Biodegradable antigen-associated PLG nanoparticlestolerize Th2-mediated allergic airway inflammationpre- and postsensitizationCharles B. Smarra,b, Woon Teck Yapc, Tobias P. Neefa, Ryan M. Pearsond, Zoe N. Huntera, Igal Ifergana, Daniel R. Gettsa,Paul J. Brycea, Lonnie D. Shead,1, and Stephen D. Millera,1

aDepartment of Microbiology-Immunology and Interdepartmental Immunobiology Center, Feinberg School of Medicine, Northwestern University, Chicago,IL 60611; bTranslational Research Program, Benaroya Research Institute at Virginia Mason, Seattle, WA 98101; cDepartment of Chemical and BiologicalEngineering, Northwestern University, Evanston, IL 60208; and dDepartment of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109

Edited by David J. Mooney, Harvard University, Cambridge, MA, and accepted by the Editorial Board March 2, 2016 (received for review March 23, 2015)

Specific immunotherapy (SIT) is the most widely used treatmentfor allergic diseases that directly targets the T helper 2 (Th2) biasunderlying allergy. However, the most widespread clinical appli-cations of SIT require a long period of dose escalation with solubleantigen (Ag) and carry a significant risk of adverse reactions,particularly in highly sensitized patients who stand to benefit mostfrom a curative treatment. Thus, the development of safer, moreefficient methods to induce Ag-specific immune tolerance is criticalto advancing allergy treatment. We hypothesized that antigen-associated nanoparticles (Ag-NPs), which we have used to preventand treat Th1/Th17-mediated autoimmune disease, would also beeffective for the induction of tolerance in a murine model of Th2-mediated ovalbumin/alum-induced allergic airway inflammation.We demonstrate here that antigen-conjugated polystyrene (Ag-PS)NPs, although effective for the prophylactic induction of tolerance,induce anaphylaxis in presensitized mice. Antigen-conjugated NPsmade of biodegradable poly(lactide-co-glycolide) (Ag-PLG) are simi-larly effective prophylactically, are well tolerated by sensitized an-imals, but only partially inhibit Th2 responses when administeredtherapeutically. PLG NPs containing encapsulated antigen [PLG(Ag)],however, were well tolerated and effectively inhibited Th2 re-sponses and airway inflammation both prophylactically and ther-apeutically. Thus, we illustrate progression toward PLG(Ag) as abiodegradable Ag carrier platform for the safe and effectiveinhibition of allergic airway inflammation without the need fornonspecific immunosuppression in animals with established Th2sensitization.

immunotherapy | tolerance | nanoparticles | allergy | Th2 cells

Allergic diseases are a growing health concern in developedcountries. T helper 2 (Th2) cells are central to the allergic

response, producing cytokines, such as IL-4, IL-5, and IL-13, toact directly and indirectly on a variety of cells, including B cells,eosinophils, basophils, mast cells, and epithelial cells, inducinginflammation and further propagating the allergic cascade (1).The primary clinical approaches to allergy consist of antigen(Ag) avoidance and symptom control by targeting acute effectormolecules with drugs, such as antihistamines, leukotriene inhibi-tors, or broad-acting glucocorticoids. However, such treatmentsfail to address the underlying Th2 bias driving allergic in-flammation. Specific immunotherapy (SIT), however, has a longhistory of treating allergies by inhibiting Th2 responses. Typicallyconsisting of gradually escalating doses of soluble Ag deliveredmucosally or s.c., SIT has been demonstrated to induce bothregulatory and Th1 responses that compete with established Th2responses (2). However, administration of soluble Ag to sensitizedpatients bears a considerable risk of adverse reactions, therebynecessitating slow dose escalation or coadministration of therapiesthat minimize anaphylaxis, such as omalizumab (3). Therefore,improved methods to induce Ag-specific tolerance safely and

effectively remain a sought-after clinical tool for the treatment ofallergic disease in presensitized subjects.We previously demonstrated the ability of i.v.-administered Ag-

conjugated apoptotic splenocytes (Ag-SPs) to induce protective tol-erance for the prevention and treatment of established Th2 responsesin murine models of allergic airway inflammation and food allergy(4). Ag-SPs induce tolerance in a variety of immune disorders, andAg-conjugated autologous leukocytes have shown initial promise inclinical trials of patients with multiple sclerosis (MS) (5). However,the need for autologous cells to be isolated and manipulated beforeeach treatment limits the broad clinical application of such atreatment. Therefore, the development of shelf-stable, tolerance-inducing Ag carriers made under good manufacturing practices(GMP) represents the next generation of Ag-specific medicine. Tothis end, we have identified nanoparticle (NP) formulations for theinduction of tolerance to prevent and treat Th1/Th17-mediatedexperimental autoimmune encephalomyelitis (EAE) (6, 7).Here, we describe the experimental progression toward the

