Nanoparticle-mediated codelivery of myelin antigen and a tolerogenic small molecule suppresses experimental autoimmune encephalomyelitis
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 considered a potential therapeutic approach for autoimmune disorders. The activation of the ligand-activated transcription factor aryl hydrocarbon receptor by 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) or other ligands induces dendritic cells (DCs) that promote FoxP3(+) Treg differentiation. Here we report the use of nanoparticles (NPs) to coadminister ITE and a T-cell epitope from myelin oligodendrocyte glycoprotein (MOG)(35)(-55) to promote the generation of Tregs by DCs. NP-treated DCs displayed a tolerogenic phenotype and promoted the differentiation of Tregs in vitro. Moreover, NPs carrying ITE and MOG(35-55) expanded the FoxP3(+) Treg compartment and suppressed the development of experimental autoimmune encephalomyelitis, an experimental model of multiple sclerosis. Thus, NPs are potential new tools to induce functional Tregs in autoimmune disorders.
Nanoparticle-mediated codelivery of myelin antigen
and a tolerogenic small molecule suppresses
experimental autoimmune encephalomyelitis
Ada Yeste, Meghan Nadeau, Evan J. Burns, Howard L. Weiner, and Francisco J. Quintana
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 review
December 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 considered
a potential therapeutic approach for autoimmune disorders. The
activation of the ligand-activated transcription factor aryl hydro-
carbon receptor by 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic
acid methyl ester (ITE) or other ligands induces dendritic cells (DCs)
that promote FoxP3
Treg differentiation. Here we report the use
of nanoparticles (NPs) to coadminister ITE and a T-cell epitope from
myelin oligodendrocyte glycoprotein (MOG)
to promote the
generation of Tregs by DCs. NP-treated DCs displayed a tolerogenic
phenotype and promoted the differentiation of Tregs in vitro.
Moreover, NPs carrying ITE and MOG
expanded the FoxP3
Treg compartment and suppressed the development of experi-
mental autoimmune encephalomyelitis, an experimental model
of multiple sclerosis. Thus, NPs are potential new tools to induce
functional Tregs in autoimmune disorders.
ultiple sclerosis (MS) is an autoimmune disease driven by
an immune response directed against antigens in the CNS
(1). The autoreactive components of the immune system are
normally 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 to
arrest the development of several experimental models of au-
toimmune disease (5). Thus, the induction of antigen-speciﬁc
tolerance is considered a promising approach for the treatment
of 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 factor
aryl hydrocarbon receptor (AhR) controls the differentiation of
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 ligands
involves the induction of tolerogenic dendritic cells (DCs). In-
deed, the activation of AhR signaling in DCs by ITE or other
ligands induces DCs that promote FoxP3
Nanoparticles (NPs) have unique features that prompted their
use in medicine. For example, NPs have been used for in vivo
tumor detection and targeting (16) and for the delivery of anti-
angiogenic compounds (17). NPs have also been used to induce
pathogen-speciﬁc immunity in vaccination regimens (18, 19). In
the context of the therapeutic management of inﬂammation, NPs
have 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 to
induce 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 located
between 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 35–55
of the myelin oligodendrocyte glycoprotein (MOG
Treg compartment and suppressed the development
of experimental autoimmune encephalomyelitis (EAE), an ex-
perimental model of MS. Thus, NPs are potential new tools for
the codelivery of T-cell antigens and tolerogenic small molecules
to induce antigen-speciﬁc Tregs in autoimmune disorders.
Construction of NPs Containing MOG
and ITE. MS and EAE are
caused by an autoimmune response directed against the CNS (1).
