Promoting tolerance to proteolipid protein-induced experimental autoimmune encephalomyelitis through targeting dendritic cells.
ABSTRACT In T cell-mediated autoimmune diseases, self-reactive T cells with known antigen specificity appear to be particularly promising targets for antigen-specific induction of tolerance without compromising desired protective host immune responses. Several lines of evidence suggest that delivery of antigens to antigen-presenting dendritic cells (DCs) in the steady state (i.e., to immature DCs) may represent a suitable approach to induce antigen-specific T-cell tolerance peripherally. Here, we report that anti-DEC205-mediated delivery of the self-peptide proteolipid protein (PLP)139-151 to DCs ameliorated clinical symptoms in the PLP-induced SJL model of experimental autoimmune encephalomyelitis. Splenocytes from treated mice were anergized to PLP139-151, and IL-17 secretion was markedly reduced. Moreover, we show directly, using transgenic CD4(+) Vβ6(+) TCR T cells specific for PLP139-151, that, under the conditions of the present experiments, these cells also became anergic. In addition, evidence for a CD4(+) T cell-mediated suppressor mechanism was obtained.
- SourceAvailable from: Jashodeep Datta[Show abstract] [Hide abstract]
ABSTRACT: Dendritic cells (DC) are professional antigen-presenting cells uniquely suited for cancer immunotherapy. They induce primary immune responses, potentiate the effector functions of previously primed T-lymphocytes, and orchestrate communication between innate and adaptive immunity. The remarkable diversity of cytokine activation regimens, DC maturation states, and antigen-loading strategies employed in current DC-based vaccine design reflect an evolving, but incomplete, understanding of optimal DC immunobiology. In the clinical realm, existing DC-based cancer immunotherapy efforts have yielded encouraging but inconsistent results. Despite recent U.S. Federal and Drug Administration (FDA) approval of DC-based sipuleucel-T for metastatic castration-resistant prostate cancer, clinically effective DC immunotherapy as monotherapy for a majority of tumors remains a distant goal. Recent work has identified strategies that may allow for more potent "next-generation" DC vaccines. Additionally, multimodality approaches incorporating DC-based immunotherapy may improve clinical outcomes.The Yale journal of biology and medicine 12/2014; 87(4):491-518.
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ABSTRACT: We recognize well the abilities of dendritic cells to activate effector T cell (Teff cell) responses to an array of antigens and think of these cells in this context as pre-eminent antigen-presenting cells, but dendritic cells are also critical to the induction of immunologic tolerance. Herein, we review our knowledge on the different kinds of tolerogenic or regulatory dendritic cells that are present or can be induced in experimental settings and humans, how they operate, and the diseases in which they are effective, from allergic to autoimmune diseases and transplant tolerance. The primary conclusions that arise from these cumulative studies clearly indicate that the agent(s) used to induce the tolerogenic phenotype and the status of the dendritic cell at the time of induction influence not only the phenotype of the dendritic cell, but also that of the regulatory T cell responses that they in turn mobilize. For example, while many, if not most, types of induced regulatory dendritic cells lead CD4(+) naïve or Teff cells to adopt a CD25(+)Foxp3(+) Treg phenotype, exposure of Langerhans cells or dermal dendritic cells to vitamin D leads in one case to the downstream induction of CD25(+)Foxp3(+) regulatory T cell responses, while in the other to Foxp3(-) type 1 regulatory T cells (Tr1) responses. Similarly, exposure of human immature versus semi-mature dendritic cells to IL-10 leads to distinct regulatory T cell outcomes. Thus, it should be possible to shape our dendritic cell immunotherapy approaches for selective induction of different types of T cell tolerance or to simultaneously induce multiple types of regulatory T cell responses. This may prove to be an important option as we target diseases in different anatomic compartments or with divergent pathologies in the clinic. Finally, we provide an overview of the use and potential use of these cells clinically, highlighting their potential as tools in an array of settings.Frontiers in Immunology 01/2014; 5:7.
