Antisense Oligonucleotides Down-Regulating Costimulation
Confer Diabetes-Preventive Properties to Nonobese Diabetic
Mouse Dendritic Cells1
Jennifer Machen,* Jo Harnaha,* Robert Lakomy,* Alexis Styche,* Massimo Trucco,*†and
Phenotypically “immature” dendritic cells (DCs), defined by low cell surface CD40, CD80, and CD86 can elicit host immune
suppression in allotransplantation and autoimmunity. Herein, we report the most direct means of achieving phenotypic imma-
turity in NOD bone marrow-derived DCs aiming at preventing diabetes in syngeneic recipients. CD40, CD80, and CD86 cell
surface molecules were specifically down-regulated by treating NOD DCs ex vivo with a mixture of antisense oligonucleotides
targeting the CD40, CD80, and CD86 primary transcripts. The incidence of diabetes was significantly delayed by a single injection
of the engineered NOD DCs into syngeneic recipients. Insulitis was absent in diabetes-free recipients and their splenic T cells
proliferated in response to alloantigen. Engineered DC promoted an increased prevalence of CD4?CD25?T cells in NOD recip-
ients at all ages examined and diabetes-free recipients exhibited significantly greater numbers of CD4?CD25?T cells compared
with untreated NOD mice. In NOD-scid recipients, antisense-treated NOD DC promoted an increased prevalence of these putative
regulatory T cells. Collectively, these data demonstrate that direct interference of cell surface expression of the major costimu-
latory molecules at the transcriptional level confers diabetes protection by promoting, in part, the proliferation and/or survival of
regulatory T cells. This approach is a useful tool by which DC-mediated activation of regulatory T cells can be studied as well as
a potential therapeutic option for type 1 diabetes. The Journal of Immunology, 2004, 173: 4331–4341.
lular level, the ? cells are subjected to cytokine-induced impair-
ment by the actions of infiltrating macrophages and T cells with
subsequent T cell-mediated destruction (1, 2). There is no question
that defects in T cell selection at the central level in the thymus and
impaired peripheral regulation of ? cell Ag-specific T cells under-
lie the etiopathogenesis of T1DM (1, 2). Dendritic cells (DC) have
proven to be integral participants in the initiation and propagation
of T1DM at multiple levels including the regulation of diabetes
onset (3–5, 6–8). DC are the primary APCs of the immune system
and as such, they control the activation of naive T cells (4, 9–11).
For full activation of naive CD4?T lymphocytes to occur, two
signals are required. The first is the presentation of the Ag to the
TCR in the context of class II MHC on DC. This will cause the
responding T cell to up-regulate the CD154 molecule (CD40 li-
gand) to its cell surface, thereby activating the initiation of the
second signal. In this process of coactivation, CD154 will interact
with the CD40 molecule at the surface of the APC resulting in the
ype 1 diabetes mellitus (T1DM),3a disorder of glucose
homeostasis, is the consequence of the autoimmune tar-
geting of pancreatic insulin-producing ? cells. At the cel-
up-regulation of CD80 and CD86 at the cell surface of the APC.
Immediately thereafter, CD80 and CD86, acting as the second sig-
nal, in the process of costimulation, will engage the CD28 mole-
cule on the T cell resulting in its full activation. In the absence of
the interactions between CD80, CD86, and CD28, the T cell will
either enter a state of functional silence, termed anergy, or will be
primed for apoptosis, perhaps in a CD95-CD95L (Fas-FasL)-de-
pendent manner (12–14). Converging lines of evidence indicate
that the phenotype of the DC cell surface can play an important
role in tolerance to self-Ags and can be manipulated to promote
allogeneic as well as autoimmune hyporesponsiveness (11, 15).
The first use of DC to prevent T1DM in NOD mice was docu-
mented by Clare-Salzler et al. (8) who demonstrated that transfer
of pancreatic lymph node DC derived from 8- to 20 wk-old NOD
mice into prediabetic NOD mice conferred significant protection
from T1DM, insulitis, and adoptive transfer of T1DM. The authors
suggested that acquisition of islet Ags by DC during insulitis may
have resulted in them acquiring a phenotype, once in the pancreatic
lymph nodes, that was able to result in the stimulation of regula-
tory immune cells which attenuated the insulitic process. Interest-
ingly, while transfer of DC isolated from nonpancreatic lymph
nodes to NOD mice was unable to affect T1DM incidence, transfer
of these DC pulsed with sonicated islets did confer protection (8).
More recently, Morel and colleagues (16, 17) have shown prolon-
gation of a diabetes-free state in NOD recipients of bone marrow-
derived syngeneic DC. Other methods of generating diabetes-sup-
pressive DC include vitamin D receptor ligands, Ag pulsing, and
IFN-? treatment (18–20).
NOD DC exhibit strong immunostimulatory capacity, underlied
by hyperactivation of NF-?B (21–23). Therefore, we proposed and
very recently showed that inhibition of NF-?B using short, double-
stranded transcriptional decoys could render NOD DC less immu-
nostimulatory and that administration of these engineered DC into
*Diabetes Institute,†Division of Immunogenetics, Department of Pediatrics, and‡De-
partment of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA
Received for publication October 9, 2003. Accepted for publication July 26, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Awards DK61328 and
DK60183 (to N.G.).
2Address correspondence and reprint requests to Dr. Nick Giannoukakis, Diabetes
Institute, University of Pittsburgh School of Medicine, Rangos Research Center, 3460
Fifth Avenue, Pittsburgh, PA 15213. E-mail address: email@example.com
3Abbreviations used in this paper: T1DM, type 1 diabetes mellitus; DC, dendritic
cell; AS-ODN, antisense oligonucleotide.
