IL-28 Supplants Requirement for TregCells in Protein s1-
Mediated Protection against Murine Experimental
Autoimmune Encephalomyelitis (EAE)
Agnieszka Rynda1,2, Massimo Maddaloni1, Javier Ochoa-Repa ´raz1,2, Gayle Callis1, David W. Pascual1*
1Veterinary Molecular Biology, Montana State University, Bozeman, Montana, United States of America, 2Department of Microbiology and Immunology, Dartmouth
Medical School, Lebanon, New Hampshire, United States of America
Conventional methods to induce tolerance in humans have met with limited success. Hence, efforts to redirect tolerogen
uptake using reovirus adhesin, protein sigma 1 (ps1), may circumvent these shortcomings based upon the recent finding
that when reovirus ps1 is engineered to deliver chicken ovalbumin (OVA) mucosally, tolerance is obtained, even with a
single dose. To test whether single-dose tolerance can be induced to treat EAE, proteolipid protein (PLP130–151) was
genetically fused to OVA to ps1 (PLP:OVA-ps1) and shown to significantly ameliorate EAE, suppressing proinflammatory
cytokines by IL-10+forkhead box P3 (FoxP3)+CD25+CD4+Tregand IL-4+CD252CD4+Th2 cells. IL-10R or IL-4 neutralization
reversed protection to EAE conferred by PLP:OVA-ps1, and adoptive transfer of Ag-specific Tregor Th2 cells restored
protection against EAE in recipients. Upon assessment of each relative participant, functional inactivation of CD25 impaired
PLP:OVA-ps1’s protective capacity, triggering TGF-b-mediated inflammation; however, concomitant inactivation of TGF-b
and CD25 reestablished PLP:OVA-ps1-mediated protection by IL-28-producing FoxP3+CD252CD4+T cells. Thus, ps1-based
therapy can resolve EAE independently of or dependently upon CD25 and assigns IL-28 as an alternative therapy for
Citation: Rynda A, Maddaloni M, Ochoa-Repa ´raz J, Callis G, Pascual DW (2010) IL-28 Supplants Requirement for TregCells in Protein s1-Mediated Protection
against Murine Experimental Autoimmune Encephalomyelitis (EAE). PLoS ONE 5(1): e8720. doi:10.1371/journal.pone.0008720
Editor: Derya Unutmaz, New York University, United States of America
Received August 27, 2009; Accepted December 9, 2009; Published January 14, 2010
Copyright: ? 2010 Rynda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by the National Institutes of Health (NIH) grant 1R01 AI-078938 and, in part, by Montana Agricultural Station and
United States Department of Agriculture Formula Funds. The VMB flow cytometry facility was, in part, supported by NIH/National Center for Research Resources,
Centers of Biomedical Excellence P20 RR-020185, and an equipment grant from the M.J. Murdock Charitable Trust. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Th17 cells are pivotal for EAE pathogenesis , although
reversible by regulatory cell intervention, including FoxP3+
CD25+CD4+T (Treg) cells producing IL-10 [2–5] and/or TGF-
b [3,6–8]. Recently, IL-13-producing Tregcells induced by an oral
recombinant Salmonella vaccine exhibiting anti-encephalitogenic
properties have also been found to treat EAE . Anti-
inflammatory Th2 cells, traditionally viewed secondary to Treg
cells, can enhance recovery from EAE and lessen EAE when
adoptively transferred into diseased animals [7,8]. Although the
Th2-type IL-4 cytokine can trigger Tregcell responses [4,5,7,9], it
can compromise Tregcell-mediated suppression of asthma ,
suggesting that Th2 cells and/or their cytokines are important
regulators of immunosuppression.
Secreted by dendritic cells (DCs) and macrophages, IL-28B
(IFNl 3) [11–13], a newly described member of IFNl family, is
known for its anti-inflammatory activity . Sharing a common
signaling pathway with anti-viral type I IFNs , IL-28’s role in
EAE has yet to be evaluated, but it can prime tolerogenic DCs in
vitro . When adapted as an adjuvant during DNA vaccination,
plasmid-encoded IL-28B reduces Tregcell numbers, but enhances
granular CD8+T cells . In this current study, we demonstrate
that protection against EAE, mediated by ps1, is conferred by the
expected IL-10-producing Tregcells; however, in the absence of
functional Tregcells, protection is mediated by IL-28-producing
Th2 cells, demonstrating for the first time that Th2 cells produce
IL-28, and endogenous IL-28 can confer protection against EAE.
Acquisition of responsiveness to myelin proteins can develop
into the autoimmune disorder, multiple sclerosis (MS) .
Current MS therapies fail to restore the unresponsiveness to these
self-antigens (Ags). While feeding myelin Ags is effective against
EAE [3,17–19], when applied to patients, oral feeding with bovine
myelin preparations was deemed unsuccessful . Thus,
conventional methods to elicit oral tolerance need to be improved.
A number of studies have sought to enhance induction of oral
tolerance by adapting liposome delivery , including oral
adjuvants  or coupling to mucosal binding molecules [23,24].
Although most of these strategies significantly ameliorate EAE,
multiple doses are required to sustain tolerance, lessening the
potency of such methods.
Past studies suggest that sustainable tolerance requires the
presence of Peyer’s patches for initial Ag sampling subsequent oral
tolerogen ingestion . Such evidence implicates the importance
of Ag-sampling microfold (M) cells to facilitate Ag uptake from the
lumenal surface. To direct tolerogen uptake, we hypothesized that
M cell adhesins could be employed to target mucosal inductive
tissues, as readily induced with a single dose of OVA fused to
PLoS ONE | www.plosone.org1January 2010 | Volume 5 | Issue 1 | e8720
reovirus ps1, [5,26]. Mucosal OVA-ps1 induced Ag-specific IL-
10+Tregand IL-4+Th2 cells capable of suppressing immunity to
OVA and ps1, even when co-administered with potent mucosal
Noting the potency of ps1-elicited tolerance, we queried if it
could be adapted to treat autoimmunity by genetically fusing two
copies of a portion of proteolipid protein (PLP) containing the
encephalitogenic sequence (PLP139–151) to OVA-ps1, termed
PLP:OVA-ps1. The described studies showed that PLP139–151-
induced EAE is ameliorated with a single nasal dose of PLP:OVA-
ps1, stimulating the induction of IL-10-producing Tregcells and
IL-4-producing FoxP3+Th2 cells. Notwithstanding that these
induced regulatory T cells were entirely protective subsequent
their adoptive transfer, and their effects were neutralized by anti-
IL-10 receptor (IL-10R) or anti-IL-4 mAb, additional analyses
sought to assess alternative regulatory T cell pathways. Functional
inactivation of PLP:OVA-ps1-primed Tregcells rendered mice to
an aggressive EAE driven by TGF-b-induced Th17 cells.
However, PLP:OVA-ps1 could re-confer protection against
EAE upon CD25 and TGF-b co-neutralization in a reversible,
IL-28-dependent fashion. Thus, these results show that ps1-based
therapeutics can stimulate multiple pathways to induce tolerance
and, importantly, can be accomplished independently of Tregcells
via IL-4+or IL-28+Th2 cells.