To induce CNS-speciﬁc Tregs, we constructed NPs containing the
AhR ligand ITE and MOG
, which contains a CD4
epitope targeted by effector and Tregs during the course of EAE
(21). By using gold particles (60 nm in diameter), we constructed
four types of NPs that were stabilized with a layer of thiol-poly-
ethylene glycol (PEG) (16): (i) unloaded NPs, (ii) NPs loaded
with ITE (NP
), (iii) NPs loaded with MOG
(iv) NPs loaded with ITE and MOG
As a ﬁrst step toward the characterization of the NPs we stud-
ied their UV–visible absorption spectra. We detected a prominent
absorption at 530 nm in unloaded gold NPs, which results from
the excitation of surface plasmon vibrations in the gold NPs (22)
(Fig. 1 B). Loading of ITE, MOG
shifted the absorption peak to 560 nm, reﬂecting the binding of
ITE and/or MOG
to the NPs (Fig. 1B). MOG
tected in NP
but n ot in NP or NP
gel electrophoresis followed by silver staining (Fig. 1C). Further
analysis by transmission EM showed that the NPs had a round
morphology 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 construct
carrying the luciferase gene under the control of an AhR-re-
sponsive promoter. We found that treatment of the reporter cell
line with ITE-containing NPs (NP
) led to the
signiﬁcant activation of the AhR-responsive promoter (Fig. 1E).
The AhR-responsive promoter, however, was not activated by
NPs that did not carry ITE (NP and NP
; Fig. 1E). Thus, ITE
loaded into NPs can be released to trigger AhR-dependent
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 by
liver enzymes. Free ITE and NP
were preincubated with
a 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.
To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
July 10, 2012
no. 28 www.pnas.org/cgi/doi/10.1073/pnas.1120611109
activate an AhR-responsive promoter was analyzed in aliquots
taken at different time points. Incubation with hepatic microsomes
for 1 h decreased the ability of free ITE to activate AhR to ∼50%
of its initial value, whereas ITE in NPs maintained its ability to
activate 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 was
observed when ITE was administered once per week (Fig. S1).
However, the weekly administration of NP-bound ITE (i.p.) led
to a signiﬁcant suppression of EAE development (Fig. S1). Taken
together, these data suggest that NPs protect ITE from enzymatic
degradation and boost its immunoregulatory activity in vivo.
Induces Tolerogenic DC. DCs play a central role in the
activation and polarization of T cells in vivo (25), so we in-
vestigated the effect of NPs on DC function. Splenic DCs were
isolated from naive mice and incubated with NPs, and NP uptake
was analyzed by transmission EM. NPs could be detected inside
DCs 1 h after their addition to the cells, and were still detectable
inside DCs 24 h later (Fig. 2A). To investigate whether the up-
take of NPs carrying ITE activates AhR signaling in DCs, we
analyzed 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 NP
, but not by
NP and NP
(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 polarize
naive T cells into effector Th1 or Th17 cells and promote the
differentiation of Tregs. Thus, we studied the effects of NPs on
the response of DCs to stimulation with the Toll-like receptor
agonist Escherichia coli lipopolysaccharide (LPS). Splenic DCs
isolated from naive mice were incubated in vitro with NPs and
activated with LPS. Incubation with empty NPs did not modify
the expression of the class II MHC or costimulatory molecules in
DCs, and did not affect their ability to activate naive T cells (Fig.
S2 A and B). However, we found that NP
showed 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 NP
to a signiﬁcant reduction in the production of the Th1 and Th17
polarizing 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 with
LPS and used to stimulate CD4
T cells, which express
a transgenic T-cell receptor that recognizes MOG
found that incubation of naive CD4
T cells with DCs and
resulted in the proliferation of the 2D2
absence of exogenous MOG
, demonstrating that NP-delivered
is presented by the DCs (Fig. 2D). Activation of 2D2
cells with DCs and NP
, however, triggered a signiﬁcantly
reduced proliferative T-cell response (Fig. 2D), which resembles the
reduced response of T cells incubated with ITE-treated DCs (11).