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ABSTRACT: Regenerative therapies that use allogeneic cells are likely to encounter immunological barriers similar to those that occur with transplantation of solid organs and allogeneic hematopoietic stem cells (HSCs). Decades of experience in clinical transplantation hold valuable lessons for regenerative medicine, offering approaches for developing tolerance-induction treatments relevant to cell therapies. Outside the field of solid-organ and allogeneic HSC transplantation, new strategies are emerging for controlling the immune response, such as methods based on biomaterials or mimicry of antigen-specific peripheral tolerance. Novel biomaterials can alter the behavior of cells in tissue-engineered constructs and can blunt host immune responses to cells and biomaterial scaffolds. Approaches to suppress autoreactive immune cells may also be useful in regenerative medicine. The most innovative solutions will be developed through closer collaboration among stem cell biologists, transplantation immunologists and materials scientists.Nature Biotechnology 08/2014; · 39.08 Impact Factor
Promoting tolerance to proteolipid protein-induced
experimental autoimmune encephalomyelitis
through targeting dendritic cells
Joel N. H. Sterna,1,2, Derin B. Keskina,1, Zenichiro Katoa, Hanspeter Waldnerb,3, Sonja Schallenbergc, Ana Andersonb,
Harald von Boehmerd,e, Karsten Kretschmerc,d,1,2, and Jack L. Stromingera,2
aDepartment of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138;bCenter for Neurologic Disease, Brigham and Women’s Hospital,
Harvard Medical School, Boston, MA 02115;cCenter for Regenerative Therapies Dresden, Dresden Technical University, 01307 Dresden, Germany;
dDepartment of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115; andeDepartment of Pathology,
Harvard Medical School, Boston, MA 02115
Contributed by Jack L. Strominger, August 9, 2010 (sent for review May 20, 2010)
In T cell-mediated autoimmune diseases, self-reactive T cells with
known antigen specificity appear to be particularly promising
targets for antigen-specific induction of tolerance without compro-
mising desired protective host immune responses. Several lines of
evidence suggest that delivery of antigens to antigen-presenting
dendritic cells (DCs) in the steady state (i.e., to immature DCs) may
represent a suitable approach to induce antigen-specific T-cell
tolerance peripherally. Here, we report that anti-DEC205–mediated
delivery ofthe self-peptide proteolipidprotein(PLP)139–151 to DCs
ameliorated clinical symptoms in the PLP-induced SJL model of ex-
perimental autoimmune encephalomyelitis. Splenocytes from trea-
ted mice were anergized to PLP139–151, and IL-17 secretion was
markedly reduced. Moreover, we show directly, using transgenic
CD4+Vβ6+TCR T cells specific for PLP139–151, that, under the con-
ditions of the present experiments, these cells also became anergic.
In addition, evidence for a CD4+T cell-mediated suppressor mech-
anism was obtained.
DEC205|multiple sclerosis|anergy|monophosphoryl lipid A|T cells
demyelination of neuronal axons, and axonal loss in the human
central nervous system (1, 2). Studies of multiple sclerosis are
facilitated by the animal model experimental autoimmune en-
cephalomyelitis (EAE) that recapitulates many aspects of the
human disease (3). Active induction of EAE is accomplished by
stimulation of T cell-mediated immunity to myelin, the insulating
phospholipid layer surrounding the neuronal axons, through im-
munization with myelin proteins or synthetic peptide antigens
derived from myelin and then emulsified in adjuvant (4). This
treatment leads to activation of autoreactive myelin-specific
CD4+T cells that circulate in the periphery of naïve animals.
Activated autoreactive T cells will cross the blood–brain barrier
(5). Within the central nervous system, local and infiltrating an-
tigen-presenting cells, such as dendritic cells (DCs) derived from
microglia, present MHC class II molecule-associated myelin
peptides to infiltrating T cells in the context of costimulation.
Myelin-specific CD4+T cells are reactivated, initiating a cascade
of neuroinflammatory responses that ultimately leads to de-
EAE can also be passively induced by adoptive transfer of pre-
activated myelin-specific T cells (6).
Although T helper 1 (Th1) cells secreting IFN-γ were consid-
recently were shown to exhibit greater pathogenicity, suggesting
that they play a more decisive role in mediating severe tissue
damage (7, 8). However, both Th1 and Th17 cells, generated with
kinetic differences and/or involved at different stages, may be
involved in development of EAE (9). In fact, the relative contri-
bution of both Th subsets was recently suggested to affect the
ultiple sclerosis is a T cell-mediated autoimmune disease
characterized by immune cell infiltration, inflammatory
anatomical location of lesion distribution between brain and
spinal cord parenchyma (10).
found in T cell-mediated autoimmune diseases such as multiple
sclerosis, appear particularly promising targets forantigen-specific
tolerance induction without compromising host immunity to in-
unwanted immunity using peptide-induced tolerance (11), in-
cluding the administration of antigens over extended periods of
time viaosmotic minipumps (12, 13).In addition, peptide antigens
can also be directly delivered to antigen-presenting cells via tar-
geting approaches. In particular, antigens delivered to different
subsets of DCs after fusion with antibodies to the endocytic
receptors DEC205 (αDEC205) or 33D1 are efficiently processed
and presented by MHC class I and class II molecules (14). This
route of antigen delivery to murine (15) or human (16) DCs is
several orders of magnitude more efficient than free peptides and
in conjunction with maturation stimuli represents an effective
method for inducing strong T-cell responses, i.e., vaccination. By
different mechanisms in different studies (15, 17–20). It may lead
to deletion of antigen-specific T cells with residual cells becoming
immunologically unresponsive, a mechanism that in one study in-
creased CD5 expression on activated T cells (17). In addition,
delivering minute amounts of peptides via αDEC205 fusion pro-
teins to steady-state immature DCs can lead to the de novo gen-
eration of antigen-specific Foxp3+Treg in vivo (18, 21).