Nasal PLP:OVA-ps1 Ameliorates EAE
Susceptible female SJL mice nasally dosed with PLP:OVA-ps1,
OVA-ps1, or PBS were subjected to conventional PLP139–151
challenge. PBS- and OVA-ps1-dosed mice developed EAE with
average clinical scores .3 at peak disease, followed by relapsing-
remitting disease and never fully recovered (Figure 1A). Mice
dosed with PLP:OVA-ps1 showed delayed development and
reduced duration of clinical disease; the average clinical scores at
peak disease were ,1. Unlike PBS- or OVA-ps1-dosed mice,
PLP:OVA-ps1-protected mice recovered completely from the
acute disease following one relapse. PLP139–151-specific delayed
type hypersensitivity (DTH) responses (Figure 1B) 2 wks after EAE
induction confirmed significant reduction of PLP139–151-specific
Figure 1. Nasal administration of PLP:OVA-ps1 protects mice from EAE. A. Mice were dosed with 100 mg of PLP:OVA-ps1, OVA-ps1, or PBS
on days 221, 214, and 27, challenged with PLP139–151peptide on day 0, and monitored daily for the development of clinical disease. PBS and OVA-
ps1-dosed mice developed fully pronounced EAE and never recovered completely. PLP:OVA-ps1-immunization prior to EAE induction significantly
delayed and ameliorated clinical onset of EAE and resulted in a complete recovery of all PLP:OVA-ps1-dosed mice. Average of 10–15 mice per group
is shown. * P,0.05 for PLP:OVA-ps1 vs. PBS. B. Anti-PLP139–151DTH response measured two weeks after EAE induction confirmed that, unlike in PBS-
or OVA-ps1-dosed mice, PLP139–151-specific Th1 response was diminished in PLP:OVA-ps1-dosed mice. Mean + SD of 10–15 mice per group is
depicted. * P,0.001 for PLP:OVA-ps1 vs. PBS. C. Mice were dosed with a single 100 mg dose of PLP:OVA-ps1 or PBS on day +6 relative to the day of
EAE induction. Unlike PBS, single dose administration of PLP:OVA-ps1 significantly inhibited the occurrence and duration of the clinical EAE. * P,0.05
for PLP:OVA-ps1 vs. PBS. D. Mice dosed with PLP:OVA-ps1 or with PBS 21 days before EAE induction were sacrificed at the peak of the disease (day
14 post-challenge), and histopathology of their spinal cords was determined by staining with luxol fast blue (LFB) and H&E. Mice dosed with PLP:OVA-
ps1 showed significant reduction in the central nervous system (CNS) tissue pathology (designated by arrows) compared to PBS-dosed mice.
* P,0.001 for PLP:OVA-ps1 vs. PBS.
IL-28 Protects against EAE
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Th1 cells by PLP:OVA-ps1-dosed mice, but not those dosed with
OVA-ps1, revealing the importance of Ag-specificity induced by
SJL mice nasally dosed with PLP:OVA-ps1 prior to EAE
induction showed minimal mononuclear cell infiltration with
reduced demyelination compared to the PBS-dosed group
(Figure 1D; Table 1). FACS analysis performed on spinal cord
cells revealed minimal infiltration of inflammatory cells (MHC
II+CD45high) into the central nervous system (CNS), and when
compared with the naive controls, only a negligible percentage of
Mac-3+macrophages was detectable in the CNS (Table 2). In
contrast, PBS-dosed mice showed significant CNS infiltration with
CD11b+Gr-1+neutrophils, CD4+TCR-b+lymphocytes, and Mac-
3+macrophages. PLP:OVA-ps1-mediated protection against
EAE is due in part to suppression of encephalitogenic cell
infiltration into the CNS.
Single Nasal Dose of PLP:OVA-ps1 Treats EAE
To investigate the impact of single PLP:OVA-ps1 dose
PLP:OVA-ps1 or with PBS six days after PLP139–151challenge.
All PBS-dosed mice displayed expected disease with the peak
average clinical score of 4 (Figure 1C). PLP:OVA-ps1-treated
mice showed delayed onset of EAE and ameliorated clinical
disease with the average clinical score of 1 at the peak of disease
(Figure 1C), implicating the therapeutic potential of ps1-delivered
SJL micewere dosed with
PLP:OVA-ps1 Confers Protection via Stimulation of Treg
and FoxP3+CD252CD4+Th2 cells
CD4+T cells isolated from PLP:OVA-ps1- and PBS-dosed
mice after EAE challenge were evaluated by flow cytometry to
determine their Tregcell composition. Nasal PLP:OVA-ps1-dosed
mice showed a significant augmentation in FoxP3+CD25+CD4+T
cells and FoxP3+CD252CD4+Th2 cells, when compared to PBS-
dosed mice (Figure 2). PLP:OVA-ps1 induced .25% of
T cells, of which .93% were FoxP3+
CD252CD4+T cells of which .18% were FoxP3+(Figure 2A;
Table S1), unlike PBS-dosed mice showing only a slight
enrichment in Tregcells (,10%) and CD252Th2 cells as naive
mice (Figure 2B). CD4+T cells from PLP:OVA-ps1-dosed mice
showed .85% Tregcells expressing IL-10 and nearly as many
CD252Th2 cells producing IL-4 and significantly more TGF-b
than those from PBS-dosed mice. Proinflammatory cytokines, IL-
17, IL-21, and IFN-c, were produced primarily by PBS-derived
CD4+T cells and only nominally by those from PLP:OVA-ps1-
dosed mice (Figure 2C). Thus, nasal administration of PLP:OVA-
ps1 induced Ag-specific, anti-inflammatory Tregand Th2 cells.
IL-10R Blockade Abolishes PLP:OVA-ps1-Derived Treg
Cells’ Protective Efficacy
To investigate the relative contribution to protection by these
PLP-specific Tregand Th2 cells, naive SJL mice were adoptively
transferred with PLP:OVA-ps1-derived Tregor Th2 cells and
treated with anti-IL-10R mAb 1 day prior and 5 days after EAE
induction. Adoptive transfer of Ag-specific Treg cells nearly
abrogated EAE, whereas, Th2 cells partially ameliorated disease
(Figure 3A; Figure S1). Anti-IL-10R mAb treatment had no effect
upon PLP:OVA-ps1-derived Th2 cells to prevent EAE; however,
IL-10R blockade in recipients given PLP:OVA-ps1-derived Treg
cells rendered them susceptible to EAE (Figure 3A).
Such pronounced clinical disease by anti-IL-10R-treated Treg
cell recipients showed enhanced proinflammatory responses
by .11- and 9-fold increases in IFN-c and IL-17, respectively,
by LN CD4+T cells when compared to IgG-treated Tregcell
recipients (Figure 3B). Likewise, IL-6 and IL-21 were augment-
ed 3- and 5-fold, respectively, when compared to lymphocytes
from Tregcell recipients treated with IgG. Consequently, Treg
cells+anti-IL-10R mAb-treated recipients did not produce IL-4,
IL-10, or IL-28, but did produce ,2-fold more TGF-b when
Table 1. Nasal administration of PLP:OVA-ps1 prior to PLP139–151challenge protects SJL mice from EAEa.
PLP:OVA-ps112/12 11.561.1*29.17* 0.560.7*0.760.4*
aSJL mice were challenged s.c. with 200 mg PLP139–151in complete Freund’s adjuvant plus 200 ng PT i.p. on days 0 and 2.
bMice were nasally immunized 14 days prior to challenge with 100 mg of PLP:OVA-ps1 or with PBS.
cNumber of mice with EAE/total in group.
dMean day 6 SD of clinical disease onset.
eMaximum (Max.) daily clinical score.
fCumulative scores (CS) were calculated as the sum of all scores from disease onset to day 26 post-challenge, divided by the number of mice in each group. * P,0.001
for PBS vs. PLP:OVA-ps1-dosed mice.
gMean score 6 SEM of inflammation: the infiltration of nucleated cells into spinal cords was scored from 0 to 4 in each mouse separately, and the mean score and SEM
were calculated. *, P,0.001 for PBS vs. PLP:OVA-ps1-dosed mice.
hMean score 6 SEM of demyelination: the demyelination in spinal cords was scored from 0 to 4 in each mouse separately, and the mean score and SEM were calculated.
*, P,0.001 for PBS vs. PLP:OVA-ps1-dosed mice.
Table 2. Nasal treatment with PLP:OVA-ps1 after EAE
inductionareduce inflammatory cell infiltrationbinto the
MHC class II+ +
Treatmentc% Infiltration % CD4+ +TCR-b+ +% CD11b+ +Gr-1+ +
% Mac-3+ +
aSJL/J mice were challenged s.c. with 200 mg PLP139–151in complete Freund’s
adjuvant plus 200 ng PT i.p. on days 0 and 2.
bResults are shown in percentage of MHC class II+CD45highcells from the total
cells in spinal cords analyzed by FACS. *, P,0.001 **, P,0.05 for PBS vs.
cMice were nasally treatment 6 days post-challenge with 100 mg of PLP:OVA-
ps1 or with PBS.