development of synthetic NPs, including antigen-conjugated poly-styrene (Ag-PS), antigen-conjugated biodegradable poly(lactide-co-glycolide) (Ag-PLG), and antigen-encapsulated poly(lactide-co-glycolide) [PLG(Ag)] particle carriers for the delivery of Ag for thesafe and effective treatment of Th2-mediated allergic airwayinflammation. Prophylactic treatment with formulations of Ag-PS,Ag-PLG, and PLG(Ag) all effectively inhibited Th2 sensitizationand subsequent airway inflammation. Ag-PS induced anaphylaxisin presensitized animals, whereas Ag-PLG and PLG(Ag) were

Significance

Allergic diseases are characterized by inappropriate inflamma-tory responses to benign environmental antigens (Ags). Primaryclinical approaches to allergic disease consist of symptom controlor administration of soluble Ag to skew the immune response toalternate phenotypes or induce immune tolerance. However,such approaches carry a significant risk of adverse events andrequire a long treatment course to achieve efficacy. In this paper,we describe the use of i.v.-administered nanoparticles as carriersof whole-protein Ag to induce tolerance safely and effectively inthe absence of nonspecific immunosuppression for the pre-vention and treatment of a mouse model of allergic asthma.

Author contributions: C.B.S., W.T.Y., D.R.G., P.J.B., L.D.S., and S.D.M. designed research;C.B.S., W.T.Y., T.P.N., R.M.P., Z.N.H., and I.I. performed research; C.B.S., W.T.Y., T.P.N., R.M.P.,Z.N.H., I.I., P.J.B., L.D.S., and S.D.M. contributed new reagents/analytic tools; C.B.S., W.T.Y.,T.P.N., R.M.P., Z.N.H., I.I., D.R.G., P.J.B., L.D.S., and S.D.M. analyzed data; and C.B.S., L.D.S.,and S.D.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. D.J.M. is a guest editor invited by the EditorialBoard.1To whom correspondence may be addressed. Email: ldshea@umich.edu or s-d-miller@northwestern.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1505782113/-/DCSupplemental.

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well tolerated. Ag-PLG partially inhibited preexisting Th2 responsesbut did not inhibit airway inflammation. Importantly, PLG(Ag) NPsinhibited both Th2 responses and airway eosinophilia in pre-sensitized mice and were not recognized by Ag-specific Ab.These studies illustrate the potential clinical development of asafe, biodegradable platform for tolerogenic Ag delivery for ef-fective abrogation of established Ag-specific Th2 responses andinhibition of allergic airway inflammation.

Materials and MethodsAnimals. Six- to eight-week-old BALB/c mice (Taconic Farms) were maintainedin specific pathogen free conditions at Northwestern University Center forComparative Medicine. All protocols were approved by the NorthwesternUniversity Institutional Animal Care and Use Committee.

Particle Preparation. PLGwith carboxylate end groups (50:50 D,L-lactide/glycolide,inherent viscosity = 0.18 dL/g) was purchased from Durect Corporation. PSparticles (Fluoresbrite Brilliant Blue carboxylate) were purchased from Poly-sciences, Inc. PLG particles with surface carboxylate groups and a negativeζ-potential were prepared using a single-emulsion/solvent evaporation methodas previously described (7). Particles were lyophilized and stored for future use.

PLG(Ag) NPs were prepared using a double-emulsion/solvent evaporationmethod. Briefly, 150 μL of 200 mg/mL aqueous Ag was added to 2 mL of20% (wt/vol) PLG in dichloromethane and emulsified by sonication. The poly(ethylene-alt-maleic acid) (PEMA) solution [10 mL of 1% (wt/vol) aqueousPEMA] was added to the emulsion and reemulsified by sonication. Theemulsion was then poured into 200 mL of 0.5% (wt/vol) aqueous PEMAunder stirring. The particles were purified, lyophilized, and stored for futureuse. Before use in vivo, the particles were rehydrated and washed three timeswith sterile PBS. The Ag dose of washed particles was determined as pre-viously described by dissolving particles in DMSO before 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde) (CQBCA) analysis (8). NP size and ζ-potential inPBS were measured using dynamic light scattering on a Zetasizer Nano ZS(Malvern Instruments).