We also observed a signiﬁcant decrease in the proliferative response
T cells activated with DCs and NP
presence of exogenously added MOG
The analysis of cytokine secretion showed that 2D2
tivation with DCs and NP
triggered the production of signiﬁ-
cant amounts of IFN-γ and IL-17, indicative of the polarization of
Th1 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 NP
(Fig. 2E). Conversely, DCs in-
showed an increased ability to promote
the differentiation of FoxP3
Tregs (Fig. 2F). Taken together,
these results are in agreement with the reported effects of AhR
activation on the ability of DCs to activate an polarize effector and
Tregs (11, 13–15), and demonstrate that NP
erogenic DCs that favor the generation of FoxP3
Fig. 1. Characterization of NPs containing ITE and
.(A) Schematic representation of NP
(B) Optical absorption obtained from NPs. (C) Gel
electrophoresis and silver staining of NPs. (D) Trans-
mission EM analysis of pegylated NPs. (E) HEK293 cells
transfected with a reporter construct coding for lucif-
erase under the control of an AhR-responsive promoter
were incubated with NPs and luciferase activity was
measured after 2 4 h. Cotransfection with a TK-Renilla
construct was used for normalization purposes. (F) AhR
activation detected after incubation of free ITE or NP
with a preparation of hepatic microsomes for 0, 10, 30,
or 60 min (*P < 0.05, **P < 0.01, and ***P < 0.001 vs.
Yeste et al. PNAS
July 10, 2012
Administration Suppresses EAE. We then studied the
effects 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 quantitative
PCR. We found a signiﬁcant up-regulation of cyp1a1 expression
in DCs isolated from NP
-treated mice, but
not in those isolated from NP- or NP
-treated mice (Fig. 3A).
We also found a signi ﬁcant activation of AhR, as indicated by
cyp1a1 expression, in splenic macrophages and B cells isolated
-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 polarizing
cytokines. DCs from NP
-treated mice showed a signiﬁ-
cant decrease in the production of IL-6 and IL-12 in response to
activation with LPS ex vivo (Fig. S3 B and C). Taken together,
these data demonstrate that ITE-containing NPs activate AhR
signaling in splenic DCs in vivo and decrease the production of
Th1 and Th17 polarizing cytokines.
Based on the effects of NP
on the activation and
antigen presenting cell (APC) function of DCs in vitro and
in vivo, and the role played by DCs in the differentiation of
encephalitogenic and Tregs (25), we studied the effects of NPs
on EAE. EAE was induced by immunization with MOG
naive B6 mice, NPs were administered weekly (6 μg per mouse)
starting on the day of EAE induction, and the animals were
monitored daily for the development of the disease. NP
administration resulted in a signiﬁcant suppression of EAE de-
velopment (Fig. 3B). Treatment with NP
amelioration in EAE symptoms, but the protective effects of NP
were not as strong as those observed with NP
difference in the protection achieved by NP
treatment was found to be signiﬁcant (Fig. 3B). Of note, the co-
administration of free MOG
on the development of EAE (Fig. S4).
EAE in B6 mice is driven by MOG
–speciﬁc Th1 and Th17
cells (21). Thus, to study the suppression of EAE by NP
we analyzed the effect of NP administration on the T-cell recall
response to MOG
. Treatment with NP
resulted in a signiﬁcant decrease in the proliferation and the pro-
duction of IFN-γ andIL-17triggeredbyMOG
in recall exper-
iments; this decrease was signiﬁcantly stronger in the NP
group (Fig. 3 C and D). No signiﬁcant effect was detected when
the respo nse to anti-CD3 stimul ation was investigated ( Fig. S5
A and B), suggesting that NP-treated mice are not s ystemically
immune-suppressed. Thus, NP
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 SJL
model of EAE induced by immunization with the 139 to 151
region of the proteolipid protein (PLP). In this model, the
chronic phase of EAE is characterized by the spreading of the
T-cell response to the PLP epitope placed between residues 178
and 191 (PLP
) (28). Thus, we tested the therapeutic effect
on SJL EAE of treatment with NP
, NPs loaded with ITE and
), and ITE and PLP
addition, an experimental group was treated with both NP
; empty NPs were used as controls. EAE was in-
duced by immunization with PLP
in naive SJL mice, and,
on day 17 after disease induction, the mice were assigned to the
Fig. 2. NP
induces tolerogenic DCs. (A)
Transmission EM analysis of uptake of NP
DCs in culture. (B) Analysis of cyp1a1 expression by
DCs coincubated with NPs 24 h after initiation of
cell cultures. (C) Quantitative PCR analysis of il6 and
il12 expression in DCs incubated in vitro with NPs
and activated with LPS for 12 h; results presented
relative to gapdh mRNA. (D–F) DCs were coincu-
bated in vitro with NPs, activated with LPS, and
used to stimulate naive 2D2
T cells. Pro-
liferation (D) and cytokine secretion (E)tothe
supernatants were analyzed at 72 and 48 h, re-
spectively. (F) The frequency of CD4
was analyzed by FACS at 72 h. Representative data
of one of three experiments that produced similar
results (*P < 0.05, **P < 0.01, and ***P < 0.001).