IL-28 Protects against EAE
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Figure 2. PLP:OVA-ps1 induces IL-10-producing Tregand IL-4-producing FoxP3+ +Th2 cells. Mice were dosed with 100 mg of PLP:OVA-ps1
A and C or with PBS B and C fourteen days before challenge with PLP139–151peptide and sacrificed 2 weeks later. A and B. Lymphocytes from head
and neck LNs (HNLNs) and spleens were isolated, cultured with 30 mg/ml of PLP139–151for 3 days, and stained for expression of extracellular (CD25,
CD4 and TGF-b), and intracellular markers (FoxP3, IL-10, and IL-4). Presented FACS plots show cells isolated from spleens with respective isotype
controls provided in inserts. Unlike PBS-dosed mice, PLP:OVA-ps1 administration induced significant enrichment in FoxP3+Tregcells and CD252Th2
cells in mice. In contrast to PBS-derived CD4+T cells, FoxP3+Tregcells from PLP:OVA-ps1-dosed mice produced predominantly IL-10, whereas
PLP:OVA-ps1-derived FoxP3+Th2 cells produced IL-4. Average percentage of 10 mice/group is depicted. * P,0.05 for PLP:OVA-ps1 vs. PBS. C.
CD25+CD4+and CD252CD4+T cells were bead-sorted from combined HNLN, MLN, and splenic lymphocytes and in vitro stimulated with plate bound
anti-CD3 and soluble anti-CD28 mAbs for 72 h. Collected supernatants were analyzed by cytokine ELISA. Negligible amounts of proinflammatory
cytokines were secreted by CD4+T cells isolated from PLP:OVA-ps1-dosed and challenged mice. Mean 6 SEM of 10 mice/group is depicted. * P,0.05
for PLP:OVA-ps1 vs. PBS.
IL-28 Protects against EAE
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compared to Tregcell recipients treated with IgG. Lymphocytes
from IgG-treated Tregcell recipients resembled PLP:OVA-ps1-
treated mice, producing anti-inflammatory cytokines, IL-4, IL-
10, and IL-28, and suppressing TGF-b production. Diseased
mice from groups treated with PBS + IgG or PBS + anti-IL-10R
mAb showed pronounced inflammatory responses evident by
augmented IL-6, IL-21, and IL-23, in addition to .5-fold
increases in IL-17 when compared to IgG-treated Treg cell
recipients (Figure 3B). IgG-treated Th2 cell recipients produced
significantly more IFN-c, at least 3-fold more IL-21 and TGF-b,
considerably less IL-10 and IL-28, but similar amounts of IL-4
when compared to IgG-treated Tregcell recipients. Lympho-
cytes from anti-IL-10R-treated Th2 cell recipients showed
inhibition of IL-4, IL-28, and 3-fold less TGF-b when compared
to IgG-treated Th2 cell recipients, and yet both groups showed
reduced EAE because of their ability to inhibit IFN-c, IL-6, and
IL-4 Neutralization Partially Reverses PLP:OVA-ps1-
Testing the relevance of IL-4 in PLP:OVA-ps1-mediated
protection against EAE, groups of mice were dosed with
PLP:OVA-ps1 or with PBS on days -14 and -7, subsequently
treating them with an anti-IL-4 mAb or rat IgG on days 21 and
+5 relative to EAE challenge. Mice dosed with PBS+IgG
developed typical EAE onset (Figure 3C). IL-4 neutralization
accelerated onset of clinical disease and amplified disease
severity in PBS-dosed mice (Figure 3C; Table S2). PLP:OVA-
ps1 + anti-IL-4 mAb-treated mice also showed earlier disease
onset and greater EAE severity than in PLP:OVA-ps1 + IgG-
dosed mice, but were less pronounced than in PBS + IgG-dosed
T cells isolated from anti-IL-4 mAb-treated and
PLP:OVA-ps1-dosed mice at the peak of the disease showed
pronounced proinflammatory cytokines, IFN-c, IL-6, and IL-17,
Figure 3. PLP:OVA-ps1 protects against EAE via IL-10-producing Tregand IL-4-producing Th2 cells. Mice were dosed with PLP:OVA-ps1,
and two weeks later, Tregand CD252CD4+Th2 cells were isolated and adoptively transferred to naive recipients. On day 0, mice were induced with
EAE and on days 21 and +5 received anti-IL-10R mAb or IgG isotype control Ab. A. In contrast to Tregcells + IgG-treated mice, mice given Treg
cells + anti-IL-10R mAb developed clinical EAE. Administration of anti-IL-10R mAb had no effect on the clinical disease in mice adoptively transferred
with PLP:OVA-ps1-derived Th2 cells. * P,0.05 vs. PBS + IgG. B. Mice were sacrificed at the peak of clinical disease, and CD4+T cells isolated from their
LNs were evaluated for production of cytokines by ELISA. Tregcells + anti-IL-10R-treated mice produced significantly more proinflammatory cytokines
and less anti-inflammatory cytokines when compared to Tregcells + IgG-treated mice. Mean and SD of 5 mice per group is depicted; * P,0.05 vs. Treg
cells + IgG. C. Mice dosed with PLP:OVA-ps1 or PBS on days 214 and 27 were injected with anti-IL-4 mAb or rat IgG on days 21 and +5. PBS + anti-
IL-4-dosed mice developed accelerated and more severe EAE than PBS + IgG-dosed mice. The disease in PLP:OVA-ps1 + anti-IL-4-dosed mice was less
severe than in PBS + IgG-dosed mice, but significantly more severe than in PLP:OVA-ps1 + IgG-dosed mice. * P,0.05 for PLP:OVA-ps1 + IgG vs.
PBS + IgG or PLP:OVA-ps1 + anti-IL-4.
IL-28 Protects against EAE
PLoS ONE | www.plosone.org5 January 2010 | Volume 5 | Issue 1 | e8720
and reduced IL-10 when compared to PLP:OVA-ps1 + IgG-dosed
mice (Figure S2). Overall, IL-4 neutralization in either PBS- or
PLP:OVA-ps1-dosed mice induced more IFN-c, when compared
to their respective IgG-treated control mice, and significantly less
IL-10 was produced in anti-IL-4 mAb-treated PLP:OVA-ps1-
dosed mice than in PLP:OVA-ps1 + IgG-protected mice.
Functional Inactivation of CD25+T Cells Abrogates
PLP:OVA-ps1-Mediated Tolerance and Stimulates
Adoptive transfer of Tregcells from PLP:OVA-ps1-primed
mice was completely protective
(Figures 3A and S1), further implicating relevance of Tregcells
for protection. Neutralization of functional Tregcells did not
affect course of EAE in PBS-dosed mice when compared to
PBS + IgG-dosed mice (Figure 4A); however, EAE accelerated
in anti-CD25 mAb-treated PLP:OVA-ps1-dosed mice, result-
ing in a notably more severe EAE (Figure 4A). The protective
regulatory and Th2-type responses normally induced by
PLP:OVA-ps1 were abated subsequent CD25 neutralization.
The impact was evident by 3-fold reductions in IL-10, loss of
IL-4, and augmented proinflammatory responses noted by 5-
fold for IFN-c, 8-fold for IL-17, and .10-fold enhancements
for IL-6 and IL-21, as well as increased TGF-b (Figure 4B).
PBS-dosed mice treated with anti-CD25 mAb or with IgG
developed classic proinflammatory responses, and LN CD4+T
cells isolated from these mice showed elevations in IFN-c, IL-
6, IL-17, and IL-21 and considerably less IL-10 and IL-4 when
compared to PLP:OVA-ps1 + IgG-dosed mice (Figure 4B).
PLP:OVA-ps1 + IgG-treated mice produced the expected
elevations in IL-4 and IL-10 and near neutralization of
proinflammatory cytokines in a PLP139–151-specific Tregcell-
dependent fashion. Moreover, the lack of protection in CD25-
neutralized PLP:OVA-ps1-dosed mice was associated with
enhanced proinflammatory cytokines and a striking induction
Figure 4. Tregcells are important for PLP:OVA-ps1-induced protection against EAE. Mice dosed with PLP:OVA-ps1 or PBS on days 214
and 27 were treated with anti-CD25 mAb or rat IgG on days 25 and 22. A. Clinical disease in PBS-dosed mice was not affected by anti-CD25 mAb.