Ag Conjugation to PLG and PS NPs. Ag was surface-conjugated to PLG or PSparticles through surface carboxylates as previously described (8). Lyophilizedparticles were rehydrated and washed three times with PBS. Peptides were thenincubated with particles and 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide(ECDI) at final concentrations of 50 mg/mL at 25 °C for 1 h. After conjugation,the particles were centrifuged at 3,000 × g for 5 min (PLG) or 16,100 × g for10 min (PS) and washed three times with PBS before injection or furtheranalysis. Ag dose and ζ-potential in PBS were measured as described above.

Allergic Airway InflammationModel.Micewere immunized i.p. with two dosesof 10 μg of ovalbumin (OVA; grade V; Sigma–Aldrich) in alum (3 mg) or alumand PBS alone. Mice were challenged for 20 min with aerosolized OVA(10 mg/mL) for three consecutive days before tissue harvest.

Analysis of Bronchoalveolar Lavage Eosinophils. Lungs were flushed with 1 mLof bronchoalveolar lavage fluid [BALF; 1 mM EDTA and 10% (vol/vol) FBS inPBS]. Total cell counts were determined, and samples were cytospun ontoslides and stained with DiffQuik (Siemens) for differential cell counts.

Airway Histology. Lungs were collected, fixed in formalin, embedded inparaffin, and stained with H&E or periodic acid–Schiff. Images are shown at amagnification of 200×.

OVA-Specific IgE. Serum was collected upon sacrifice, and OVA-specific IgE(OVA-IgE) was quantified by sandwich ELISA. Anti-mouse IgE (BD Biosciences)was used as the capture Ab, and biotinylated OVA (prepared using an EZ-LinkSulfo-NHS-LC-Biotin kit from Pierce) was used as a secondary reagent. Thequantity of OVA-IgE was determined by a standard curve generated usingpurified mouse OVA-IgE from the TOe hybridoma.

Ag-Specific Recall Responses. Spleens andmediastinal lymph nodes (LNs) wereharvested, processed into RBC-free single-cell suspensions, an incubated with25 μg/mL OVA in serum-free HL-1 culture medium at 37 °C for 48 h, andpulsed with 1 μCi of tritiated thymidine ([3H]TdR) per well for the last 24 h ofculture. Proliferation was determined by [3H]TdR incorporation as detectedby a Topcount Microplate Scintillation Counter (PerkinElmer).

Cytokine Quantification. BALF and supernatants from recall response cultures(harvested at 48 h) were assayed for IL-4, IL-5, IL-13, IL-10, IL-17, and IFN-γ bymagnetic Milliplex MAP multiplex assay (Millipore).

TemperatureMeasurements.Temperaturewasmeasured for 1 h after treatmentusing either s.c.-implanted temperature transponders (BioMedic Data Systems)or a rectally inserted temperature probe attached to an Oakton Temp-10 typeK thermocouple thermometer.

OVA-Ig Binding in Vitro. Fiftymicrograms of particleswas incubatedwith 1 μg/mLOVA-IgE or 10 μg/mL OVA-IgG1 (Sigma–Aldrich) for 30 min at 4 °C, washed, andincubated with 2.5 μg/mL biotinylated anti-IgE (BioLegend) or biotinylatedanti-IgG1 (BD Biosciences), followed by washing and incubation with 2.5 μg/mLstreptavidin-FITC (eBioscience). After a final wash, fluorescence was measuredon a BD FACSCanto (BD Biosciences).

Statistics. Statistics were performed on GraphPad Prism 5 software using aStudent t test or one-way ANOVA.

ResultsAg-Linked PS NPs Prevent Induction of Allergic Airway Inflammation.We previously demonstrated that Ag-SPs induce protective tol-erance for the prevention and treatment of established Th2 re-sponses in murine models of allergic airway inflammation andfood allergy (4). Ag-SPs administered i.v. rapidly undergo apo-ptosis (9) and localize to the splenic marginal zone, where theyinduce the up-regulation of IL-10 and coinhibitory moleculesby antigen-presenting cells (APCs) (10). Ag-PS NPs have beenidentified as surrogates mimicking many of the properties andmechanisms of Ag-SPs for the induction of tolerance to preventand treat Th1/Th17-driven EAE (6). These particles are mosteffective at diameters of ∼500 nm and are decorated on the sur-face with carboxyl groups to facilitate the attachment of Ag bypeptide bond formation induced by the chemical cross-linkerECDI and to facilitate tolerogenic uptake by the macrophagereceptor with collagenous structure (MARCO) scavenger re-ceptor (6). We therefore sought to determine the efficacy of Ag-PS for the modulation of Th2-driven allergic airway inflammation.PS particles had a diameter of 532.4 ± 4.96 nm. Measurementof protein coupling showed 0.50 ± 0.12 nmol of OVA was at-tached per dose of OVA-PS NPs, which had a ζ-potential of −17.3 ±0.5 mV (Table S1).Similar to prior studies with Ag-SP (4), we administered Ag-