www.pnas.org/cgi/doi/10.1073/pnas.1120611109 Yeste et al.
different experimental groups and treated with NPs (6 μg per
mouse); the animals were treated weekly during the duration of
the experiment. We observed that treatment with NP
signiﬁcant effect in the development of EAE (Fig. 3E). We also
found that the administration of NP
had a transient
beneﬁcial effect on the development of EAE; however, the ad-
ministration of NP
led to a signiﬁcant
reduction in the EAE score, maximal EAE score, and the
number of relapses after the initiation of NP treatment (Fig. 3E
and Table S1). Treatment with NP
alone had no signiﬁ-
cant effect on the chronic phase of EAE. Taken together, these
data suggest that a combination of NPs targeting several relevant
T-cell reactivities can be engineered to control epitope spreading
and chronic inﬂammation in established CNS autoimmunity.
Tregs Mediate Suppression of EAE by NP
. We (9–11)
and others (13, 14, 29–31) have shown that AhR activation can
expand the FoxP3
compartment, so we investigated the
effect of ITE on FoxP3
. We found that the suppression of
EAE development by AhR activation with NP
sociated with a signiﬁcant increase in the frequency of CD4
Tregs in the spleen (Fig. 4B) and the blood (Fig. 4B).
administration suppresses the encephalito-
genic Th1/Th17 (Fig. 3D) whereas it promotes the generation of
We then performed transfer experiments to study the role of
Tregs in the suppression of EAE triggered by
. EAE was induced in Foxp3
mice carrying a GFP
reporter in the foxp3 gene (32), the mice were treated with NP or
, and CD4
T cells were isolated and transferred into
naive mice. We found that naive recipients could be protected
from the development of EAE by the transfer of 5 × 10
cells isolated from NP
-treated mice, but not with cells
isolated from NP-treated mice (Fig. 4C). Removal of the
Treg fraction abrogated the protective effect
of the transferred cells (Fig. 4C), suggesting that NP
Tregs are responsible for the control
of the encephalitogenic T-cell response.