PLP:OVA-ps1 + anti-CD25-treated mice developed severe EAE, and all succumbed to the disease. Averaged clinical scores for 10 mice per group are
shown. * P,0.05 for PLP:OVA-ps1 + anti-CD25- and PBS + IgG- vs. PLP:OVA-ps1 + IgG-treated mice. B. CD4+T cells were cultured with feeder cells
and PLP139–151peptide for 72 h. Functional inactivation of Tregcells in PLP:OVA-ps1-dosed mice resulted in induction of proinflammatory CD4+T
cells, producing IFN-c, IL-6, IL-17, IL-21, and TGF-b. Importantly, CD4+T cells obtained from PLP:OVA-ps1-dosed anti-CD25 mAb-treated mice
produced more IL-13 in LNs and spleens and more TGF-b in LNs than in any other experimental group. Mean + SEM from 5 mice per group is shown.
*, P,0.05 for PLP:OVA-ps1 + anti-CD25 vs. PLP:OVA-ps1 + IgG and PBS + IgG.
IL-28 Protects against EAE
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TGF-b and CD25 Co-Neutralization Restores
ps1-Mediated Protection against EAE Independent
Tregcell neutralization exacerbated EAE, negating the protec-
tive capacity of PLP:OVA-ps1, resulting in enhanced TGF-b
production (Figure 4B) and implicating a proinflammatory role of
this cytokine in EAE. To address TGF-b’s participation in EAE
development subsequent CD25 neutralization, PLP:OVA-ps1-
and PBS-dosed mice were treated in vivo with anti-TGF-b mAb,
anti-CD25 mAb, both, or IgG (Figure 5A). No difference in onset
or disease severity was observed between mice dosed with PBS and
treated with the different combinations of mAbs, except those mice
dosed with PBS + anti-TGF-b mAb recovered sooner from the
acute disease (Figure 5B). In contrast to PLP:OVA-ps1 + anti-
CD25 mAb-treated mice, which developed a very aggressive
disease, mice given PLP:OVA-ps1 + any of the remaining Ab
treatments developed only very mild disease with the average peak
clinical score of ,1.5; all of these mice recovered from acute EAE
(Figure 5C). Thus, co-neutralization of CD25 and TGF-b in
PLP:OVA-ps1-treated mice resembled ameliorated disease, as
seen in PLP:OVA-ps1 + IgG-dosed mice. These results showed
that mice functionally neutralized of their CD25+Tregcells in the
presence of tolerogen develop a more aggressive, TGF-b-
dependent EAE. The suppressive activity of PLP:OVA-ps1 could
only be restored upon co-neutralization of TGF-b. These findings
corroborate the results in Figure 3B in which minimal to no TGF-
b was detected in PBS-treated mice, suggesting that TGF-b has a
minimal role in PLP-mediated EAE.
Restoration of Tolerance by PLP:OVA-ps1 upon Co-
Neutralization of CD25 and TGF-b Is IL-28-Dependent
Examination of cytokine profiles conducted 10 days post-EAE
challenge revealed that PLP139–151-restimulated mononuclear cells
from PBS-dosed groups produced elevated amounts of IL-6, IL-
17, IL-21, and IL-23, and very little to no anti-inflammatory IL-4
and IL-10 (Figure S3). When compared to PLP:OVA-ps1 + IgG-
treated mice, PLP:OVA-ps1 + anti-CD25 mAb + anti-TGF-b
mAb-treated mice showed a slight reduction in IL-4, but a marked
7.3-fold reduction in IL-10, which was consistent with the lack of
functional IL-10-producing Tregcells. In addition, this treatment
also reduced IL-22 by 7.7-fold and showed inhibition of IL-6, IL-
17, IL-21, and IL-23 (Figure S3). IL-28 production was preserved
by this treatment; however, when compared to diseased
PLP:OVA-ps1 + anti-CD25 mAb-treated mice, IL-28 was
enhanced 16.4-fold, but with no change in IL-10, implicating
that in the absence of functional Treg cells, PLP:OVA-ps1-
induced protection can occur via Th2-type cells. IL-28 was not
produced in diseased mice treated with either PBS plus the various
mAbs, or with PLP:OVA-ps1 + anti-CD25 mAb, showing the
anti-inflammatory property of IL-28 in EAE. Lack of differences in
IL-4-secretion between mice dosed with PLP:OVA-ps1 + IgG or
PLP:OVA-ps1 + anti-TGF-b + anti-CD25 mAb further suggests
that the presence of Tregcells is not required for IL-4 production
(Figure 5D; Figure S3).
Although Tregcells were absent in mice treated with anti-CD25
plus anti-TGF-b mAbs, upon tolerance induction with PLP:OVA-
ps1, alternative regulatory T cells were induced evident by the
expression of FoxP3 by CD252CD4+T cells (Table 3). In fact, the
inclusion of anti-TGF-b mAb in the treatment paradigm restored
elevated FoxP3 expression by CD252CD4+T cells to levels
similar to those obtained from PLP:OVA-ps1 + IgG-dosed mice.
In contrast, PLP:OVA-ps1 + anti-CD25 mAb-treated mice
displayed a 67% reduction in FoxP3+CD252CD4+T cells when
compared to PLP:OVA-ps1-protected mice (Table 3). PLP:OVA-
ps1 + anti-TGF-b mAb treatment showed only a 20% reduction
in FoxP3+CD252CD4+T cells; these mice still retained their Treg
Aside from TGF-b, IL-23 also has been shown to activate Th17
cells [27–29]. To determine the necessity of IL-23 for induction of
EAE in PLP:OVA-ps1 + anti-CD25 mAb-treated mice, mice
were co-treated with anti-IL-23p19 serum. Neutralization of IL-23
had no effect on clinical onset and severity of EAE (Figure 6).
Endogenous IL-28 Protects against EAE in the Absence of
To investigate the role of IL-28 in the PLP:OVA-ps1-mediated
protection against EAE in the absence of functional Tregcells,
mice dosed with PLP:OVA-ps1 or PBS were treated with anti-IL-
28 rabbit serum (RS), normal RS (NRS), and/or with mAbs to
CD25 and TGF-b (Figure 7A). Treatment of PBS- or PLP:OVA-
ps1-dosed mice with anti-IL-28 RS did not significantly alter EAE
when compared to their respective control groups dosed with NRS
(Figure 7B). Neutralization of IL-28 in concert with co-neutralized
CD25 and TGF-b rendered PLP:OVA-ps1-dosed mice suscep-
tible to EAE development, presumably due to the significant
reduction in FoxP3+Th2 cells (Figure 7C), reaffirming the
importance of IL-28-mediated protection in EAE.
As before, CD4+T cells from PLP:OVA-ps1 + NRS- and
PLP:OVA-ps1 + anti-CD25 + anti-TGF-b-treated mice produced
predominantly anti-inflammatory cytokines (Figure 7D). Although
PLP:OVA-ps1 + anti-IL-28-dosed mice conferred protection
against EAE (Figure 7B), upon peptide restimulation, their CD4+
(Figure 7D), and these mice expressed fewer FoxP3+Th2 cells
(Figure 7C), unlike PLP:OVA-ps1 + NRS-treated mice that were
protected against EAE, but without inducing proinflammatory
cytokines. Such differences in protection were not attributed to IL-
10 since these remained unchanged in both groups. Neutralization
of IL-28 in PBS or PBS + anti-CD25 + anti-TGF-b-dosed mice did
not significantly affect proinflammatory cytokine production by
CD4+T cells; however, secretion of IL-4 and TGF-b was
significantly reduced in these mice when compared to PBS +
NRS-dosed mice. Independent of treatment, all PBS-dosed mice
showed only marginal production of anti-inflammatory cytokines.