an encephalogenic peptide of the myelin oligodendrocyte glyco-
protein (MOG), a minor myelin component, to DCs in vivo pre-
vents EAE induction by subsequent injection of the same peptide
in complete Freund’s adjuvant (CFA) in C57BL/6 mice (17). In
this model, pretreatment with large doses of the free peptide in
the absence of adjuvants also leads to protection from subsequent
challenge. Here, we report experiments with αDEC205-mediated
targeting of the autoantigen of the proteolipid protein peptide
(PLP139–151) (derived from a major myelin constituent) in the
Author contributions: H.W., H.v.B., K.K., and J.L.S. designed research; J.N.H.S., D.B.K., Z.K.,
and S.S. performed research; H.W. and A.A. contributed new reagents/analytic tools; and
J.N.H.S. and J.L.S. wrote the paper.
The authors declare no conflict of interest.
1J.N.H.S., D.B.K., and K.K. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: firstname.lastname@example.org, karsten.
email@example.com, or firstname.lastname@example.org.
3Present address: Department of Microbiology and Immunology, Pennsylvania State Uni-
versity College of Medicine, Hershey, PA 17033.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| October 5, 2010
| vol. 107
| no. 40www.pnas.org/cgi/doi/10.1073/pnas.1010263107
in which free peptide administration does not lead to protection.
in the steady state with nanogram amounts of a peptide that
generates autoimmunity efficiently ameliorates disease by pro-
moting tolerance. In the present case, the amelioration of disease
results both from induction of T-cell anergy and by generation of
suppressor T cells.
Dendritic Cell Targeting of Proteolipid Protein-Derived Peptide via
αDEC205 Fusion Antibodies. To target the encephalogenic antigen
to DCs, recombinant proteins consisting of amino acids 139–151
of proteolipid protein (PLP139–151) fused either to the C ter-
minus of the Ig heavy chain of cloned αDEC205 (αDEC205/PLP)
or to the GL117 isotype control antibody (GL117/PLP) were
produced. To confirm that the antigenic peptide delivered by the
αDEC205 fusion antibody was properly processed and presented,
purified splenic CD11c+DCs from SJL mice were incubated
for 3 h with various concentrations of either αDEC205/PLP or
GL117/PLP control antibodies. After unbound antibodies were
transgenic mice (22, 23).3H-thymidine incorporation at day 4 of
the culture demonstrated that DCs preincubated with αDEC205/
PLP fusion antibody induced vigorous proliferation of these
transgenic T cells compared with GL117/PLP isotype control
antibody or in the absence of a specific antigen (Fig. S1A).
In addition, a PLP139–151-specific T-cell line was established
by immunizing SJL mice with PLP139–151 and restimulating
times at 2-wk intervals in vitro. The CD4+T-cell line obtained
exhibited an activated surface marker phenotype (CD25+,
CD69+, CD45+, CD30+, GITR+, CTLA4+, CD71low, or
CD62Llow) and secreted high amounts of IL-17 (10,300 pg/mL)
pg/mL), and TNF-α (278 pg/mL). Coculture of the PLP139–151
a dose-dependent manner (Fig. S1B). In contrast, preincubation
of DCs with either GL117/PLP isotype control antibody or
αDEC205 antibody fused to an irrelevant antigen [peptide 107–
119 of hemagglutinin (HA), αDEC205/HA] induced little pro-
liferation. Proliferation was accompanied by an ∼10-fold in-
crease in IFN-γ secretion only after treatment with αDEC205/
PLP (Fig. S1C).
Immunization or Preimmunization with αDEC205/PLP Ameliorates EAE
Induced by Either Adoptive Transfer of a PLP139–151-Specific T-Cell
Line or by Immunization with PLP139–151. The PLP139–151-specific
splenic T-cell line discussed above was adoptively transferred
into naïve SJL mice to passively induce EAE, and then the mice
PLP fusion antibodies, equivalent to ∼20 ng of PLP139–151. All
recipients were injected with pertussis toxin (PT) the following
day. As expected, mice that received PLP139–151-specific T cells
and the GL117/PLP isotype control antibody rapidly developed
severe EAE with a maximal mean score of 4 on day 28 of this
experiment (Fig. 1). In contrast, mice that received PLP139–151-
specific T cells followed by αDEC205/PLP exhibited a sub-
stantially delayed onset of disease with a low maximal mean score
of 1 on day 28. Thus, αDEC205-mediated targeting of nanogram
amounts of PLP139–151 efficiently interfered with the passive
induction of EAE by adoptive transfer of highly encephalitogenic
T cells with the same antigen specificity.