PS i.v. 1 wk before each of two i.p. immunizations with OVA inalum adjuvant followed by airway challenge with aerosolizedOVA. Similar to tolerance induced by OVA-SP, OVA-PS inhibitedproduction of OVA-IgE compared with control lysozyme-PS par-ticles or no particle treatment (Fig. S1A). Additionally, OVA-PSinhibited airway inflammation as measured by eosinophil in-filtration in BALF (Fig. S1B) and corroborated by airway histology(Fig. S1C). Analysis of BALF cytokines showed significant in-hibition of the Th2-associated cytokines IL-4, IL-5, IL-13, and IL-10without the induction of IFN-γ or IL-17 associated with skewing toTh1 or Th17 phenotypes (Fig. S1D). OVA restimulation of cellsfrom mediastinal LN further demonstrated the tolerization ofOVA-specific Th2 cells. Cells from OVA-PS recipients exhibitedsignificantly reduced proliferation (Fig. S1E) and Th2 cytokineproduction (Fig. S1F) following ex vivo OVA restimulation. To-gether, these data indicate that Ag-PS NPs are effective Ag carriersfor the prophylactic inhibition of Th2 responses.

Ag-Linked Biodegradable PLG NPs Prevent Induction of AllergicAirway Inflammation. Despite the successful tolerance inducedby Ag-PS particles in allergic airway inflammation and othermodels, the nonbiodegradable nature of PS renders it unap-pealing for clinical use. Our recent studies have identified NPscomposed of PLG as a biodegradable alternative to PS effectivefor the prevention and treatment of EAE (6, 7). The PLG par-ticles had diameters of 543.8 ± 14.82 nm and a carboxylatedsurface to facilitate peptide bond formation by ECDI chemistryfor the attachment of Ag and tolerogenic uptake by APCs. Due tocarboxylation, the ζ-potential of the particles was −55.2 ± 3.8 mV.

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Measurement of protein conjugated to OVA-PLG particlesshowed 0.13 ± 0.06 nmol of OVA was attached per dose with apostcoupling ζ-potential of −11.5 ± 0.2 mV (Table S1). To de-termine the prophylactic potential of these particles in allergicairway inflammation, Ag-PLG was administered 1 wk before eachof two immunizations with OVA in alum adjuvant, followed byairway challenge with aerosolized OVA. OVA-PLG significantlyinhibited production of OVA-IgE compared with control myelinbasic protein (MBP)-PLG particles (Fig. 1A). Additionally, OVA-PLG significantly inhibited airway inflammation as measured byBALF eosinophilia (Fig. 1B), which was further borne out byairway histology (Fig. 1C). Analysis of BALF cytokines showedsignificant inhibition of IL-4, IL-5, IL-13, and IL-10 without in-duction of IFN-γ or IL-17 (Fig. 1D). OVA restimulation of me-diastinal LN cells further demonstrated the tolerization of OVA-specific Th2 cells. Cells from OVA-PLG recipients exhibitedtrends, albeit nonsignificant, toward reduced proliferation (Fig.S2A) and significant reduction of Th2 cytokine production (Fig.S2B) in response to OVA restimulation. Together, these dataindicate that Ag-PLG NPs are effective Ag carriers for the pro-phylactic inhibition of allergic airway inflammation.

Ag-Linked PS NPs Induce Anaphylactic Temperature Drop inPresensitized Mice. Given the striking inhibition of Th2 responsesby prophylactic treatment with Ag-PS, we tested whether Ag-PSwould be a viable Ag carrier for therapeutic tolerance. Ag-PS hadpreviously been shown to treat ongoing EAE by inhibiting re-sponses to spread epitopes (6). We opted to replicate the two-dosetolerance regimen that was effective for therapeutic inhibition ofTh2 responses with Ag-SP (4). However, our first execution of thisprotocol resulted in significant adverse reactions in OVA/alum-sensitized OVA-PS recipients, including labored breathing and lossof mobility, resulting in the deaths of three of five mice followingthe second dose of OVA-PS. Given these reactions in Th2-sensi-tized animals, we hypothesized that Ag presented on OVA-PS–induced an anaphylactic response. Indeed, OVA-PS–treated miceexhibited a significant reduction in body temperature comparedwith control MBP-PS–treated animals (Fig. S3A). This reactionmay be due to recognition of OVA on the particle surface byOVA-IgE, which significantly bound OVA-PS particles in vitro, asdid OVA-IgG1 (Fig. S3 D and E). This anaphylactic response, inconjunction with the nonbiodegradable nature of PS, contra-indicated Ag-PS for further development as a therapeutic treatment.