These studies demonstrate IL-28 can be induced to supplant
protective IL-10+Tregcells. To our knowledge, this is the first such
report describing IL-28’s protective capacity against inflammatory
Tolerance is the active inability to respond to self or a defined Ag
and represents a method to abolish self-reactivity to ultimately
enable protection against autoimmune disease. A caveat in
preventing or treating autoimmune diseases in humans is being
able to successfully adapt tolerance methods used in various animal
models. In a double-blind phase III clinical trial to test the feasibility
of inducing oral tolerance to myelin basic protein and PLP, high
doses of myelin Ags were administered, and although TGF-b-
secreting CD4+T cells were induced , no differences in MS
outcomes between placebo and treated were observed .
Notwithstanding, alternative methods of mucosal delivery, particu-
larlysublingual,haveproveneffective inhumansto induce tolerance
to alleviate allergies to dust mites and grass pollens [32–34].
Rendering tolerance via sublingual delivery of these allergens
requires contactwith the oral mucosa since immediateswallowing of
allergens diminishes the tolerogenic capacity . To a limited
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extent, targeting auto-Ags to the mucosal epithelium has been tested
using cholera toxin subunit B (CT-B) to improve diabetes  and
EAE  in mice. However, CT-B also behaves as a mucosal
adjuvant [37,38], resulting in conflicting outcomes when Ags
chemically coupled to CT-B produce potent Ag-specific immunity
[36,39] or induce tolerance [35,40]. Since chemical modification
Figure 5. Abated protection against EAE in anti-CD25 mAb-treated PLP:OVA-ps1-dosed mice is restored upon TGF-b co-
neutralization. A. Experimental design for neutralization of TGF-b and CD25. B. PBS-dosed mice, independent of treatment, developed expected
course of EAE. C. Treatment with anti-CD25 mAb abrogated PLP:OVA-ps1-mediated protection against EAE, but concomitant treatment with anti-
TGF-b and anti-CD25 mAbs restored PLP:OVA-ps1-induced protection. Mean of 10 mice per group is shown. B and C. * P,0.05 vs. IgG-dosed mice.
D. HNLN CD4+T cells on day 10 post-EAE induction were cultured with PLP139–151peptide for 72 h. PBS-dosed mice independent of Ab treatment
produced elevated proinflammatory cytokines IFN-c, IL-17, and IL-21, and little to no IL-4, IL-10, IL-13, and TGF-b. However, levels of IFN-c and IL-17
produced by CD4+T cells were diminished in PBS + anti-TGF-b-treated mice when compared to PBS + IgG- treated mice. PLP:OVA-ps1-protected
mice treated with IgG, anti-TGF-b mAb, or anti-TGF-b + anti-CD25 mAbs produced enhanced IL-4 and IL-28, but minimal proinflammatory cytokines.
Additionally, mice dosed with PLP:OVA-ps1 + anti-CD25 + anti-TGF-b mAbs produced significantly more IL-13 and IL-4 than diseased PLP:OVA-ps1 +
anti-CD25-dosed mice. Mean + SEM of 5 mice per group is shown * P,0.05 for the PLP:OVA-ps1 + anti-TGF-b + anti-CD25 vs. PLP:OVA-ps1 + IgG and
PLP:OVA-ps1 + anti-CD25.
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can render a tolerogen immunogenic, the alternative approach of
genetically fusing PLP139–151to CT-B maintains its ability to remain
tolerogenic and suppress EAE . Despite its success, this
approach again requires multiple nasal administrations to maintain
efficacy against EAE. These collective studies implicate the potential
of induced tolerance if given by a suitable mucosal route, allowing
for tolerogen uptake. Thus, there have been limited attempts to test
the potential of microbial adhesins in humans to facilitate tolerance
induction, let alone, with ps1.
To enable this latter possibility and to take advantage of ps1’s
adhesive properties [5,41], OVA-ps1 was modified with two
copies of immunodominant T cell epitope PLP130–151, resulting in
a functional PLP:OVA-ps1, which, when given nasally, protected
susceptible SJL mice against EAE. PLP:OVA-ps1 protection
resulted in the diminution of proinflammatory CD4+T cells and
the stimulation of CD4+T cells producing regulatory and anti-
inflammatory cytokines. The related protein lacking PLP peptide,
OVA-ps1, was not protective, further showing the tolerogenic
responses being Ag-specific. Importantly, the therapeutic effect
conferred by PLP:OVA-ps1 could be rendered with as low as a
single 100 mg dose, similar to that shown for OVA-specific
tolerance . While tolerance to encephalitogenic proteins or
peptides when applied mucosally can be shown, these generally
require multiple and/or large Ag doses to confer protection
against EAE [3,17,19,20,42].
Although PLP:OVA-ps1 is a derivative of OVA-ps1, this was
done to facilitate monitoring of B cell responses subsequent
mucosal delivery of this tolerogen since the PLP139–151 T cell
epitope produced weak to no Ab responses in PBS-treated,
PLP139–151-challenged mice (data not shown). The intent of this
work is to establish the potential of ps1-based therapeutics for
MS, and subsequent work will fashion a ps1-based fusion
tolerogen bearing relevant human T and B cell epitopes for MS.
PLP:OVA-ps1-mediated protection was facilitated by IL-10-
producing FoxP3+Treg cells and supported by IL-4-producing
Th2 cells. IL-10R blockade abolished
protection mediated by PLP:OVA-ps1-derived Tregcells, allow-
ing for uninhibited Th1- and Th17-type cytokine production with
concomitant reductions in IL-4 and IL-28, resulting in attrition to
EAE. Such findings regarding IL-10’s importance in protection
[2,4,17,43,44]. Likewise, ps1-mediated tolerance could not be
established in IL-102/2mice presumably due to the failure in the
generation of FoxP3+Tregcells .
IL-4 neutralization only partially altered PLP:OVA-ps1-
mediated protection against EAE evident as reduced IL-10
production with concomitant stimulation of proinflammatory
Th1- and Th17- type cytokines. Investigating their relative
contributions, adoptive transfer of PLP:OVA-ps1-primed Treg
cells prior to PLP139–151peptide challenge conferred complete
protection against EAE, but transfer of PLP:OVA-ps1-primed
CD252CD4+Th2 cells, while not conferring complete protection,
significantly delayed the disease onset and reduced disease severity.
In agreement with our previous study in which OVA-ps1-
mediated tolerance was supported by IL-4-producing Th2 cells,
FoxP3+CD252CD4+Th2 cells, .80% of which produced IL-4.
The anti-inflammatory potential of IL-4 has been well character-
ized [1,45], and stimulation of regulatory FoxP3+CD252CD4+T
cells has been shown by us and others [4,5,7,9]. Alternatively, it is
plausible that PLP:OVA-ps1-induced FoxP3+Th2 cells are
undergoing conversion to the Treg cells, since conversion of
FoxP3+CD252CD4+T cells potently inhibited CD4+T cells’
expansion in vivo and proliferation in vitro [9,46]. It is reported
that IL-4 can supplement suppressive function of TGF-b-secreting
what othershave shown
Table 3. In vivo neutralization of TGF-b and CD25 induces
FoxP3 expression of PLP:OVA-ps1-primed Th2 cells.
PLP:OVA-ps1 + IgG
PLP:OVA-ps1 + anti-TGF-b
PLP:OVA-ps1 + anti-CD25
PLP:OVA-ps1 + anti-TGF-b + anti-CD25
aSJL/J mice were challenged s.c. with 200 mg PLP139–151in complete Freund’s
adjuvant plus 200 ng PT i.p. on days 0 and 2, and were nasally dosed 14 and 7
days prior to EAE challenge with 100 mg of PLP:OVA-ps1 or with PBS and
treated as described in Fig. 5.
bAverage percentages 6 SD of FoxP3+CD252CD4+T cells as a fraction of CD4+T
cells from 3 mice per group are depicted, *, P,0.001, **, P,0.05 vs. PLP:OVA-
ps1 + anti-CD25,1, P,0.05 vs. PLP:OVA-ps1 + anti-TGF-b˜.