The Journal of Immunology
Copyright © 2004 by The American Association of Immunologists, Inc.0022-1767/04/$02.00
NOD prediabetic mice could prevent the development of diabetes
(24). Attractive as this approach is, we nevertheless do have con-
cerns that are under investigation that NF-?B blockade may inter-
fere with functions crucial to DCsurvivalinvivothatmayimpacton
the persistence of the immunosuppressive effect of these DC in NOD
mice. Hence, as a second, and complementary approach, we decided
to engineer DC in a way where the expression of only the costimu-
latory molecules CD40, CD80, and CD86 would be suppressed at the
cell surface. We based our rationale on the many studies demonstrat-
ing the effectiveness of CD80/CD86-CD28 blockade in generating
immune hyporesponsiveness to alloantigens and in preventing auto-
our approach limits the cell population that is targeted, because the
treatment is performed ex vivo and does not involve systemic dis-
semination of a protein which, in the instance of CTLA4-Ig and
anti-CD40L have exhibited nonspecific and toxic effects (53, 54).
DCs with a mixture of antisense oligonucleotides (AS-ODN) target-
ing the CD40, CD80, and CD86 transcripts confers specific suppres-
sion of the respective cell surface proteins. We further demonstrate
that a single injection of these engineered DC into syngeneic predi-
abetic female NOD mice significantly delays the incidence of T1DM
without affecting the response of T cells from diabetes-free DC
recipients to alloantigen. Furthermore, there was no evidence of
insulitis in the diabetes-free recipients. In NOD-scid recipients, we
show that ODN-treated NOD DC administration in cotransfer with T
cells promotes an increased prevalence of CD4?CD25?CD62L?T
cells. The use of AS technology specifically targeting the transcripts
of key DC cell surface proteins involved in T cell activation and
regulation could be a useful technique to study DC:T cell interactions
promoting immunoregulatory cell networks and as a potential means
of T1DM cell therapy.
Materials and Methods
Female C57BL/6, NOD/LtJ (H2g7), NOD-scid, and C3H/HeJ (H2k) mice
were purchased from The Jackson Laboratory (Bar Harbor, ME) and
housed under pathogen-free conditions. The ?-actin-GFP transgenic mouse
was bred on the C57BL/6 background and was propagated in our mouse
colony. All animal experimentation was conducted in compliance with the
Animal Research Care Committee of the Children’s Hospital of Pittsburgh.
Abs to immune cells were purchased from BD Biosciences (San Diego,
CA) and were used as the direct FITC, PE, CyChrome, or allophycocyanin
fluorochrome conjugates. The clones used were as follows: CD40 (clone
3/23), CD11c (clone HL3), CD86 (clone GL1), CD80 (clone 16-10A1),
CD4 (clone RM4-5), CD25 (clone 7D4), and CD62L (MEL-14). Anti-
insulin and anti-glucagon Abs were purchased from DakoCytomation
(Carpinteria, CA). Isotype- and species-matched irrelevant monoclonal or
polyclonal Abs (where appropriate) were used as controls. The NIT-1 cell
line was obtained from American Type Culture Collection (CRL-2055;
Manassas, VA) and propagated as described by the repository. Phospho-
rothioate-modified ODNs were synthesized by the University of Pittsburgh
DNA Synthesis Facility and HPLC purified. Cell culture reagents (serum
and media) were purchased from Invitrogen Life Technologies (Gaithers-
burg, MD). Beadlyte multiplex fluorescence cytokine detection kits were
purchased from Upstate Biotechnology (Lake Placid, NY) and fluores-
cence-based proliferation as well as phagocytosis probes from Molecular
Probes (Eugene, OR). Recombinant murine cytokines and immune cell
enrichment columns were purchased from R&D Systems (Indianapolis,
IN). All other biochemicals were purchased from Sigma-Aldrich (St.
DC propagation and treatment with AS-ODN
DC were propagated from bone marrow progenitors of 5- to 8-wk-old
female C57BL/6 or NOD mice in GM-CSF and IL-4 as outlined by Ma et
al. (24) and originally described in Fu et al. (55, 56). Briefly, bone marrow
was obtained from the femurs and tibiae of female NOD mice. The RBC
were lysed using a commercially available reagent (Red Blood Cell Lysing
Buffer; Sigma-Aldrich) and the bone marrow cells were plated in 24-well
multiwell plates at 2 ? 106cells/ml in R-10 medium (RPMI 1640/10%
heat-inactivated FBS/50 ?M 2-ME/1% sodium pyruvate/1% nonessential
amino acids/1% penicillin-streptomycin solution (Invitrogen Life Technol-
ogies) with the addition of 4 ng/ml GM-CSF and 1000 U/ml IL-4 (R&D
Systems). Two days later, the nonadherent cells were removed and a 1:1
volume of conditioned medium, fresh R-10 medium, and cytokines were
added to the adherent cells. Three days later, the loosely adherent cells
were gently agitated and harvested. By FACS analysis, ?85% of these
cells are routinely DC with class II MHC, CD11c, CD80, CD40, and CD86
positivity (57). The AS-ODN mixture consisted of phosphorothioate-mod-
ified ODNs each targeting the 5? end of the CD40, CD80, and CD86 pri-
mary transcripts. The sequences are: CD40 AS-ODN, 5?-CAC AGC CGA
GGC AAA GAC ACC ATG CAG GGC A-3?; CD80 AS-ODN, 5?-GGG
AAA GCC AGG AAT CTA GAG CCA ATG GA-3?; CD86 AS-ODN,
5?-TGG GTG CTT CCG TAA GTT CTG GAA CAC GTC-3?. The AS-
ODN were HPLC purified and resuspended in PBS. DC were treated
18–24 h in 10% heat-inactivated FBS/RPMI 1640 with a mixture of 3.3
?M CD40 AS-ODN, 3.3 ?M CD80 AS-ODN, and 3.3 ?M CD86 AS-
ODN (the mixture is collectively referred to as AS-ODN in this study). The
cells were then washed extensively in PBS and subsequently used in cul-
ture or in vivo. Ag uptake and processing capacity were assessed using the
Vybrant Phagocytosis Assay reagent as described by the manufacturer
(Molecular Probes). Uptake of fluorescent bioparticles was measured in a
fluorescence microplate reader at 480 nm excitation/520 nm emission (Vic-
tor2; PerkinElmer Instruments, Boston, MA).