To determine whether preimmunization of SJL mice with
αDEC205/PLP also ameliorated disease induced in mice immu-
nized with unconjugated PLP139–151, SJL mice were either left
untreated or treated with a single injection of 1 μg of αDEC205/
PLP or GL117/PLP isotype control antibody at day minus 10 or
151 in CFA followed by 200 ng PT (PLP139–151/CFA/PT) the
next day, and mice were monitored daily for 30 d for clinical signs
of EAE (Fig. 2 A–C). When EAE was induced in naïve SJL mice
(i.e., without pretreatment), all of the mice developed clinical
symptoms between days 9 and 10 and rapidly progressed to severe
and B). Similarly, pretreatment with the GL117/PLP control
resulted in severe EAE with scores of 3.6–4.4 on days 16–18.
Deaths of 40–60% of the mice occurred in these experiments.
Thus, pretreatment with GL117/PLP control antibody did not re-
sult in an amelioration of diseaseprogression andseverity and was
comparable to non-pretreated mice. Other control antibodies,
αDEC205 itself (NLDC-145), and recombinant αDEC205/
HA107-119 also had no significant effect on the disease course.
By contrast, mice pretreated with 1 μg αDEC205/PLP showed
consistently delayed onset of disease by up to 5 d, with maximal
scores of 1.4–1.7 on days 16–23 (Fig. 2 A and C) (only two
mortalities were observed). This reduction was seen when
αDEC205/PLP was administered 10 or 15 d before induction of
EAE (Fig. 2 A and B) but in one experiment appeared less ef-
fective when administered at day 20. Thus, the treatment pre-
vented disease when administered 23 d before disease onset in
controls. However, administration of αDEC205/PLP at the same
time as immunization with PLP139–151/CFA/PT did not in-
terfere with onset or severity of EAE, possibly due to the rapid
conversion of immature to mature DCs by immunization.
Moreover, coadministration at day 10 of 1 μg αDEC205/PLP with
10 μg monophosphoryl lipid A (MPLA), a low-toxicity derivative
of LPS with potent proinflammatory activity that leads to DC
maturation and activation (24), completely abrogated the bene-
ficial effect of αDEC205/PLP alone on PLP139–151/CFA/PT-
induced EAE (Fig. 2C).
Effect of αDEC205-Mediated Targeting on Pathogenic IL-17–Producing
TCells.To determine whether αDEC205/PLP-mediated targeting
interfered with early antigen-specific T-cell induction, SJL mice
were either left untreated or treated with a single injection of
1 μg of αDEC205/PLP or GL117/PLP control mAb 10 d before
immunization with PLP139–151/CFA/PT. Total splenocytes that
contained both antigen-presenting cells and T cells isolated from
mice at day 17, either without pretreatment or pretreated with
GL117/PLP control antibody, proliferated vigorously to various
PLP139–151 concentrations in vitro, whereas little proliferation
was seen after pretreatment with αDEC205/PLP even in re-
sponse to nonphysiologically high peptide concentrations (Fig.
3A). Thus, αDEC205 targeting in vivo reduced either the num-
pathogenic PLP139–151-specific T cells. PLP139–151-specific T-cell lines were
generated as described in Materials and Methods and 5 × 106cells were
adoptively transferred into naïve SJL/J mice i.v. into the tail veins. One day
later, mice were immunized i.p. with either 1 μg of αDEC205/PLP mAb (n = 5)
orGL117/PLP mAb(n= 5),andthenPT(200 ng) was injected i.v. onday 3.Mice
were monitored for30d.αDEC205/PLP-treated micewere protected,whereas
the GL117/PLP-treated mice developed severe disease (P < 0.02 at 30 d).
αDEC205/PLP ameliorates EAE induced by adoptive transfer of
Stern et al.PNAS
| October 5, 2010
| vol. 107
| no. 40
bers of antigen-specific T cells or their proliferative capacity
tested in vitro.
To address this question in more detail, the number of path-
ogenic IL-17–secreting cells in splenocytes from SJL mice that
were either left untreated or pretreated with a single injection of
1 μg of recombinant αDEC205/PLP, GL117/PLP control, or ir-
relevant αDEC205/HA fusion mAb followed by PLP139–151/
CFA/PT immunization 10 d later, was determined. ELISPOT
analysis at day 17 using total splenocytes and overnight restim-
ulation with varying concentrations of PLP139–151 in vitro
showed that αDEC205/PLP resulted in an ∼2- to 3-fold reduc-
tion in the number of cells secreting IL-17 compared with mice
that were not pretreated (P < 0.02) or were pretreated with
αDEC205/HA (P < 0.03) (Fig. 3 B and C). Pretreatment with
GL117/PLP seemed to increase the number of IL-17 secreting
cells in the spleen (P < 0.004).