Ag-Linked Biodegradable NPs Are Nonanaphylactic and Inhibit Th2Responses but Not Airway Inflammation in Presensitized Mice. Wenext sought to determine the safety profile of OVA-PLG inpresensitized mice. Like OVA-PS, OVA-PLG was significantlybound by OVA-IgE and OVA-IgG1 in vitro, although signifi-cantly more OVA-IgG1 was bound to OVA-PLG than to OVA-PS (Fig. S3 D and E). However, unlike OVA-PS–treated mice,animals treated with OVA-PLG exhibited no adverse reactions ortemperature drop following particle administration (Fig. S3B).Having identified OVA-PLG as safe to administer to pre-

sensitized recipients, we next determined its ability to inhibitestablished Th2 responses. Following the second dose of OVA-PLG, mice were challenged with aerosolized OVA to induceairway inflammation. Similar to results seen with OVA-SP (4),OVA-PLG significantly enhanced OVA-IgE production com-pared with controls (Fig. 2A). OVA-PLG had no effect on airwayeosinophilia (Fig. 2B), although analysis of BALF cytokinesshowed reduced levels of Th2-associated cytokines in OVA-PLG–treated mice compared with lysozyme-PLG controls (Fig.2C). Ex vivo restimulation of LN cells from OVA-PLG recipientsdemonstrated a significant reduction in proliferation (Fig. 2D).However, a significant reduction in Th2 cytokines was not ob-served in these recall cultures (Fig. 2E). Altogether, these datashow a partial reduction of established Th2 responses by OVA-PLG, but no significant reduction in allergic airway inflammation.

Ag-Encapsulated Biodegradable NPs Are Nonanaphylactic and InhibitBoth Th2 Responses and Airway Inflammation in Mice treated Pre- orPostsensitization. To be clinically feasible for the treatment ofallergic disease, tolerogenic Ag carriers must be both well tol-erated and efficacious in subjects with established Th2 sensiti-zation. With these criteria, the data presented thus far rule outcovalently linked Ag-PS and Ag-PLG at these doses as unsafeand ineffective, respectively, for the treatment of established Th2responses. However, our group has recently investigated the useof PLG(Ag). PLG(Ag) allows for the potential delivery of largeramounts of Ag while retaining a free particle surface for furthermodification. Such particles are effective for the treatment of avariety of immune disorders, including EAE and allograft re-jection, and can be prepared and lyophilized in advance to min-imize end-user manipulation (11). PLG(Ag) had a diameter of703.5 ± 84.28 nm. PLG(OVA) particles had a ζ-potential of −41.9 ±12.9 mV and were loaded with 1.18 ± 0.81 nmol of OVA perdose (Table S1). Ag release studies demonstrated a slow release of

Fig. 1. Prophylactic treatment with OVA-PLG inhibits Th2-induced airway inflammation. Naive female BALB/c mice (n = 5) were treated i.v. with 1.25 mg ofOVA-PLG or control MBP-PLG on days −7 and +7 relative to i.p. immunization with 10 μg of OVA in 3 mg of alum or alum alone on days 0 and +14 before aerosolchallenge with 10 mg/mL OVA for 20 min on days +28–30 and sample collection on day +31. (A) Concentration of serum OVA-IgE was determined by sandwichELISA. (B) Lungs were flushed with BALF, total cell counts were determined, and samples were cytospun onto slides before DiffQuik staining for differential cellcounts of bronchoalveolar lavage eosinophils. (C) Lungs were fixed in formalin and stained with periodic acid–Schiff. (D) Cytokines from BALF supernatant wereanalyzed by Milliplex. Results are mean ± SEM and are representative of four separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Rx, treatment.