Flow cytometry analysis
All the FACS analyses were performed in a FACSVantage SE flow cy-
tometer with FACSDiva version 2.2.1, capable of eight-color, multiparam-
eter discrimination (BD Biosciences). In every FACS analysis, cells were
stained with propidium iodide to exclude dead cells. The initial cell pop-
ulations were selected based on forward and side scatter properties specific
for DC or T cells. By forward and side scatter, we excluded debris and
clumped cells and by propidium iodide we excluded dead cells from all
analyses. The initial gate was set around the remaining cells. In T cell
populations, wherever we aimed at discriminating putative T regulatory
cells, we gated CD4?cells and analyzed this population for CD25 and
CD62L positivity. Results were recorded as dot plots in QuadStat analyses.
In vitro phenotype of DC treated with AS-ODN and Ag loading
AS-ODN-treated DC were stimulated with 25 ?g/ml LPS (Sigma-Aldrich)
or 50 ng/ml recombinant murine CD40L (R&D Systems) for 18–24 h. The
culture supernatant was collected and assayed for NO production using the
Griess assay and profiled for cytokine secretion using the Beadlyte multi-
plex assay system (Upstate Biotechnology) in a Luminex Fluorescence
Analyser (Luminex, Austin, TX). In parallel, the cells were stained with
fluorescence-conjugated Abs against CD40, CD80, and CD86. T cell pro-
liferation was measured in cocultures of irradiated splenocytes and spleen-
isolated and column-enriched (R&D Systems) T cells from 5- to 8-wk-old,
diabetes-free, diabetic female NOD or T cells from the spleen of C3H/HeJ
females (5–10 wk of age). Proliferation was measured after 5 days in cul-
ture using the CyQuant fluorescence reagent (Molecular Probes). Cytokine
production was measured in the culture supernatants of the cocultures at 5
days using the Beadlyte-Luminex assay. All assays were performed in
triplicate on at least two different occasions.
Examination of DC phenotype following in vivo transfer of
CFSE-labeled or GFP-positive (GFP?) DC into
We first labeled control and AS-ODN-treated NOD DC (from 7-wk-old
female donors) with CFSE (Molecular Probes) as directed by the manu-
facturer. Cells (2 ? 106) were injected i.p. into age-matched female recip-
ients. At weekly intervals, for 3 wk, we harvested the spleens from indi-
vidual recipients and examined the levels of CD80 and CD86 in recovered
CFSE?cells by FACS using Abs for CD80 and CD86 (BD Biosciences).
As a complementary approach, we obtained DC from the bone marrow
progenitors of GFP transgenic mice. In these mice, the GFP transgene is
under the control of the chicken ?-actin promoter. These mice were gen-
erated on a C57BL/6 background. They express GFP in almost all tissues
including monocytes and DC. The DC were treated ex vivo with AS-ODN
or PBS vehicle and 2 ? 106cells were injected i.p. into nontransgenic
C57BL/6 sex-matched recipients. At weekly intervals, for 3 wk, the spleens
of individual recipients were harvested and single cells were stained with
CD80 and CD86 Abs. CD80 and CD86 levels were analyzed by FACS in
DC administration to NOD mice, diabetes monitoring, and
immune profiling of DC recipients
NOD DC (2 ? 106–3 ? 106) (control, AS-ODN-treated, or ? cell Ag/AS-
ODN cotreated) in PBS were injected by i.p. route into 5- to 8-wk-old
female NOD mice. The ? cell Ag was in the form of a lysate obtained from
the NOD-derived NIT-1 insulinoma cell line. Given that multiple autoan-
tigens have been identified for diabetes, we chose to coadminister NIT-1
lysate which in principle should contain all known (and unknown) autoan-
tigens. The NIT-1 cell line is derived from NOD transgenic mice where the
SV40 large T-Ag is expressed from the rat insulin gene promoter. These
transgenic NOD mice display ? cell adenoma and these ? cells have been
immortalized as the NIT-1 line (58). The choice of the NIT-1 cell line over
NOD islets as source of ? cell Ag was also due to the significant logistics
we previously experienced in the isolation of islets (24) that involved hun-
dreds of mice to produce enough lysate to realistically provide Ags at
reasonable levels (100 islets usually isolated per mouse; 1000 islet cells per
islet on average). The cell line was easily grown to very large numbers in
a short time period. Second, the NIT-1 cell line shares almost identical
phenotype with normal ? cells, expresses class I MHC in response to
IFN-?, but does not express class II MHC at the cell surface (58). More
importantly, sera from diabetic NOD mice strongly stained NIT-1 cells, but
no staining was observed when sera from prediabetic or diabetes-resistant
NOD were used (58). Moreover, sonicated NIT-1 membranes injected i.v.
into 5-wk-old NOD mice prevented T1DM (59). Finally, CD8?T cells
from NOD mice were able to recognize and destroy NIT-1 cells in
Diabetes incidence was ascertained twice weekly in tail vein blood by
electronic sampling (One-Touch; LifeScan Technologies, Milpitas, CA).