CD4+T Cells from αDEC205/PLP-Pretreated Mice Control EAE Induc-
tion After Adoptive Transfer. Did αDEC205/PLP-mediated tar-
geting also result in induction of regulatory T cells (Treg)? To
address the question, SJL mice were either untreated or pre-
treated with either 1 μg αDEC205/PLP or GL117/PLP (Fig. 4A).
In one of the experiments, as a positive control, an additional
group of SJL mice was coimmunized with 500 μg of the synthetic
shown to ameliorate PLP139–151-induced EAE by the genera-
tion of IL-10–secreting Tr1-like Tregs (25, 26). Disease was in-
duced 10 d later by PLP139–151/CFA/PT administration. After
an additional 10 d, splenic CD4+T cells from all four groups were
purified using magnetic beads, and 5 × 106cells were i.v. trans-
ferred into naïve SJL mice. EAE was induced in recipients the
following day by PLP139–151/CFA/PT immunization. Recipients
adoptively transferred with 5 × 106CD4+T cells from mice
without pretreatment or pretreated with GL117/PLP developed
severe EAE with mean maximum scores of 3.2–3.6 on days 16–18
(Fig. 4). As expected, adoptive transfer of CD4+T cells from poly
(F,Y,A,K)n pretreated mice efficiently prevented EAE induction
in recipient SJL mice. Similarly, CD4+T cells from αDEC205/
PLP-treated mice also significantly ameliorated EAE with a mean
maximum score of 2.0 on days 16–18 (P = 0.003 compared with
the control groups). Strikingly, symptoms ameliorated in the
treated groups (but not in the untreated groups) so that, from day
23 onward, basically no signs of EAE were detectable (Fig. 4).
Thus,thegenerationofregulatoryCD4+Tcells alsoplayeda role
in amelioration of EAE after administration of αDEC205/PLP.
Effects of αDEC205/PLP on Pathogenic Vβ6+TCR Transgenic T Cells.
Splenocytes and lymph node cells from Vβ6+TCR CD4+T cells
recognizing PLP139–151 obtained from 5B6 transgenic B10.S
mice (22, 23) were adoptively transferred into rag−/−B10.S(I-As)
mice. Mice were treated with 1 μg of either αDEC205/PLP or
GL117/PLP. Splenocytes and lymph nodes were harvested 10 d
later, and CD4+T cells were separated using anti-CD4 magnetic
beads. Cells from the mice that had been injected with αDEC205/
PLP exhibited limited proliferation and reduced IL-17 production
but unchanged IFN-γ production in response to in vitro restim-
ulation, in comparison with PLP139–151-specific CD4+T cells
from GL117/PLP-treated recipients (P < 0.006) (Fig. 5 A–C).
Thus, αDEC205/PLP targeting in vivo contributed to ameliora-
tion of EAE by reducing the number of antigen-specific patho-
genic IL-17–producing T cells and their proliferative capacity in
vitro. In addition, Foxp3+cells in the CD4+T cell populations
were enumerated by FACS (Fig. 5 D). The percentage of Foxp3+
cells among CD4+cells in αDEC205/PLP and GL117/PLP pre-
treated mice was 15% in each case under these conditions. Anti-
DEC205/PLP did not result in detectable conversion of CD4+
Foxp3−T cells to Foxp3+cells. The percentage of these cells in
normalB10.S micethat havebeenshown toexpress ahigh levelof
CD4+CD25+Tregs (27) averaged 6.1%, and in B10.S mice
bearing the Vβ6 TCR transgene, it averaged 8.3%. Thus, ho-
background likely accounts for the increased numbers found in
both αDEC205/PLP- and αGL117/PLP-treated mice. A smaller
specific conversion to Foxp3+CD4+T cells induced by αDEC205/
PLP treatment (18) would not have been detected. CD5 was found
to be expressed in a previous study of αDEC205/MOG35–55 treat-
expressed on the isolated anergized Vβ6+CD4+T cells from
αDEC205/PLP-treated mice shown here.
(A and B) EAE was induced in SJL/J mice by immunizing with PLP 139–151
with or without preadministration of 1 μg of fusion mAbs (αDEC205/PLP
mAb, αDEC205/HA mAb, αDEC205 mAb, or GL117/PLP mAb) in sterile PBS on
either (A) day minus 10 or (B) day minus 15. Mice were then immunized with
75 μg of PLP139–151 in CFA s.c. on day 0 followed by PT (200 ng) i.v. on day 1.