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OVA from PLG(OVA) particles in PBS over the course of 5 d(Fig. S4). Similar to OVA-PS and OVA-PLG, prophylactictreatment with PLG(OVA) significantly inhibited allergic sensiti-zation as measured by serum OVA-IgE, airway inflammation, andTh2 recall responses (Fig. 3 and Fig. S5). Treatment of OVA/alum-sensitized mice with PLG(OVA) induced no observedadverse reactions and no change in core body temperature (Fig.S3C). Unlike the case with Ag-coupled particles, neither OVA-IgE nor OVA-IgG1 bound PLG(OVA) in vitro (Fig. S3 D andE). As with other OVA carriers, postsensitization PLG(OVA)treatment significantly increased production of OVA-IgE com-pared with controls (Fig. 4A). Strikingly, however, PLG(OVA)treatment significantly decreased inflammatory airway eosino-philia (Fig. 4 B and C). This reduction in airway inflammation was

associated with significant inhibition of Th2 cytokines in the BALFof PLG(OVA) recipients without concurrent induction of IFN-γor IL-17 (Fig. 4D). Restimulation of mediastinal LNs with OVAsimilarly demonstrated a significant reduction in proliferation andTh2 cytokines in PLG(OVA) recipients (Fig. 4 E and F).

DiscussionWe previously demonstrated the ability of i.v.-administered Ag-SP to induce tolerance in models of autoimmunity, transplant,and allergy (4, 10, 12). Ag-SP uses natural tolerogenic pathways,targeting apoptotic cells to the splenic marginal zone via scavengerreceptor-mediated uptake, resulting in induction of IL-10 and PD-L1 on APCs and, ultimately, T-cell anergy and regulatory T-cell(Treg) activation (10). The use of Ag-coupled leukocytes in

Fig. 2. Treatment of presensitizedmicewith OVA-PLGdoes not inhibit Th2-induced airway inflammation.Naive female BALB/c mice (n = 5) were treated i.v. with1.25 mg of OVA-PLG or control lysozyme-PLG on days+21 and +28 relative to i.p. immunization with 10 μgof OVA in 3 mg of alum or alum alone on days 0 and+14 before aerosol challenge with 10 mg/mL OVA for20 min on days +31–33 and sample collection on day+34. (A) Concentration of serum OVA-IgE was de-termined by sandwich ELISA. (B) Lungs were flushedwith BALF, total cell counts were determined, andsamples were cytospun onto slides before DiffQuikstaining for differential cell counts of bronchoalveolarlavage eosinophils. (C) Cytokines from BALF superna-tant were analyzed by Milliplex. (D) Mediastinal LNswere harvested and cultured with 25 μg/mL OVA. At48 h, cells were pulsed with [3H]TdR for the final 24 h ofculture before harvest. (E) Before [3H]TdR pulsing,supernatant from LN cultures was reserved and cy-tokines were analyzed by Milliplex. Results aremean ± SEM and are representative of two separateexperiments. **P < 0.01; ***P < 0.001.

Fig. 3. Prophylactic treatment with PLG(OVA) inhibits Th2-induced airway inflammation. Naive female BALB/c mice (n = 5) were treated i.v. with 2.5 mgPLG(OVA) or control PLG(LYS) on days −7 and +7 relative to i.p. immunization with 10 μg of OVA in 3 mg of alum or alum alone on days 0 and +14 beforeaerosol challenge with 10 mg/mL OVA for 20 min on days +28–30 and sample collection on day +31. LYS, lysozyme. (A) Concentration of serum OVA-IgE wasdetermined by sandwich ELISA. (B) Lungs were flushed with BALF, total cell counts were determined, and samples were cytospun onto slides before DiffQuikstaining for differential cell counts of bronchoalveolar lavage eosinophils. (C) Lungs were fixed in formalin and stained with H&E. (D) Cytokines from BALFsupernatant were analyzed by Milliplex. Results are mean ± SEM and are representative of three separate experiments. *P < 0.05; **P < 0.01.

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patients with MS appears promising (5), but complexities associ-ated with leukocyte manipulation led us to test alternative Agcarriers for the promotion of tolerance. Ag-PS and biodegradableAg-PLG NPs have been identified as suitable Ag carriers for theinduction of tolerance in EAE (6, 7). To our knowledge, this reportis the first report of i.v.-administered Ag-PS, Ag-PLG, and PLG(Ag)NPs for the induction of tolerance in a Th2-mediated allergy model.Ag-PS particles induced effective tolerance for the prevention