Confirmation of diabetes was noted upon two consecutive readings of
blood glucose ?280 mg/dL. At various time points, DC recipients were
euthanized, pancreata, lymph nodes, and spleens were isolated. Pancreata
were fixed in 4% paraformaldehyde and embedded in paraffin. Multiple
sections (4 mm) were subsequently stained with anti-insulin and glucagon
Abs (DakoCytomation) followed by secondary probing with biotin-conju-
gated secondary Abs followed by diaminobenzidine chromogen visualiza-
tion. Parallel sections were also stained with H&E. T cells from mesenteric
and inguinal lymph nodes as well as spleen were isolated using column
enrichment (R&D Systems) and used in coculture proliferation/cytokine
profiling experiments with NOD (5–8 wk of age) or C3H/HeJ (5–8 wk of
age) irradiated bone marrow-derived DC as stimulators. T cells from DC-
treated, diabetes-free NOD mice were cultured overnight in the presence of
Con A (5 ?g/ml) and the supernatants were then probed for cytokine se-
cretion profiles using the Beadlyte assay (Upstate Biotechnology) in the
Luminex multiplex fluorescence-based detection system.
NOD DC and T cell cotransfer into NOD-scid recipients
PBS- or AS-ODN-treated NOD DC (1 ? 106–2 ? 106) (from 5- to 8-wk-
old females) were injected i.p. into sex and age-matched NOD-scid recip-
ients. Twenty-four to 48 h later, an equal amount of splenic T cells from 5-
to 8-wk-old female NOD mice was injected i.v. Five days later, the mes-
enteric and popliteal lymph nodes and spleen were harvested. The lymph
nodes were pooled and single cells were isolated (from spleen and pooled
lymph nodes) over a T cell enrichment column (R&D Systems). The cells
were cultured overnight and the supernatant collected for cytokine profiling
using the Beadlyte assay system. In parallel, T cell phenotype was analyzed
by FACS where CD4?-gated cells were reanalyzed for CD25 and CD62L
GraphPad Prism version 4.0 (San Diego, CA) was used to analyze the data
where appropriate. Kaplan-Meier log-rank analysis was used for survival
data and unpaired ANOVA or Student’s t test (where appropriate) were
applied to the data obtained from in vitro studies.
AS-ODN treatment confers a CD80lowCD86lowphenotype to
NOD DC in vitro and in vivo and prevents production of
IL-12p70, TNF-?, and NO
Because NF-?B is an important transcription factor in many sig-
naling pathways, perhaps crucial for DC function not involving T
cell activation, we chose to assess the potential of a less “global”
method of maintaining DC in an immature state characterized by
a phenotype of low cell surface levels of costimulatory molecules
(CD40, CD80, and CD86). We reasoned that, by using specific
short AS-ODN targeting the transcripts for the mouse CD40,
CD80, and CD86, we would be able to mimic the immature state
conferred by the NF-?B ODN directly by interfering with crucial
regulators of T cell activation (CD40, CD80, and CD86). We first
tested the ability of a number of ODN targeting different regions of
the CD40, CD80, and CD86 to inhibit NOD DC cell surface ex-
pression of these proteins in response to LPS stimulation in cul-
ture. Of 27 ODNs each targeting different sequences of the primary
transcript (5? end, exon-intron, 3? end), we selected the ones yield-
ing the greatest suppressive effect on cell surface expression, as
assessed by FACS, for subsequent studies. Of all ODNs, the ones
with the greatest effect were those targeting sequences at the 5? end
(Fig. 1A). Despite the presence of LPS stimulation, cell surface
expression of CD40, CD80, and CD86 were specifically sup-
pressed in DC treated with each of the ODN. Although LPS is a
powerful maturation signal in vitro, the most relevant maturation
signal in vivo would be ligation of CD40 by CD40L. To examine
the effects of CD40L on the phenotype of DC in culture, we added
bioactive recombinant trimeric CD40L (50 ng/ml) to NOD DC
treated with PBS or with the AS-ODN. After a period of 24–36 h,
the supernatants were collected and examined for cytokine profile
and NO production, given that NO production is a feature of ma-
turing DC. The cells were analyzed by FACS for CD80 and CD86
cell surface expression. In Fig. 1, B and C, we demonstrate that
CD40L was able to up-regulate CD80 and CD86 in control DC but
not in AS-ODN-treated DC. Also, we show that NO, TNF-?, and
IL-12p70 production was significantly suppressed in AS-ODN-
treated DC exposed to CD40L compared with untreated DC (Fig.
1C). Although there is no evidence that ODN treatment of DC
impairs their capacity to uptake Ag, we proceeded to formally
examine this possibility using the Vybrant Phagocytosis assay sys-
tem (Molecular Probes) where cell fluorescence depends on the
uptake and processing of exogenously supplied Escherichia coli
bioparticles whose fluorescence is quenched outside the cell due to
trypan blue inclusion in the assay buffer. Fig. 1E demonstrates that
AS-ODN DC fluorescence is identical to that of untreated DC
when the cells are pulsed with the Vybrant bioparticles.