Appearance of clinical signs of EAE was monitored daily, and disease severity
was scored as described in Materials and Methods. Mean EAE scores for 5–10
mice in each group are shown. The majority of mice in groups immunized
with PLP139–151 that had received αDEC205 mAb, αDEC205/HA mAb, or
GL117/PLP mAb were dead by day 12 whereas disease was ameliorated in
those that received αDEC205/PLP. (A) On day 15, αDEC205/PLP mAb (n = 5) vs.
GL117/PLP mAb (n = 5) (P < 0.01). (B) On day 15, αDEC205/PLP mAb (n =
13) vs. GL117/PLP mAb (n = 9) (P < 0.001). All scoring was performed double
blind. The data shown are representative of three to six separate experi-
ments. (C) Effect of preimmunization with fusion mAbs together with MPLA.
MPLA (10 μg) was administered together with either αDEC205/PLP mAb (n =
8) or GL117/PLP (n = 5) i.p. 10 d before induction of EAE with PLP139–151 in
CFA s.c. and PT (200 ng) i.v. as above. Mice that received MPLA + αDEC205/
PLP mAb were not significantly different from controls (P > 0.05). A repre-
sentative of two independent experiments is shown. All scoring was per-
formed double blind.
Effect of preadministration of αDEC205/PLP on disease course.
| www.pnas.org/cgi/doi/10.1073/pnas.1010263107Stern et al.
Lack or loss of tolerance to several self-molecules that have been
identified as target antigens in autoimmune diseases is one of
the key events promoting autoimmunity such as multiple scle-
rosis or type I diabetes. Despite many studies in both rodents and
humans to stimulate tolerogenic mechanisms using various pro-
tocols of antigen administration with antigens in different
pharmaceutical forms (e.g., peptides or whole antigens) and
testing diverse administration routes, robust data demonstrating
clinical benefits are not yet available (18). Recent studies in mice
have also indicated that repeated administration of free antigens
can induce fatal autoimmune responses (30). In this context, the
ability to target minute amounts of antigens to steady-state im-
mature DCs in vivo has promise as an approach to obtain antigen-
specific immunological tolerance.
In earlier studies of immunological tolerance induced by tar-
different mechanisms have been reported. In earlier studies using
an artificial system in which HA was the target antigen, the in-
duction of immunological unresponsiveness by deletion of autor-
(18, 21). In the only previous study using a known autoantigen,
MOG35–55-induced EAE in C57BL/6 mice was ameliorated by
pretreatment at day minus 7 with αDEC205/MOG35–55 (17). In
the present experiment, αDEC205/PLP139–151 fusion mAb was
is induced by PLP139–151 in SJL mice. Anti-DEC205–mediated
targeting of low nanogram amounts of the immunodominant
PLP139–151 efficiently ameliorated EAE induced either by im-
munization with PLP139–151 or by adoptive transfer of PLP139–
151-specific T cells (Fig. 2). It is important to note that, in the
PLP139–151-induced EAE model in SJL mice, pretreatment with
to protection from disease induced by subsequent challenge with
peptide/CFA/PT, in contrast to the MOG35–55-induced EAE
model in C57BL/6 mice (17). Thus, the fact that αDEC205 tar-
geting is several magnitudes more efficient in inducing T-cell
responses compared with free peptide administration does not
explain the tolerogenic effect of small amounts of αDEC205/PLP
fusion antibodies in the PLP-induced EAE model.
In an attemptto define the mechanism of PLP139–151-induced
tolerance, we showed that αDEC205-mediated targeting in-
terfered with early antigen-specific T-cell induction in peripheral
numbers of pathogenic antigen-specific IL-17–producing T cells
(Fig.3).Inaddition, andconsistent withprevious reports (15), the
remaining cells exhibited an anergic phenotype upon restim-
fusion antibodies. After 10 d, mice were immunized with PLP139–151 followed by i.v. PT as described in Fig. 1. Seventeen days after disease induction,
splenocytes were removed and challenged with a titration of PLP139–151. On day 4 of the proliferation assay, cells were pulsed with3[H]-thymidine; 16 h
later, proliferative response was measured as cpm. (B) IL-17 ELISPOT analysis of mouse splenocytes isolated on day 17. Splenocytes were plated onto precoated
plates as described in protocols from eBioscience’s IL-17 ELISPOT kit and stimulated with 10 μg/mL PLP139–151. Unstimulated wells were used as controls. A
representative of two independent experiments is shown. (C) Quantification of IL-17 ELISPOT. Statistics: none vs. αDEC205/PLP mAb (P < 0.02); αDEC205/PLP
mAb vs. GL117/PLP mAb (P < 0.006). Spots per million were calculated by multiplying the average of triplicate wells (2 × 105cells) by 5-fold.