Figure 6. IL-23-independent induction of Th17 cells in Tregcell-depleted PLP:OVA-ps1-dosed mice. SJL mice were nasally dosed with
PLP:OVA-ps1 or PBS on days 214 and 27 relative to EAE induction. Mice were i.p. injected with anti-CD25/rat IgG and anti-IL-23p19 rabbit serum
(RS) or normal rabbit serum (NRS). Neutralization of IL-23 did not suppress clinical disease in Tregcell-inactivated PLP:OVA-ps1-dosed mice. Mean of 5
mice per group is shown. * P,0.05 vs. PLP:OVA-ps1 + anti-CD25-treated mice.
IL-28 Protects against EAE
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regulatory Th3 cells . IL-4 has also been implicated in
triggering expression of FoxP3 on naive peripheral CD252CD4+
T cells , and when secreted by Th2 cells, IL-4 supports the
inhibition of PLP139–151-specific proinflammatory responses .
Surprisingly, IL-4 deficiency has not been associated with
increased susceptibility to autoimmune diseases, suggesting a
supportive role for IL-4 in suppression of inflammatory responses
against self-Ags . Therefore, consistent with the supportive role
Figure 7. IL-28 is responsible for protection against EAE in Tregcell-depleted PLP:OVA-ps1-dosed mice. A. Mice were nasally
dosed with 100 mg of PLP:OVA-ps1 or with PBS on days 214 and 27 relative to EAE induction (day 0). Mice were treated with anti-CD25 mAb
on days 25, and 22; anti-TGF-b mAb on days 21 and +5, and/or with anti-IL-28 RS on days 23 and +4, relative to EAE induction. B. In
contrast to PLP:OVA-ps1 (+ NRS)- or PLP:OVA-ps1 + anti-IL-28 RS-treated mice, mice treated with PLP:OVA-ps1 + anti-CD25 mAb or PLP:OVA-
ps1 + anti-CD25 mAb + anti-TGF-b mAb + anti-IL-28 RS developed pronounced EAE. C. Pooled lymphocytes from MLNs and HNLNs at the
peak of EAE were stained for FACS and analyzed for expression of FoxP3. In contrast to PLP:OVA-ps1-protected mice that received NRS, anti-
IL-28 RS, or combination of anti-CD25 and anti-TGF-b mAbs, mice that received PLP:OVA-ps1 + anti-CD25 mAb + anti-TGF-b mAb + anti-IL-28
RS or PLP:OVA-ps1 + anti-CD25 mAb showed significantly reduced percentages of FoxP3+Th2 cells. B and C. * P,0.001, ** P,0.05 vs.
PLP:OVA-ps1 + anti-CD25,1P,0.05 vs. PLP:OVA-ps1 + anti-IL-28. D. Pooled HNLN and MLN CD4+T cells were evaluated for production of
proinflammatory and anti-inflammatory cytokines by ELISA. CD4+T cells from mice treated with PLP:OVA-ps1 + anti-IL-28 RS produced
similar amounts of anti-inflammatory cytokines and slightly elevated amounts of proinflammatory cytokines when compared to PLP:OVA-ps1
(+ NRS)-dosed mice. In contrast to PLP:OVA-ps1-dosed mice, CD4+T cells from mice dosed with PLP:OVA-ps1 + anti-CD25 + anti-TGF-b mAbs
+ anti-IL-28 RS displayed cytokine profile that closely resembled PLP:OVA-ps1 + anti-CD25-treated mice. Mean and SD of 5 mice per group
are depicted; * P,0.05 vs. PLP:OVA-ps1-dosed mice.
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of IL-4 in induction of Tregcells , possibly IL-4 neutralization
downregulates IL-10-producing Tregcells in PLP:OVA-ps1-dosed
Inquiry into the role of Th2 cytokines to ps1-dependent
tolerance found that the functional inactivation of Tregcells greatly
impaired the PLP:OVA-ps1-induced protection against EAE.
Anti-CD25 mAb treatment relinquished the tolerogenic property
of PLP:OVA-ps1, making it more immunostimulatory, as evident
by the increased disease severity and enhanced production of TGF-
b, IL-6, IL-17, IL-21, and IFN-c with concomitant reductions in
IL-4, IL-10, and IL-28. Although IL-13 was enhanced, our
preliminary studies implicated lack of proinflammatory function
for this cytokine in Tregcell-depleted PLP:OVA-ps1-dosed mice.
In contrast to concomitant inhibition of CD25 and IL-23,
simultaneous neutralization of TGF-b and CD25 reestablished
PLP:OVA-ps1-induced protection, suggesting that anti-CD25
mAb-induced Th17 inflammatory responses in PLP:OVA-ps1-
dosed mice are mediated via TGF-b rather than IL-23. In addition
to its established proinflammatory role [28,29], TGF-b can be a
major regulatory cytokine secreted by Tregcells, and its production
has been linked to potent suppression of EAE [3,7].
Protected PLP:OVA-ps1 + anti-TGF-b-treated mice showed a
modest, although significant, decrease in FoxP3+Th2 cells when
compared to PLP:OVA-ps1 + IgG-dosed mice, but these cells
remained significantly enhanced when compared to PLP:OVA-
ps1 + anti-CD25-treated diseased mice. Although TGF-b can
support conversion of CD252CD4+T cells to Treg cells via
enhanced FoxP3 expression [9,49], our data showed that CD25
neutralization upon PLP:OVA-ps1 treatment results in enhanced
TGF-b responses reminiscent of TGF-b’s inflammatory proper-
ties, perhaps because of the increased presence of IL-6  and/or
Mice co-neutralized of the Tregcells and TGF-b, but dosed with
PLP:OVA-ps1, reestablished tolerance evidenced by the inhibi-
tion of Th1 and Th17 cells and the enhanced production of IL-4,
IL-13, and IL-28. Reversion of IL-10 production was not evident
presumably because of neutralization of Tregcells. Concomitant
neutralization of TGF-b, CD25, and IL-28 reversed the Th2 cell-
dependent tolerance by PLP:OVA-ps1 treatment, resulting in
EAE and reaffirming the novel role for IL-28 in protection against
EAE in the absence of Tregcells. Combined with their enhanced
expression of FoxP3, these results suggest an alternative regulatory
pathway that can be induced by PLP:OVA-ps1, but independent
of conventional Tregcells. In some cases, minimal levels of IL-10
were still being secreted by CD4+T cells in PLP:OVA-ps1 + anti-
CD25 + anti-TGF-b-dosed mice. Consequently, the possibility of
potential synergistic or priming effect by IL-10 upon IL-28 cannot
be excluded to account for the observed protection against EAE in
the absence of functional Tregcells and proinflammatory TGF-b.
A growing body of evidence suggests that type I and II IFNs can
induce proinflammatory potential of IL-10 by switching the
balance of IL-10 STAT activation from Stat3 to Stat1 [51,52].
Type III IFNs share functional and structural similarities with type
I IFNs, including the Jak-STAT signaling pathway [11,13,14,53];
therefore, it is plausible that in the presence of IL-28 the nominal
amounts of IL-10 produced by the CD4+T cells in PLP:OVA-ps1
+ anti-CD25+anti-TGF-b-dosedmiceareinfact proinflammatory.
The role for IL-28 has not yet been evaluated in EAE, although IL-
28 is known for its anti-inflammatory activity , and it has been
shown to prime tolerogenic DCs in vitro .
In summary, we showed that even a single 100 mg dose of ps1-
based nasal vaccine in an Ag-specific fashion protected mice
against EAE. The ps1-based vaccine protected against EAE via
various mechanisms, including activation of IL-10-producing
FoxP3+Tregcells and IL-4-secreting FoxP3+Th2 cells. In the
absence of Treg cells, ps1-based protection against EAE was
associated with an increased expression of FoxP3 on CD252CD4+
T cells producing IL-28, which, to our knowledge, is the first
report describing regulatory role of IL-28-producing CD4+T cells
conferring protection against EAE. These results show that a single
low dose nasal tolerance mediated by genetically modified ps1
can be successfully applied to prevent and/or treat autoimmune
Materials and Methods
All animal care and procedures were in accordance with
institutional policies for animal health and well-being, and
approved by MSU Institutional Animal Care and Use Committee.