A single injection of AS-ODN-treated NOD DC into prediabetic
NOD mice prolonged the time to diabetes onset
Our previous studies indicated that ODN-engineered DC were ca-
pable of prolonging allograft survival and time to diabetes onset
(24, 61). To extend those studies we wanted to determine whether
AS-ODN DC could prolong the time to diabetes onset. Indeed,
significant prolongation of diabetes onset time was observed in
female NOD mice given a single injection of 2 ? 106AS-ODN-
treated DC (injection at 5–8 wk of age) but not in untreated mice,
untreated-DC recipients, NIT-1 lysate-treated DC, or those admin-
istered DC cotreated with AS-ODN and NIT-1 lysate (Fig. 2A). Up
to 45 wk following the injection, 4 of the original 20 NOD recip-
ients given a single i.p. injection of AS-ODN-treated DC remained
diabetes-free (blood glucose ?200 mg/dL).
4333The Journal of Immunology
Continued on next page
Though diabetes, as measured by glucose levels, was consider-
ably delayed in AS-ODN DC-recipient NOD mice, we wished to
determine whether this was paralleled by prevention or reduced
grade of insulitis—the histological readout of diabetes prevention.
We examined the histology of pancreata of NOD recipients of
AS-ODN-treated DC who had no signs of diabetes at 35 wk of age.
We could find no sign of insulitis in H&E sections (Fig. 2B, left
panel) and we were able to visualize normal insulin and glucagon
content as well (Fig. 2B, middle and right panels). Twenty-two
individual islets were visualized from each one of 47 pancreatic
the presence of 10 ?M ODN (mixture of CD40, CD80, and CD86 ODN, each at 3.3 ?M final concentration in culture). Untreated DC stimulated with 25
?g/ml LPS overnight exhibited robust up-regulation of CD40, CD80, and CD86 (?). AS-ODN pretreatment prevented LPS-triggered up-regulation of
costimulatory molecules at the DC cell surface (f). These data are representative of four independently performed experiments. AS refers to AS treatment
(shown at the top of each panel). The y-axis indicates mean fluorescence intensity of cells gated on forward and side scatter properties of DC and negative
for propidium iodide staining. B, FACS analysis of CD80 and CD86 levels on the cell surface of AS-ODN NOD DC treated with rCD40L. NOD DC
obtained from bone marrow progenitors of 5- to 8-wk-old NOD mice were treated with 10 ?M ODN (AS-ODN) or PBS vehicle as control. rCD40L was
added at a final concentration of 50 ng/ml for a period between 18 and 24 h. CD40L addition was unable to stimulate the cell surface expression of CD80
or CD86 to levels higher than control. The data are representative of three independently performed experiments. C, Graph representation of the data in
B. D, Cytokine production by NOD DC treated with CD40L. Bone marrow-derived NOD DC were treated with control PBS vehicle or AS-ODN DC.
rCD40L was added subsequently at a final concentration of 50 ng/ml for a period of 18–24 h. Multiplex-based cytokine profiling of the culture supernatants
following the CD40L addition demonstrated that AS-ODN-treated DC were refractive to CD40L-stimulated NO production and TNF-? and exhibited
suppressed capacity to produce IL-12p70. These data are representative of three independent experiments. E, Ag uptake and processing capacity of
AS-ODN DC. Bone marrow-derived NOD DC (1 ? 105) were treated with control PBS vehicle or AS-ODN DC and then pulsed with the Vybrant
Phagocytosis reagent. Twenty-four hours later, cell fluorescence was quantitated microfluorometrically. The graph bars denote fluorescence intensity (in
arbitrary units) and the error bars denote the SEM of triplicate determinations.
A, FACS analysis of AS-ODN-treated NOD DC. Bone marrow-derived DC from 8-wk-old female NOD mice were cultured overnight in
4335The Journal of Immunology
sections of two diabetes-free mice with identical results (no insu-
litis). These data indicate that injection of the AS-ODN NOD DC
was able to prevent the infiltration of immune cells that otherwise
impair ? cell function and that promote ? cell destruction, at least
in these mice.
T cells from diabetes-free NOD recipients of AS-ODN DC
responded to alloantigens in culture
Many immunoregulatory protocols induce systemic immunosup-
pression. To determine whether our approach acted at a systemic
level, we asked whether T cells from “protected” NOD mice would
respond to allogeneic stimulation. In Fig. 3A, we demonstrate the
results of T cell proliferation showing that DC from bone marrow
progenitors of allogeneic C3H/HeJ mice were able to stimulate the
proliferation of T cells from the spleen of “protected” NOD mice
to levels identical with proliferation of NOD DC-stimulated C3H/
HeJ spleen-derived T cells. These data indicate that alloreactivity
was maintained in NOD recipients of AS-ODN DC-treated, “pro-
tected” NOD mice and that the diabetes suppression was due to a
more precise, yet-to-be fully understood mechanism.
TH1-type cytokine production was suppressed in splenocytes
obtained from diabetes-free recipients of AS-ODN DC compared
with splenocytes from diabetic NOD mice.
Having ascertained that systemic immunosuppression was not at
the root of our AS-ODN DC effect, we asked whether AS-ODN
DC in vivo shifted the balance of T cell immune responses from
TH1 to TH2. Many studies demonstrate that type 1 diabetes is
characterized by a TH1-type immune response and that a shift to
TH2 is often associated with prevention or prolongation of time-
to-onset (62–64). To determine whether the protection conferred
by the AS-ODN DC was due to the predominance of a TH2-type
immune environment, we examined the cytokine secretion profile
of splenocytes obtained from diabetes-free NOD recipients of the
AS-ODN DC. In Fig. 3B, we show that at 31 wk of age, compared
with a diabetic NOD mouse (22 wk of age), there were lower
levels of TNF-? and IFN-? in the supernatants of Con A-stimu-
lated T cells obtained from the spleen of a “protected” NOD re-
cipient of AS-ODN DC. There were no significant differences in
the levels of all other cytokines when compared with a diabetic
NOD mouse at 22 wk of age.