Effect of αDEC205/PLP on splenocyte proliferation and number of IL-17–producing cells. (A) SJL/J mice were preimmunized with 1 μg of different
mAb preimmunized mice ameliorates induction of PLP139–151-induced EAE.
Two independent experiments are presented (A and B). (A) The 5 × 106CD4 +
T cells enriched from splenocytes from SJL mice preimmunized on day 10 i.p.
with a 1 μg fusion mAbs (αDEC205 /PLP mAb or GL117/PLP mAb) were adop-
75 μg of PLP139–151 in CFA and PTi.v.the following day. Controls received PBS
injections. Mean disease scores of five mice/group are shown. At days 20–21,
additionalidentical experiment isshowninwhichSJL micewerepreimmunized
at day 10 i.p. with 1 μg of αDEC205/PLP mAb only. Mice were monitored for
clinical signs of EAE for 30 d. All scoring was performed double blind.
Adoptive transfer (ATx) of CD4+T cells from anti-DEC205/PLP139–151
Stern et al.PNAS
| October 5, 2010
| vol. 107
| no. 40
ulation in vitro. It is likely that both deletion and induction of an
anergicphenotype inpathogenicTcells contributedto αDEC205/
PLP-mediated amelioration of EAE.
In addition, however, adoptively transferred CD4+Tcells from
αDEC205/PLP-treated mice efficiently prevented EAE induction
in recipients (Fig. 4 A and B). These data point toward an addi-
tional dominant T-cell suppressive mechanism of immunological
However, this experiment does not make clear to what extent de
novo generation or expansion of preexisting Foxp3−expressing
CD4+Tregs or IL-10 secreting T cells, or conversion of patho-
to disease amelioration.
To approach the latter possibility, pathogenic CD4+Vβ6+T
cells were adoptively transferred to B10.S rag−/−mice. After
treatment with αDEC205/PLP, splenocytes or lymph node cells
were markedly anergic to PLP139–151 and had severely reduced
IL-17 production but little or no change in IFNγ secretion. This
experiment may reinforce the relative importance of IL-17 in the
pathogenesis of EAE in this model system (31). A high level of
Foxp3+CD4+Vβ6+T cells was seen after treatment with control
GL117 mAb, and no further increase was found after treatment
with αDEC205/PLP. Thus, no evidence of specific conversion
couldbedetected undertheconditions ofthepresent experiment.
These experiments demonstrate that αDEC205/PLP139–151
151-specific T cells. In addition, evidence of T-cell suppression
was obtained, although induction of neither IL-10 secretion
norFoxp3+T cells was seen.In a previous study (17), MOG35–55
induced EAE was ameliorated by αDEC205/MOG35-55. In ad-
dition to these two autoantigens, MBP85–99 has also been shown
to induce EAE, and all have been shown to be potentially im-
portant in multiple sclerosis (32, 33). Conceivably, a combination
of these three αDEC205 fusion proteins could represent a thera-
peutic modality for this disease.
Materials and Methods
Mice. Six- to 12-wk-old female SJL/J (H-2s) mice were purchased from the
Jackson Laboratory. Vβ6+PLP139–151-specific 5B6 TCR transgenic mice on
the rag−/−B10.S (B10/I-As) background along with nontransgenic rag−/−
B10.S mice were previously described (22). All animals were maintained at
the animal facilities of Harvard University according to the animal protocol
guidelines of Harvard University.
Recombinant Fusion Antibody Production. Double-stranded DNA fragments
coding for PLP139–151 with spacer residues on both sides were constructed
using synthetic oligonucleotides as described previously (34) using the fol-
lowing oligonucleotides: PLP-1 forward, 5′-cta gcg aca tgg cca aga agg aga
cag tct gga ggc tcg agg agt tcg gta ggt tca caa aca ggC AT; PLP-1 reverse, 5′-
CAG GC Tat gcc tgt ttg tga acc tac cga act cct cga gcc tcc aga ctg tct cct tct tgg
cca tgt cg; PLP-2 forward, 5′- AGC CTG GGC AAA TGG CTG GGC CAT CCG GAT
AAATTTtattatgac ggtaggacatgataggc; PLP-2reverse,5′-ggccgcctatcatgt
cct accgtcata ata AAA TTTATC CGG ATGGCC CAG CCA TTT GCC(the PLP139–
151 peptide-encoding nucleotide sequence split between the two sets of
oligonucleotides is shown in uppercase letters). DNA fragments were added
in-frame to the C terminus of the heavy chains of cloned NLDC-145 (αDEC205)
targeting the rat IgG2a, constant regions of the original NLDC-145 and iso-
type control antibodies were replaced with mouse IgG1 constant regions,
which carry point mutations interfering with Fc receptor binding (35). The
plasmid vectors of the IgH chain cDNA of the cloned NLDC-145 (pDEC-IgH)
and GL117 (GL117/10-IgH) and their respective IgL-k light chain cDNA (pDEC-
IgL-k and pGL117/10-IgL-k) were kindly provided by M. C. Nussenzweig (The
Rockefeller University, New York, NY). The plasmid vectors containing the
cDNA of amino acids 107–119 of HA (HA107-119) added to the C terminus of
cloned αDEC205 and III/10 control antibody have been described previously
(18). Hybrid antibodies were produced using the FreeStyle MAX 293 expres-
sion system (Invitrogen) according to the manufacturer’s recommendations.