Preparation of PLP:OVA-ps1
PLP:OVA-ps1 was constructed using the OVA-ps1 backbone
. Two copies of PLP peptide (PLP130–151; QAHSLERVC
(RHRHVDCSGRNLTTLPPGLQE) were synthesized as a single
cDNA fragment containing restriction enzyme sites 59 and 39
termini (GenScript Corp.). The synthetic cDNA fragment was
amplified by PCR and cloned into pUC19. The 59 and 39 primers
encoded EcoRI sites, and 59 primer encoded an ATG initiation
codon embedded into an optimal Kozak’s sequence. PCR
amplified PLP peptides were ligated with the 59 terminus of
OVA-ps1 in a pPICZ B vector (Invitrogen Corp.) bearing a his-
tag carboxy terminus for protein purification (Invitrogen), referred
to as PLP:OVA-ps1. The junction between the PLP139–151
epitopes and the OVA-ps1 featured a flexible linker (Gly-Arg-
Pro) to minimize steric hindrance between the components. The
resulting construct was sequenced and expressed in the yeast Pichia
pastoris, according to the manufacturer’s directions (Invitrogen
Corp.). Recombinant proteins were extracted from yeast cells by a
bead-beater (Biospec Products) and purified on a Talon metal
affinity resin (BD Biosciences, Palo Alto, CA), according to
manufacturer’s instructions. Proteins were assessed for purity and
quality by Coomassie-stained polyacrylamide gels and by Western
blot analysis using a polyclonal rabbit anti-ps1 (produced in-
house) or a polyclonal rabbit anti-OVA Ab (Sigma-Aldrich). All
recombinant proteins migrated as a single band with the expected
Female six wk old SJL mice were obtained from Frederick
Cancer Research Facility, National Cancer Institute, and The
Jackson Laboratories. All mice were maintained at Montana State
University Animal Resources Center under pathogen-free condi-
tions in individual ventilated cages under HEPA-filtered barrier
conditions and were fed sterile food and water ad libitum. The mice
were free of bacterial and viral pathogens, as determined by
antibody screening and histopathological analysis of major organs
Tolerance Induction, PLP:OVA-ps1 Treatment, and EAE
For tolerance induction, mice (5–10 mice/group) were nasally
dosed up to three times with 50–100 mg of PLP:OVA-ps1 or
OVA-ps1 before or 6 days after EAE challenge, as described in
the text. Control groups were treated with PBS or equivalent
amounts of OVA-ps1. PLP:OVA-ps1 or OVA-ps1 was
administered nasally, as previously described .
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For EAE induction, mice were challenged s.c. with 200 mg of the
encephalitogenic PLP peptide (PLP139–151; HSLGKWLGHPDKF;
Global Peptide Services; HPLC-purified to .90%) in 200 ml .
On days 0 and 2 post-challenge, mice received i.p. 200 ng of
Bordetella pertussis toxin (PT; List Biological Laboratories). Mice were
monitored and scored daily for disease progression : 0, normal;
1, a limp tail; 2, hind limb weakness; 3, hind limb paralysis; 4,
quadriplegia; 5, death.
Measurement of Delayed-Type Hypersensitivity (DTH)
To measure OVA- or PLP139–151- specific DTH responses ,
OVA or PLP139–151(10 mg) was injected into the left ear pinna,
and PBS alone (20 ml) was administered to the right ear pinna as a
control. Ear swelling was measured 24 h later with an electronic
digital caliper (World Precision Instruments). The DTH response
was calculated as the increase in ear swelling after antigen
injection following subtraction of swelling in the control site
injected with PBS.
Histological Evaluation of Spinal Cords
For histological evaluation of tissue pathology, spinal cords were
removed 14 days after challenge and fixed with neutral buffered
formalin (VWR International), embedded into paraffin, and
sectioned at 5 mm. Cross sections of spinal cords were stained with
H&E for pathological changes and inflammatory cell infiltration,
and adjacent sections with luxol fast blue (LFB) for loss of myelin.
Pathological manifestations were scored separately for cell infiltrates
and demyelination. Each H&E section was scored from 0 to 4: 0,
normal; 1, cell infiltrate into the meninges; 2, one to four small focal
perivascular infiltrates; 3, five or more small focal perivascular
infiltrates and/or one or more large infiltrates invading the
parenchyma; 4, extensive cell infiltrates involving 20% or more of
the white matter [7,54]. In each LFB stained section, myelin was
also scored from 0 to 4: 0, normal; 1, one small focal area of
demyelination; 2, two or three small focal areas of demyelination; 3,
one to two large areas of demyelination; 4, extensive demyelination
involving 20% or more of white matter [7,54].
Spleens, mesenteric lymph nodes (MLNs), and head and neck
LNs (HNLNs) were aseptically removed 14 days after EAE
induction from PBS-, PLP:OVA-ps1- and OVA-ps1-dosed mice.
Lymphocytes were prepared, as previously described , and
resuspended in complete medium (CM) . Lymphocytes were
cultured in 24-well tissue plates at 56106cells/ml in CM alone or
with PLP139–151peptide (30 mg/ml) for 3–5 days at 37uC. The
supernatants were collected by centrifugation and stored at
280uC. Capture ELISA was employed to quantify, on triplicate
sets of samples, the levels of IFN-c, IL-4, IL-6, IL-10, IL-13, IL-17,
and TGF-b produced by lymphocytes, as previously described .
For detection of IL-21, IL-22, and IL-28, microtiter wells were
coated with 2 mg/ml of purified goat anti-mouse IL-21 Ab, goat
anti-mouse IL-22 Ab, or anti-mouse IL-28B mAb (clone 244716),
respectively (all R&D Systems). For detection of IL-23, wells were
coated with 8 mg/ml of anti-mouse IL-23p19 (clone G23-8,
eBioscience). After blocking with PBS +1% BSA for 2 h at 37uC,
washed wells were incubated with cell culture supernatants at 4uC
for 24 h. After washing, 0.5 mg/ml biotinylated goat anti-mouse
IL-21 Ab, biotinylated goat anti-mouse IL-22 Ab, biotinylated
anti-mouse IL-28B mAb (clone 244707) (all R&D Systems), or
biotinylated anti-mouse IL-12 and IL-23 (p40) mAb (clone C17.8,
eBioscience) was added, respectively, for 90 min at 37uC.
Following washing, 1:500 HRP-goat anti-biotin Ab (Vector
Laboratories) was added for 1 h at room temperature (RT). After
washing, ABTS peroxidase substrate (Moss, Inc.) was added to
develop the reaction. Production of cytokines by unstimulated cells
set as a background was subtracted from all measurements.
Lymphocytes from the HNLNs, MLNs, and spleens were
isolated 14 days after challenge, and single cell suspensions were
prepared, as described above . To obtain lymphocytes from
spinal cords, mice were perfused through the left ventricle with
20 ml of ice cold sPBS, and spinal cords were removed by flushing
the vertebral canal with media and prepared, as previously
Cells were stained for FACS analysis using conventional
methods. Leukocyte gates were set within the forward and side
scatter profiles to exclude resting microglia cells in the spinal cord
preparations. Neutrophils and macrophages were analyzed by
forward and side scatter profiles and using fluorochrome-
conjugated mAbs for SK208 (7,8), CD11b, Gr1, and Mac-3 (BD
Pharmingen). T cell subsets were analyzed using fluorochrome-
conjugated mAbs for CD4, CD25, TCRb, CD8, GITR, CCR6
(all from BD Pharmingen), OX-40 (CD134; clone OX-86)
(eBioscience), and biotinylated TGF-b(R&D Systems). Intracellu-
lar staining for FoxP3 was accomplished using FITC-, Cy-, or PE-
anti-FoxP3 mAb (clone FJK-16s; eBioscience), FITC, or PE-anti-
IFN-c Ab, PE, or APC- anti-IL-10 and anti-IL-4, and PE-anti-
CTLA-4 (CD152) (all from BD Pharmingen). Bound fluorescence
was analyzed with a FACS Canto (BD Biosciences).