Persistence of low level B7 levels on AS-ODN DC in vivo
Throughout these experiments, it was unclear whether the AS-
ODN-treated DC maintained the same cell surface levels of CD80
and CD86 in vivo or whether these levels changed following ex-
ogenous administration. Two complementary approaches were
used to address this question. In the first, we examined the levels
of CD80 and CD86 on spleen cells derived from NOD recipients
of CFSE-labeled syngeneic DC. The CFSE?cells obtained from
AS-ODN DC-administered ODN recipients exhibited the same
levels of CD80 and CD86 as did the CFSE?cells from control
DC-administered recipients 1 wk following injection (Fig. 3C). As
a complementary approach, we transferred GFP-transgenic DC
that were treated with PBS or with AS-ODN in culture into syn-
geneic recipients. One week later, we harvested the spleens of the
recipients and examined the levels of CD80 and CD86 in GFP?
populations by FACS. In Fig. 3D, we show that there were no
changes in the cell surface levels of CD80 and CD86 on AS-ODN-
treated GFP?DC indicating that these cells maintained the same
levels of CD80 and CD86 in vivo, as they did before the injection.
These studies were followed up and in Fig. 3, E and F, we dem-
onstrate that AS-ODN-treated DC persist for 3 wk while the num-
ber of CFSE?and GFP?cells recoverable from control DC-
treated mice declined by 3 wk after exhibiting a significant
increase in CD86 levels by 2 wk in vivo. This increase of CD86
was observed only in NOD recipients and not in the C57BL/6
syngeneic recipients of GFP?control DC.
An increased prevalence of CD4?CD25?cells was observed in
the splenocytes of diabetes-free AS-ODN DC NOD recipients
Although preliminary, we proposed that the “protective” nature of
the AS-ODN-modified DC involved the generation and/or survival
of regulatory cell activity in the splenocyte fraction resulting in the
suppression of activity and/or modulation of the viability of the
diabetogenic immune cell populations. A number of investigators
have identified a population of T cells that possess regulatory
activity and can prevent a number of autoimmune disorders (65–67).
The cell surface phenotype that these cells all appear to share is
CD4?, CD25?. Although it is not yet clear whether these are the
specific cells which confer regulation, this population does have
activity (cellular or soluble) which fulfills this function (68, 69,
70–73). Therefore, we wished to compare the prevalence of this cell
subtype in the splenocytes of “protected” NOD mice with that in
untreated and control DC-treated NOD mice from time of adminis-
tration to the time of diabetes onset. By FACS analysis using fluo-
rescently conjugated anti-CD4 and anti-CD25 Abs, we have deter-
mined the profile in Table I. It appears that the protective effect of the
bone marrow progenitors of 6- to 8-wk-old female NOD mice were prop-
agated in GM-CSF/IL-4 and further treated with AS-ODN with or without
NIT-1 lysate as described. PBS-resuspended cells (in 200 ?l; 2 ? 106)
were injected i.p. into syngeneic age- and sex-matched NOD recipients.
Blood glucose was measured by electronic sampling of tail vein blood
beginning at 15 wk of age. AS-ODN DC ? DC treated with a mixture of
CD40, CD80, CD86 AS-ODN, each oligo at 3.3 ?M; AS-ODN ? NIT1
DC ? DC cotreated with the AS-ODN mixture and NIT-1 lysate. p ?
0.012, AS-ODN DC recipients vs AS-ODN ? NIT-1 DC recipients,
Kaplan-Meier log rank test. B, Immunohistochemistry of pancreata from
diabetes-free recipients of AS-ODN DC. For immunohistochemistry, the
sections were fixed in paraffin, treated to block peroxidase, and incubated
with nonfat dried milk. The slides were then incubated with anti-insulin or
anti-glucagon Ab followed by an isotype-reactive biotinylated secondary
Ab. Avidin-HRP was then added followed by diaminobenzidine after
which a brown color could be observed. No evidence of insulitis was ob-
served with insulin and glucagon readily detectable.
A, Diabetes incidence in NOD recipients of DC. DC from
4336 DIABETES-SUPPRESSIVE DCs
AS-ODN injection in NOD mice may be partially due to the gener-
ation/survival/activation of T cells within the CD4?CD25?compart-
ment. Interestingly, control DC also confer some degree of increased
CD4?CD25?cell prevalence, although these numbers are far less
than those obtained in spleen of AS-ODN DC. Furthermore, in the
cohort studied, control DC-treated NOD mice all became diabetic.
Although these data support a DC-based mechanism for
CD4?CD25?T cell expansion, we cannot yet exclude the possibility
that other cell types may be involved in transducing the effects of the
recipients of AS-ODN-treated syngeneic DC; C3H Tc, T cells isolated from spleen of age-matched C3H-HeJ mice; C3H DC, DC obtained from bone
marrow of C3H mice; NODpr Tc, T cells isolated from spleen of NOD recipients of AS-ODN-treated DC. Data are from a representative proliferation assay
with triplicate cocultures. No significant differences between cocultures (Student’s two-tailed t test). B, Cytokine production by T cells from a diabetes-free
NOD recipient of AS-ODN DC. Splenic T cells from 22-wk-old diabetic mice and a 31-wk-old NOD recipient of AS-ODN-treated DC (diabetes-free) were
stimulated with Con A for 18 h. The supernatants were probed using the Multiplex Beadlyte assay system for cytokine profile. Key is at the bottom right
of the figure. Cytokine identities are at the top of each bar pair. C, B7 levels on exogenously administered DC in NOD recipients. CFSE-labeled DC derived
from prediabetic female NOD bone marrow progenitors were recovered 1 wk following i.p. injection (2 ? 106DC originally administered). CD80/CD86
double-positive cells were ascertained in the CFSE?cell population by FACS analysis of labeled splenocytes. Data are representative of two different DC
administrations. D, B7 levels on exogenously administered GFP?DC in C57BL/6 recipients. DC propagated from bone marrow of GFP?transgenic
C57BL/6 mice were recovered 1 wk following i.p. injection (2 ? 106DC originally administered) into C57BL/6 recipients. CD80/CD86 double-positive
cells were ascertained in the GFP?cell population by FACS analysis of labeled splenocytes. Data are representative of two different DC administrations.