In brief, suspension cultures of FreeStyle 293-F cells were maintained in
serum-free FreeStyle 293 expression medium and transiently transfected
with plasmid vectors of the respective IgH chain and Igk chain cDNA using
FreeStyle MAX reagent. The original anti-DEC205 antibody NLDC-145 (with-
out peptide tag), which was included in some experiments as a control, was
produced by hybridoma cells in serum-free Hybridoma medium (Invitrogen).
All antibodies were purified on prepacked HiTrapTM Protein G HP columns
(Amersham Biosciences). Protein concentrations were determined specto-
presence of full-length recombinant fusion protein were verified by SDS/
PAGE with an IgG1/IgLκ antibody as a reference.
Effect of Fusion Antibodies on the Induction of EAE. For preimmunization, SJL/J
mice were immunized with 1 μg i.p. of fusion antibodies (αDEC205/PLP mAb,
αDEC205/HA mAb, αDEC205 mAb alone, or GL117/PLP mAb) either 10 or 15
transgenic (tg) T cells. Splenocytes were isolated from B10.S mice that carry
a transgenic TCR 5B6 recognizing PLP139–151 presented on I-As. Splenocytes
were enriched for Vβ6+CD4+tg T cells using Miltenyi CD4-positive selection
kits (purity ∼89%). (A and B) The 10 × 106T cells were injected i.v. into naïve
B10.S rag−/−mice along with 1 μg i.p. of fusion antibodies (αDEC205/PLP
mAb or GL117/PLP mAb). Splenocytes (A) and axillary lymph nodes (B) were
removed 10 d later. Single cell suspensions were stimulated with PLP139–151
for 4 d, and3H-thymidine incorporation was measured. The αDEC205/PLP
mAb-treated Vβ6+CD4+tg T cells did not proliferate in response to PLP139–
151 peptide, whereas Vβ6+CD4+tg T cells treated with GL117/PLP mAb
proliferated (P < 0.03). (C) Vβ6+TCR 5B6 tg CD4+T cells were stimulated by
cross-linking using plate-bound CD3 and CD28 mAb coated overnight to
detect cytokine production. Supernatants from the proliferation assay were
removed 3 d after stimulation, and cytokines were measured by Luminex
assay as described in Materials and Methods. IL-17 was significantly reduced
upon administration of 1 μg of αDEC205/PLP mAb compared with a control
group treated with 1 μg of GL117/PLP mAb (P < 0.005). (D) Splenocytes used
were obtained in A. FACS analysis of gated CD4+cells stained for in-
tracellular Foxp3 was carried out using CD4-FITC and Foxp3-PE.
Effect of αDEC205/PLP on adoptively transferred CD4+Vβ6+TCR 5B6
| www.pnas.org/cgi/doi/10.1073/pnas.1010263107 Stern et al.
d before inducing EAE. Six- to 10-wk-old female mice were immunized s.c.
with 75 μg of PLP139–151 emulsified in CFA; 200 ng PT (List Biological
Laboratories) was given i.v. on the day after immunization. The mice were
monitored for clinical signs of EAE, and they were scored from 0 to 5: 1, limp
tail; 2, hind limb paralysis; 3, complete hind limb paralysis; 4, four limbs
paralyzed; 5, moribund. All scoring was performed double blind.
Details of the proliferation assay, cytokine measurements, and adoptive
transfer experiments are included in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank M. Nussenzweig (The Rockefeller Univer-
sity, New York) for providing the plasmid vectors of the IgH and respective
Igκ light chain cDNA of cloned anti-DEC205 NLDC-145 and III/10 isotype
control recombinant antibodies and T. Koenig (Kretschmer Laboratory) for
excellent technical assistance in recombinant antibody production. This work
was supported by grants from the National Institute of Allergy and Infec-
tious Diseases (AI049524) and from the National Multiple Sclerosis Society
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| October 5, 2010
| vol. 107
| no. 40