of Th2 sensitization, but induced a profound anaphylactic tem-perature drop in Ag-sensitized animals. This reaction was un-expected because Ag-SPs were well tolerated when administeredto animals with significant Ag-specific IgE on board (4, 13).Moreover, the process of ECDI coupling Ag to carboxylated NPs,similar to allergoid generation by aldehyde fixation, was expectedto obscure conformational epitopes recognized by Fce receptor(FceR)-bound OVA-IgE (14). OVA-PS and OVA-PLG werebound by OVA-specific IgE and IgG1, which both can mediateanaphylaxis via cross-linking of FceRI and FcγRIII, respectively(15), but only OVA-PS induced anaphylaxis in presensitized mice.Binding of mAbs in vitro is nonphysiological and difficult to cor-relate directly to in vivo cross-linking of FcR. The lack of ana-phylaxis in response to OVA-PLG may be due to increasedbinding of OVA-IgG1 to OVA-PLG vs. OVA-PS particles be-cause IgG1 binding to low-dose Ag can inhibit anaphylaxis bycross-linking inhibitory FcγRIIb and inhibiting IgE binding (15).Ag-PLG effectively inhibited Th2 sensitization, but only par-

tially inhibited established Th2 responses and did not inhibitairway eosinophilia. Eosinophilia despite reduced airway IL-5may be due to the lack of reduction in LN-derived IL-5 becauseperipheral, but not airway-specific, expression of IL-5 has been

demonstrated to be required for the induction of airway eosin-ophilia (16). A similar phenotype was seen with postsensitizationtreatment with Ag-SP, in which airway Th2 cytokines wereinhibited but Ag-specific IgE and airway eosinophilia were ex-acerbated (4). Interestingly, postsensitization treatment withPLG(Ag) significantly inhibited both Th2 responses and airwayinflammation. Because PLG(OVA) particles carry more OVAper dose than OVA-PLG particles, we hypothesize that improvedAg loading, resulting in increased Ag dosage, is important to thetherapeutic efficacy of PLG(Ag), which will be optimized to de-termine minimal effective Ag dose in future studies. Considerablevariability in loading can be observed from batch-to-batch pro-duction of PLG(Ag), and the in vivo studies were performed witha minimal loading of 0.79 ± 0.05 nmol of OVA per dose. En-hanced efficacy could also be related to the more negativelycharged surface of PLG(OVA) vs. Ag-PLG. Highly charged par-ticles are rapidly opsonized by serum proteins upon injection, fa-cilitating APC uptake and Ag delivery, although the opsonizingprotein corona varies depending on the charge polarity (17). Wereported that, independent of opsonization, particle uptake viascavenger receptors, such as MARCO, occurs in a charge-dependent fashion (6, 18). As demonstrated in Table S1, a con-founding factor in conjugating different Ags to particles is thevariable surface charge imparted by those Ags. This variationpresents a caveat in the use of control Ags, such as lysozyme,that result in more neutrally charged particles. Additionally,different Ags would be expected to have distinct release kineticsfrom PLG(Ag) in solution, with factors such as Ag size, charge,and hydrophobicity affecting the release rate (19). Differentialbinding of OVA-IgE and OVA-IgG1 to the surface of OVA-PLG

Fig. 4. Treatment of presensitized mice with PLG(OVA) inhibits Th2-induced airway inflammation. Female BALB/c mice (n = 5) were treated i.v. with 2.5 mgPLG(OVA) or control PLG(LYS) on days +28 and +42 relative to i.p. immunization with 10 μg of OVA in 3 mg of alum or alum alone on days 0 and +14 beforeaerosol challenge with 10 mg/mL OVA for 20 min on days +56–58 and sample collection on day +59. (A) Concentration of serum OVA-IgE was determined bysandwich ELISA. (B) Lungs were flushed with BALF, total cell counts were determined, and samples were cytospun onto slides before DiffQuik staining fordifferential cell counts of BAL eosinophils. (C) Lungs were fixed in formalin and stained with periodic acid–Schiff. (D) Cytokines from BALF supernatant wereanalyzed by Milliplex. (E) Mediastinal LNs were harvested and cultured with 25 μg/mL OVA. At 48 h, cells were pulsed with [3H]TdR for the final 24 h ofculture. (F) Before [3H]TdR pulsing, supernatant from LN cultures was reserved and cytokines were analyzed by Milliplex. Results are mean ± SEM of threeseparate experiments. *P < 0.05; **P < 0.005; ***P < 0.001.