E, Persistence of exogenously administered DC in NOD recipients. CFSE-labeled DC derived from prediabetic female NOD bone marrow progenitors were
recovered 1, 2, and 3 wk following i.p. injection of 2 ? 106DC from spleen. CD80- and CD86-positive cells were ascertained in the CFSE?cell populations
by FACS analysis of labeled splenocytes. F, Persistence of exogenously administered GFP?DC in C57BL/6 recipients. DC propagated from bone marrow
of GFP?transgenic C57BL/6 mice were recovered from spleen 1, 2, and 3 wk following i.p. injection of 2 ? 106DC into C57BL/6 recipients. CD80- and
CD86-positive cells were ascertained in the GFP?cell population by FACS analysis of labeled splenocytes.
A, Alloantigen reactivity of T cells from diabetes-free NOD recipients of AS-ODN DC. NODpr DC, DC obtained from bone marrow of NOD
4337 The Journal of Immunology
Adoptive cotransfer of NOD AS-ODN DC and NOD T cells into
NOD-scid recipients results in an increased prevalence of
CD4?CD25?CD62L?T cells in spleen
As a first approach to understanding the potential mechanism(s) by
which the AS-ODN may be acting to prolong the diabetes-free
state and whether the association between AS-ODN DC adminis-
tration and increased numbers of CD4?CD25?T cells was caus-
ally linked, we examined the prevalence of CD25?CD62L?cells
in CD4?T cell populations from spleen of NOD-scid recipients of
control and AS-ODN NOD DC. We first administered 1 ? 106–
2 ? 106DC from 5- to 8-wk-old female NOD mice into age- and
sex-matched NOD-scid recipients i.p. Three days later, we injected
1 ? 107purified splenic T cells from 5- to 7-wk-old female NOD
mice. In Fig. 4, we demonstrate a significant increase in the num-
ber of total splenic CD4?CD25?as well as CD25?CD62L?cells
in the splenic CD4?-enriched cell component of NOD-scid recip-
ients of AS-ODN DC compared with untreated DC 1 wk following
the T cell transfer. Culture supernatant from splenic and lymph
node T cells obtained from all these NOD-scid recipients did not
reveal any detectable levels of IL-4 or IL-10 by Beadlyte cytokine
profiling (data not shown).
By suppressing costimulatory molecule expression at the cell sur-
face using AS-ODN targeting the primary transcripts of CD40,
CD80, and CD86, we have achieved a specific means of engineer-
ing host DCs into potentially “tolerogenic” effectors. Our data are
comparable with the outcomes presented in a recently published
approach targeting the NF-?B transcriptional pathway in DC using
transcriptional decoy ODNs (24). In contrast to the transcriptional
decoy approach, direct targeting of the costimulatory transcripts
aimed at specific down-regulation of the costimulatory proteins
avoids the potential of interfering with NF-?B-sensitive pathways
in DC that may be relevant for survival and in vivo function/
persistence of the exogenously administered DC. Although we
have not exhaustively determined the effect of the AS-ODN treat-
ment on the transcription of every single gene in DC, preliminary
data do not suggest any particular detrimental effects on survival
or promiscuous and nonspecific inhibition of cell function/gene
transcription (data not shown). Most impressively, by a single ad-
ministration of AS-ODN DC, we have conferred diabetes protection
to NOD mice, although not all recipients remained diabetes-free in-
definitely. Interestingly, diabetes incidence was no different in NOD
mice administered DC cotreated with NIT-1 lysate and AS-ODN than
untreated recipients or mice that received untreated DC. The latter
observation is in contrast to data shown by Feili-Hariri et al. (16, 74)
and the reasons for this are currently under investigation.
We have shown herein that the exogenously administered DC
are detectable and viable following i.p. administration for up to 3
wk. Although no changes are evident in the cell surface levels of
CD80 and CD86 of AS-ODN-treated DC across 3 wk following
administration, significant changes especially of CD86, are ob-
served on control DC following exposure to an in vivo autoim-
mune environment (the NOD mouse). In these recipients, control
DC CD86 was observed to be increased at 2 wk following exog-
enous administration and was dramatically reduced along with
CD80 by 3 wk. Its increased level may underlie a time period in
which autoimmune processes could be recapitulated in exogenous
DC NOD mouse transfer models. Additionally, it appears that un-
treated DC become scarce by 3 wk following exogenous admin-
istration, suggesting a time frame of exogenously supplied DC
survival in vivo. In contrast, AS-ODN DC appear to persist at 3 wk
at numbers and with a B7 phenotype similar to that observed at 1
wk following exogenous administration. The maintenance of
nearly identical B7 levels on AS-ODN DC at 1 and 3 wk following
exogenous transfer could also suggest one possible mechanism of
immunoregulation where the persistence of DC with low or absent
B7 in an environment poised for autoreactivity can engage coun-
terreceptors present exclusively on regulatory T cells promoting
their expansion and/or survival. A number of recent studies sup-
port such a potential mechanism (75, 76). Indeed, the ability of
AS-ODN DC to suppress diabetes onset may involve a direct effect
of the AS-ODN DC on the expansion of CD25?CD62L?cells
from CD4?precursors, and/or their enhanced survival. The failure
to observe increases of these same cells in NOD-scid recipients of
control DC as well as the absence of any differences in the prev-
alence and numbers of single CD4?or single CD8?cells between
NOD-scid recipients of control and AS-ODN DC argues against
homeostatic expansion as the basis for the increased prevalence of
the CD4?CD25?CD62L?cells (data not shown).