In Vivo Neutralization of IL-4 and Blockade of IL-10R
To inhibit IL-4 in vivo, mice dosed with PLP:OVA-ps1 on days
214 and 27 before EAE challenge were given i.p. 1.0 mg of anti-
IL-4 mAb (clone 11B11; ATCC) on day 21 before challenge, and
on day +5 after EAE challenge with PLP139–151. Control mice
received i.p. injection of 1.0 mg purified rat IgG Ab (AbD
To inhibit IL-10 receptor function, mice were i.p. injected with
0.5 mg of anti-IL-10R mAb (clone1B1.3A, BioXCell), or IgG
isotype control Ab at the day of an adoptive transfer and 6 days
later (day 21 and +5 relative to the day of EAE induction). Mice
were induced with EAE on day 0, as described above.
In Vitro T Cell Assays
To assess cytokine production by Tregcells and effector T cells,
CD25+CD4+and CD252CD4+T cells (26105) were stimulated in
vitro with anti-CD3 mAb-coated wells (10 mg/ml; BD Pharmin-
gen) and a soluble anti-CD28 mAb (5 mg/ml; BD Pharmingen) for
5 days (final volume of 300 ml in a 48-well plate). Capture ELISA
was used to quantify triplicate sets of samples to measure cytokine
Adoptive Transfer Studies
Following PLP:OVA-ps1 immunization, total CD4+T cells
from spleens, HNLNs, and MLNs were obtained (negative CD4+
T cell isolation kit, Dynal Biotech ASA). CD25+CD4+and
CD252CD4+T cells were isolated from total CD4+T cells with
.95% and 99% purity, respectively, by positive selection using
CELLection Biotin Binder Kit (Dynal Biotech; Invitrogen) and
biotin-conjugated anti-mouse CD25 (PC61, eBioscience), accord-
ing to manufacture’s instructions. To test PLP:OVA-ps1-primed
Tregcell efficacy, 66105CD252CD4+T cells or CD25+CD4+T
cells were i.v. injected into naı ¨ve recipients. The group of control
IL-28 Protects against EAE
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mice was i.v. injected with sPBS. One day after the adoptive
transfer of T cell subsets, mice were challenged with PLP139–151
and evaluated for the clinical symptoms.
In Vivo Inactivation of CD25 and Neutralization of TGF-b
Mice were nasally dosed with PLP:OVA-ps1 or PBS on days
214 and 27 relative to the EAE induction with PLP139–151. To
functionally inactivate CD25+CD4+T cells, the same mice were
given i.p. 0.5 mg anti-CD25 mAb (clone PC 61.5.3; ATCC TIB-
222) on days 25 and 22 before EAE induction. As a control,
groups of PLP:OVA-ps1- or PBS-dosed mice received 0.5 mg of
purified rat IgG on the same days before EAE challenge. All mice
were monitored daily for development of EAE.
To neutralize TGF-b in vivo, mice dosed with PLP:OVA-ps1
or PBS and treated with anti-CD25 mAb or rat IgG were i.p.
injected with an additional 0.5 mg of anti-TGF-b mAb (clone
1D11.16.8, ATCC) on days 21 and +5 relative to the day of EAE
To neutralize IL-28, mice were i.p. injected on days 23 and +4
relative to the EAE induction with 1 ml of anti-IL-28 RS
(developed in-house by immunizing rabbits with recombinant
IL-28B; R&D Systems) or with NRS as control.
In Vivo Neutralization of IL-23
To block IL-23 in vivo, mice dosed with 100 mg of PLP:OVA-
ps1 on day 214 and 27 before EAE challenge were given i.p.
0.5 ml of anti-IL-23p19 rabbit serum (RS, made in-house) on day
21 before challenge, and 0.25 ml of anti-IL-23 RS on days 1 and
5 after EAE challenge with PLP139–151. Control mice received i.p.
injection of an equal amount of NRS (Jackson ImmunoResearch
The ANOVA followed by posthoc Tukey test was applied to
show differences in clinical scores in treated vs. PBS mice. The
student t test was used to evaluate the differences between
variations in cytokine level production, and P-values ,0.05 are
indicated, unless specified otherwise.
Characterization of CD4+T cells from combined LNs
and spleens of naı ¨ve, PBS- and PLP:OVA-ps1-dosed SJL micea.
Found at: doi:10.1371/journal.pone.0008720.s001 (0.04 MB
PLP:OVA-ps1-mediated protection against EAEa.
Found at: doi:10.1371/journal.pone.0008720.s002 (0.03 MB
CD25+CD4+Tregcells are important for PLP-OVA-
ps1-induced protection against EAE. Mice dosed with PLP:OVA-
In vivo neutralization of IL-4 partially reverses
ps1 on day 0 and 7, were sacrificed on day 14. CD25+CD4+(Treg)
cells or CD252CD4+(Th2) cells from these mice were adoptively
transferred into naive recipients induced with EAE 24 h later.
Transfer of Tregcells entirely protected mice from development of
EAE, but Th2 cells also significantly delayed and improved
severity of EAE. Averaged clinical scores from 2 experiments (10
mice/group) are shown. *, P,0.05 vs. PBS-dosed mice.
Found at: doi:10.1371/journal.pone.0008720.s003 (0.18 MB TIF)
tion against EAE. Mice (5/group) dosed with PLP:OVA-ps1 or
PBS on day 214 and 27 were injected with anti-IL-4 mAb or rat
IgG on day 21 and +5. CD4+T cells isolated from HNLNs,
MLNs, and spleens of these mice at the peak of the disease (day 14
post challenge) were incubated with feeder cells and PLP139–151
peptide for 72 h. Cultured supernatants were analyzed for
cytokine production by ELISA. Results show cytokine production
by cultured CD4+T cells corrected over the cytokine production
by unstimulated cells. PBS and PLP:OVA-ps1-dosed mice treated
with anti-IL-4 mAb produced more IFN-c and less IL-10 than
their respective IgG-treated controls. PLP:OVA-ps1 + anti-IL-4-
dosed mice produced less IL-10 and more IFN-c, IL-6 and IL-17
than PLP:OVA-ps1 + IgG-dosed mice. PBS + anti-IL-4 -treated
mice produced less IL-21 that mice dosed with PBS + IgG.
*, P,0.05 for PLP:OVA-ps1 + anti-IL-4 vs. PBS + IgG or
PLP:OVA-ps1 + IgG, and PBS + IgG vs. PBS + anti-IL-4.
Found at: doi:10.1371/journal.pone.0008720.s004 (0.34 MB TIF)
IL-4 contributes to PLP:OVA-ps1-induced protec-
production by PLP:OVA-ps1-tolerized mice depleted of Tregcells.
Total lymphocytes isolated from the HNLNs of anti-CD25 and/or
TGF-b-treated mice (described in Fig. 5D legend) on day 10 post
EAE induction were cultured with PLP139–151peptide for 72 h.
PBS-dosed mice independent of Ab treatment produced enhanced
amounts of proinflammatory cytokines IL-6, IL-17, IL-21, and
IL-23, and little to no IL-4, IL-10, and IL-28. PBS + anti-TGF-b +
anti-CD25-treated mice showed elevated IL-22 production.
PLP:OVA-ps1-protected mice treated with IgG, anti-TGF-b
mAb or with anti-TGF-b + anti-CD25 mAbs, produced enhanced
amounts ofIL-4andIL-28 and little tono ofIL-6, IL-17,IL-21,and
IL-23. Mean 6 SEM of 5 mice per group is shown * P,0.05 for the
PLP:OVA-ps1 + anti-TGF-b + anti-CD25 vs. PLP:OVA-ps1 +
IgG and PLP:OVA-ps1 + anti-CD25.
Found at: doi:10.1371/journal.pone.0008720.s005 (0.74 MB TIF)
TGF-b is responsible for proinflammatory cytokine
We thank Nancy Kommers for her assistance in preparing this manuscript.
Conceived and designed the experiments: AR MM JOR DWP. Performed
the experiments: AR JOR GC. Analyzed the data: AR DWP. Contributed
reagents/materials/analysis tools: AR MM GC. Wrote the paper: AR
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