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vs. PLG(OVA) may explain the differences in postsensitizationefficacy between OVA-PLG and PLG(OVA) (i.e., binding ofOVA-specific Abs to OVA-PLG may target particles to non-tolerogenic uptake pathways).Similar to Ag-SP (4), both Ag-PLG and PLG(Ag) induce in-

creased Ag-specific IgE when administered to presensitized an-imals, perhaps because memory B cells respond to Ag encounterwith increased production of Ag-specific Ig, in the absence ofT-cell help (20). Oral SIT therapy resulted in increased Ag-specific IgE for months following treatment, returning to base-line levels only at 12 mo (21). It is unknown whether the IgEinduced by antigen-associated nanoparticles (Ag-NPs) will simi-larly decrease over time, and therapeutic use of Ag-NP tolerancevia targeting of allergen-specific Th2 cells may have to be used incombination with anti-IgE. Indeed, clinical use of SIT combinedwith omalizumab (IgE-specific IgG1) was safer and more effica-cious than SIT alone (3).The precise mechanisms by which Ag-NPs mediate tolerance

have yet to be elucidated. Ag-SPs act by a variety of mechanisms,including induction of IL-10 and inhibitory molecules, such asPD-L1 and CTLA-4 on splenic marginal zone APCs, leading toanergy, depletion of Ag-specific T cells, and induction of Ag-specific Tregs (10, 22). Ag-PS infused i.v. localizes to the splenicmarginal zone, inducing T-cell anergy and disease protection inthe Th1/Th17-mediated EAE model of MS in an IL-10/Treg-dependent fashion (6). This effect is size-dependent, because500-nm particles are more effective than smaller or larger parti-cles. Five hundred-nanometer Ag-PLG particles protect fromEAE (7), and i.v.-infused PLG(Ag) associates with APCs in thespleen and liver, inducing up-regulation of PD-L1 and establishingPD-1–dependent tolerance to prevent and treat EAE.Other studies have used PLG(Ag) to modulate allergic re-

sponses. Given orally, s.c., or i.p., PLG(Ag) inhibits Ag-specific IgEand hypersensitivity reactions marked by skewing toward Th1 re-sponses (23, 24), which inhibit Th2 responses (2). Indeed, s.c. ad-ministration of Ag-SP and Ag-PLG is also immunogenic, enhancingTh1/Th17-mediated responses in EAE, as opposed to the tolero-genic response achieved with i.v. delivery (7, 10). Ag-NPs inhibitTh2 responses without Th1/Th17 skewing because i.v. injection ofPLG(Ag) induces protective and therapeutic tolerance in Th1/Th17-mediated EAE models and transplant rejection (11). Weposit that abrogation of immune responses as induced by PLG(Ag)

is preferable to the induction of a competitive inflammatoryphenotype and represents a “true” state of tolerance. A recentreport demonstrated that i.v. or s.c. administration of PLG(Ag)containing rapamycin induces tolerance in models of autoim-munity and allergic hypersensitivity (25). Upon s.c. administra-tion, the rapamycin particles localized to draining LNs, whereasour i.v.-administered PLG(Ag) particles localize with APCs inthe liver and splenic marginal zone. Similar to i.v. PLG(Ag),rapamycin-bearing particles induced tolerance both pre- andpostsensitization, but unlike PLG(Ag) in our study, post-sensitization treatment did not induce production of Ag-specificIgE. Without rapamycin, these particles were immunogenic, in-dicating a fundamental difference in the mechanism of actionfrom the 500-nm particles used in our studies, perhaps due totheir smaller size or differences in surface charge leading to thetargeting of different APCs or uptake pathways. In our PLG(Ag)platform, i.v. administration directing Ag toward more naturallytolerogenic APCs in the liver and splenic marginal zone targetingis crucial, whereas s.c. administration targets Ag to immunoge-nic, Th1-promoting microenvironments (6, 10).Our data indicate biodegradable PLG(Ag) induces effective

tolerance in a safe manner for the treatment of established Th2responses in a model of allergic airway inflammation. Ag-NPsrepresent an evolution from Ag-SPs toward a more convenient,standardizable Ag carrier that can be efficiently manufacturedunder GMP conditions. Ag-PS is an effective Ag carrier for theinduction of prophylactic tolerance, but it is nonbiodegradableand presents Ag to sensitized cells in an anaphylactic manner.Biodegradable Ag-PLG is similarly effective prophylactically butonly partially inhibits established Th2 responses. PLG(Ag) is alsocapable of being loaded with multiple Ags or drugs while leavingthe particle surface available for the attachment of additionalimmunomodulatory or targeting molecules. Relevant to clinicalapplication, i.v. PLG(Ag) is well tolerated in sensitized mice,inhibiting established Th2 responses and airway eosinophiliawithout skewing toward Th1 or Th17 phenotypes.

ACKNOWLEDGMENTS. This study was supported, in part, by NIH Grants EB-013198 (to S.D.M. and L.D.S.) and NS-026543 (to S.D.M.) and by a grant fromthe Dunard Fund (to S.D.M.). C.B.S. was supported by Predoctoral FellowshipTL1R000108 from the NIH National Center for Research Resources and theNational Center for Advancing Translational Sciences.

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