucose 6-phosphate, pH 7.4, at37 °C for different periods of time.

Purification of DCs. DCs were purified from the spleens of naive B6 mice byusing CD11c+ magnetic beads according to the manufacturer’s instructions (Mil-tenyi). To generate bone marrow-derived DCs, bone marrow cells were isolatedfrom the femurs of naive mice and cultured for 5 d in the presence of IL-4 (10ng/mL) and GM-CSF (10 ng/mL). On day 5, the cells were purified with CD11c+

magnetic beads (Miltenyi), incubated with NPs, and stimulated with LPS.

Real-Time PCR. RNA was extracted from cells by using an RNA Easy Mini Kit(Qiagen), cDNA was prepared as recommended, and real-time PCR wasperformed by using an ABI7500 cycler (Applied Biosystems). All values areexpressed as fold increase or decrease relative to the expression of GAPDH.

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Fig. 4. FoxP3+ Tregs mediate the suppression of EAE byNPITE+MOG. (A and B) Frequency of CD4+ Foxp3+ Treg insplenocytes (A) and blood (B) from NP-treated mice immu-nized with MOG35–55 in CFA. (C) CD4+ or CD4+FoxP3:GFP− Tcells (5 × 106) were purified from NP- or NPITE+MOG-treatedmice and transferred into naive B6 mice, and, 24 h later, EAEwas induced in the recipients with MOG35–55. The course ofEAE is shown as the mean EAE score ± SEM (n = 5–10 miceper group). Representative data of one of at least threeexperiments that produced similar results (*P < 0.05, **P <0.01, and ***P < 0.001 vs. NP-treated mice).

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T-Cell Differentiation in Vitro. CD4+ T cells were activated with bone-marrow-derived cells or DCs at a 3:1 (100,000:30,000) T-cell-to-DC ratio, and activatedwith MOG35–55 (20 μg/mL) as described (11).

Cell Proliferation and Cytokine Production. Cells were cultured in serum-freeX-VIVO 20 media (BioWhittaker) for 72 h. During the last 16 h, cells werepulsed with 1 mCi of [3H]thymidine (PerkinElmer), followed by harvesting onglass fiber filters and analysis of incorporated [3H]thymidine in a β-counter(1450 Microbeta Trilux; PerkinElmer). Culture supernatants were collectedafter 48 h, and cytokine concentration was determined by ELISA by usingantibodies to IFN-γ and IL-17 from BD Biosciences.

FACS. For intracellular cytokine staining, cells were stimulated in culture me-dium containing phorbol 12-myristate 13-acetate (50 ng/mL; Sigma-Aldrich),ionomycin (1 μg/mL; Calbiochem), andGolgiStop (BD Biosciences) for 4 h. Afterstaining of surface markers, cells were fixed and permeabilized as describedand incubated with cytokine-specific antibodies (1:100) at 25 °C for 30 min.

NP Administration and EAE Induction. NPs were administered i.v. or i.p. (6 μgper mouse). EAE was induced by s.c. immunization with 100 μg of theMOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) in complete Freund adju-vant (CFA) and administration of 150 ng of pertussis toxin (Sigma-Aldrich)i.p. on days 0 and 2 as described (10). Clinical signs of EAE were assessed ac-cording to the following score: 0, no signs of disease; 1, loss of tone in the tail;2, hind limb paresis; 3, hind limb paralysis; 4, tetraplegia; and 5, moribund.

Statistical Analysis. Statistical analysis was performed by using Prism software(GraphPad). P values <0.05 were considered significant.

ACKNOWLEDGMENTS. The authors thank Deneen Kozoriz for cell sorting.This work was supported by National Institutes of Health Grants AI075285(to F.J.Q.), AI093903 (to F.J.Q.), and AI435801 (to H.L.W.); National MultipleSclerosis Society Grant RG4111A1 (to F.J.Q.) and a National Multiple SclerosisSociety Pilot Grant (to F.J.Q.); and the Harvard Medical School Office forDiversity and Community Partnership (F.J.Q.).

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