NPs are being actively studied as tools for the modulation of the
immune response. Most of these studies, however, are focused on
the induction of pathogen-speciﬁc effector immunity in the context
of vaccine development (18, 19). Although the generation of anti-
gen-speciﬁc Treg is considered a promising approach for the
treatment of autoimmune disorders (6), the use of NPs to induce
antigen-speciﬁc tolerance and treat autoimmune disorders remains
largely unexplored. Here we describe NPs designed to coadminister
atissuespeciﬁc antigen (i.e., MOG
) and an AhR ligand (i.e.,
ITE) to induce tolerogenic APCs that promote the differentiation
of 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 antigen
coupled to red blood cells (38) have also been developed to expand
antigen-speciﬁc Tregs, and their translational relevance is now be-
ing investigated in clinical trials. However, compared with other
methods for antigen delivery, an advantage of NPs is their ability to
codeliver 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/
Fig. 3. NP
suppresses EAE. (A)
cyp1a1 expression in splenic DCs from
NP-treated mice. (B) EAE was induced
by immunization of naive B6 mice
, and NPs were admin-
istered i.p. weekly from the day of
immunization until the termination of
the experiment. The course of EAE is
shown as the mean EAE score ± SEM
(n = 10 mice per group) and also as
the linear regression curves of the
disease for each group (Right). (C)
Proliferative response to MOG
splenocytes taken from NP-treated
animals immunized with MOG
CFA. Cell proliferation is indicated as
the mean cpm ± SEM in three to ﬁve
mice per group. (D) Cytokine secretion
triggered by MOG
taken from NP-treated animals im-
munized with MOG
in CFA. (E)
EAE was induced by immunization of
naive SJL mice with PLP
NPs were administered i.p. weekly
from day 17 until the termination of
the experiment. The course of EAE is
shown as the mean EAE score ± SEM
(n = 5 mice per group) for the whole
observation period, and also as the
linear regression curves of the disease
for each group from day 30 until the
termination of the experiment (Right).
Representative data of one of at least
three experiments that produced sim-
ilar results (*P < 0.05, **P < 0.01, and
***P < 0.001 vs. NP-treated mice;
0.05 vs. NP
Yeste et al. PNAS
July 10, 2012
MHC NPs induced CD8
Tregs that suppress the diabetogenic
T-cell response, restoring normoglycemia in WT and humanized
nonobese diabetic mice (39). Although the work by Tsai et al.
showed for the ﬁrst time the feasibility of using NPs to reestablish
immune tolerance, the use of these NPs in humans is limited by its
reliance on recombinant HLA/peptide complexes. However, it is
conceivable that the use of peptide/MHC NPs in combination
with the NPs described in the present study will promote the gen-
Tregs, resulting in more robust
immune 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 to
control the development of tolerogenic DCs that promote the
differentiation 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 also
be capable of inducing antigen-speciﬁc tolerance. Thus, future
studies 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), and
also as therapeutic tools. Nanoliposomes have been used to
deliver CNS antigens (41) or to deplete macrophages (42) and
control disease progression in different EAE models. More re-
D,L-lactic-coglycolic acid) NPs were used to deliver
a peptide designed to interfere with the activation of PLP
specﬁcic T cells (43). Although these poly(
acid) 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 of
the autoimmune T-cell response to other epitopes. These results
are in agreement with those of Robinson et al., who demon-
strated that the successful treatment of ongoing EAE in the SJL
model using tolerogenic DNA vaccines requires targeting other
myelin antigens in addition to PLP
(44). Taken together
with 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 in
MS. In combination with methods for the high-throughput
characterization of the autoimmune response (33, 45–47), NPs
might provide a new tool to codeliver tissue-speci ﬁc antigens and
tolerogenic small molecules to generate antigen-speciﬁc Tregs
suited to the individual needs of patients with MS.
Mice and Reagents. C57BL/6 mice were purchased from Jackson Laborat ories
and kept, together with 2D2
(27) and FoxP3
(32) mice, in a pathogen-free
facility at the Harvard Institutes of Medicine. All experiments were carried
out in accordance wit h the guidelines of the standing committee of animals
at Harvard Medical School. ITE was purchased from Sigma-Aldrich and from
NP Preparation. NPs were produced by using PBS solution, 60 nm tannic acid-
stabilized gold particles at a concentration of 2.6 × 10
particles per milliliter
(Ted Pella), methoxy-PEG-SH (molecular weight, ∼5 kDa; Nektar Therapeu-
tics), ITE (Tocris Bioscience), and MOG
Freshly prepared solut ions of ITE (3.5 mM), MOG
(600 μg/mL), or ITE and
(3.5 mM and 600 μg/mL, respectively) were added drop by drop to
a rapidly mixing gold colloid at a 1:6 ITE solution:colloid volume ratio, which
facilitates 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 gold
particle 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 or
in the thickness of the polymer coating layer (16). After an additional 30 min
incubation 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 at
room 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% OsO
, washed in water three times, and incubated in 1% aqueous uranyl
acetate for 30 min followed by two washes in water and subsequent de-
hydration in grades of alcoho l [5 min each; 50%, 70%, 95% (vol/vol), twice at
100%]. Cells were removed from the dish in propylene oxide, pelleted at
1,000 × g for 3 min, and inﬁltrated for 2 h in a 1:1 mixture of propylene
oxide and TAAB Epon (Marivac). The samples were then embedded in TAAB
Epon and polymerized at 60 °C for 48 h.
Ultrathin sections (approximately 60 nm) were cut on a Reichert Ultracut-S
microtome, picked up onto copper grids stained with lead citrate, and ex-
amined in TecnaiG
Spirit BioTWIN, and images were recorded with an AMT
2k CCD camera.
Gel Electrophoresis. NPs (3.5 μg per lane) and MOG
(1, 0.1, and 0.01 μg)
were run by using NuPAGE 4% to 12% 1.0-mm Bis-Tris gels (Invitrogen), and
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 (New
England Biolabs). Tk-Renilla was used for standardization.
Microsomal Degradation. ITE or NP
was incubated with mouse hepatic
microsomes (2 mg/mL ; Sigma-Aldrich) in a reaction buffer containing NAPDH
(1 mM), MgSO
(8 mM), KCl (45 mM), and 3.3 glucose 6-phosphate, pH 7.4, at
37 °C for different periods of time.
Puriﬁcation of DCs. DCs were puriﬁedfromthespleensofnaiveB6miceby
magnetic beads according to the manufacturer’s instructions (Mil-
tenyi). To generate bone marrow-derived DCs, bone marrow cells were isolated
from the femurs of naive mice and cultured for 5 d in the presence of IL-4 (10
ng/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 was
performed by using an ABI7500 cycler (Applied Biosystems). All values are
expressed as fold increase or decrease relative to the expression of GAPDH.
Fig. 4. FoxP3
Tregs mediate the suppression of EAE by
.(A and B) Frequency of CD4
splenocytes (A) and blood (B) from NP-treated mice immu-
nized with MOG
in CFA. (C) CD4
cells (5 × 10
) were puriﬁed from NP- or NP
mice and transferred into naive B6 mice, and, 24 h later, EAE
was induced in the recipients with MOG
. The course of
EAE is shown as the mean EAE score ± SEM (n =5–10 mice
per group). Representative data of one of at least three
experiments that produced similar results (*P < 0.05, **P <
0.01, and ***P < 0.001 vs. NP-treated mice).
www.pnas.org/cgi/doi/10.1073/pnas.1120611109 Yeste et al.
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 activated
(20 μg/mL) as described (11).
Cell Proliferation and Cytokine Production. Cells were cultured in serum-free
X-VIVO 20 media (BioWhittaker) for 72 h. During the last 16 h, cells were
pulsed with 1 mCi of [
H]thymidine (PerkinElmer), followed by harvesting on
glass ﬁber ﬁlters and analysis of incorporated [
H]thymidine in a β-counter
(1450 Microbeta Trilux; PerkinElmer). Culture supernatants were collected
after 48 h, and cytokine concentration was deter mined by ELISA by using
antibodies 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), and GolgiStop (BD Biosciences) for 4 h. After
staining of surface markers, cells were ﬁxed and permeabilized as described
and 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 μg
per mouse). EAE was induced by s. c. immu nization w ith 100 μgofthe
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 Multiple
Sclerosis Society Grant RG4111A1 (to F.J.Q.) and a National Multiple Sclerosis
Society Pilot Grant (to F.J.Q.); and the Harvard Medical School Ofﬁce for
Diversity and Community Partnership (F.J.Q.).
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Yeste et al. PNAS
July 10, 2012