Despite the prolonged time to diabetes onset in a significant
number of AS-ODN recipients, many NOD mice eventually be-
came diabetic. The most obvious reason for failure of persistence
with a single injection of AS-ODN DC is the limited lifespan of
exogenously administered DC in vivo, a possibility that is sup-
ported by the data presented in Fig. 3, E and F. A number of other
spleen T cells. Three days following i.p. PBS injection, control DC or AS-ODN DC injection (2 ? 106cells), 1 ? 107splenic NOD T cells were injected i.v. One
week later, the recovered splenic T cells were analyzed by FACS for CD4 and CD25 prevalence. The data are representative of prevalence in three different
NOD-scid recipients. The cell population was selected based on forward and side scatter. Also shown are the FACS dot plots of the Ab isotypes and the percentage
of gated cells that are double positive. B, CD25?CD62L?cell prevalence in a CD4?T cell population obtained from spleen of NOD-scid mice reconstituted with
NOD DC and prediabetic NOD spleen T cells. Three days following i.p. PBS injection, control DC or AS-ODN DC injection (2 ? 106cells), 1 ? 107splenic
NOD T cells were injected i.v. One week later, the recovered splenic CD4?T cells (magnetic bead column enriched) were analyzed by FACS for CD25 and
CD62L prevalence. The data are representative of prevalence in three different NOD-scid recipients. The cell population was selected based on forward and side
scatter. Also shown are the FACS dot-plots of the Ab isotypes and the percentage of gated cells that are double positive.
A, CD4?CD25?cell prevalence in a T cell population obtained from spleen of NOD-scid mice reconstituted with NOD DC and prediabetic NOD
Table I. Prevalence/frequency of CD4?CD25?T cells in NOD
splenocytes by FACS analysisa
% CD4?CD25?Cells in Splenocytes (mean
(untreated)Control DCAS-ODN DC
NOD at 4 wk of age
NOD at 12 wk of age
NOD at 20 wk of age
NOD at 35 wk of age
0.78 ? 0.06b
0.75 ? 0.11c0.82 ? 0.04c1.22 ? 0.30d
0.50 ? 0.09e1.19 ? 0.03e4.81 ? 0.16f
4.41 ? 1.40h
aSplenocytes were obtained from groups of control DC or AS-ODN DC-treated
mice and T-cells were obtained using T-cell enrichment columns. CD4?CD25?cells
were ascertained by FACS analysis.
bn ? 5 for control NOD mice (no DC administration).
cn ? 2 for all groups; insulitis evident in control and control DC-treated mice.
dn ? 3.
en ? 2.
fn ? 2.
gAll untreated and control DC-treated mice were diabetic by this age.
hn ? 2; no evidence of insulitis.
4339 The Journal of Immunology
studies indicate that exogenously supplied DC have a lifespan be-
tween 7 and 14 days (77, 78). Assuming that the DC effect is
directly suppressive (i.e., DC:autoreactive T cell interaction), the
exhaustion of the exogenous DC population conferring suppres-
sive activity would explain the lack of persistence. If so, multiple
dosings could theoretically prolong the effect or achieve indefinite
protection. This would also be valid if regulatory T cell expansion
was dependent on DC persistence; recent studies appear to support
this possibility. Especially exciting are the data by Steinman and
colleagues (75, 76) who have just recently shown that DC can directly
induce the expansion of CD4?CD25?T cells in vivo which possess
Ag-specific suppressive capacity. We have data that are similar to
those published by Yamazaki et al. (76) demonstrating that AS-ODN-
treated DC derived from the DO11.10 TCR transgenic mouse pro-
mote suppressed Ag-specific T cell proliferation in vivo and a con-
comitant increase in the prevalence of CD4?CD25?T cells in vitro
that could underlie the in vivo suppression (our unpublished data).
Although these and a number of other similar studies favor the in-
duction of regulatory T cells by DC with immunosuppressive activity,
a role for NK-T or other cell populations cannot be ruled out (79, 80).
Additionally, if the DC effect is indirect, it would be of interest to
determine how immunoregulatory DC induce different regulatory im-
mune cell populations and how they promote the persistence of these
secondary cellular networks.
Our studies show that AS-ODN DC-treated, diabetes-free NOD
mice exhibited a complete absence of insulitis. However, it is pos-
sible that this may not be the case in all diabetes-free recipients of
AS-ODN. A study of a significantly larger population of diabetes-
free mice administered AS-ODN may reveal varying degrees of
insulitis and these infiltrating (or peri-islet) cells will need to be
phenotyped if such observations are indeed made. AS-ODN DC
administration was associated with an increase in CD4?CD25?cell
numbers in the splenocytes of diabetes-free NOD recipients. Further-
more, AS-ODN DC administration to NOD mice was associated with
a progressive increase in the prevalence of CD4?CD25?T cells with
increasing age and was not observed in untreated NOD or control
DC-treated NOD. Our data in NOD-scid mouse recipients of
AS-ODN DC and T cells support the hypothesis that these DC may
directly promote the proliferation/survival/activity of CD4?CD25?
cells with immunoregulatory capacity. We are currently determining
whether CD4?CD25?cells generated in NOD-scid mice by AS-
NOD T cells into secondary NOD-scid recipients. The number of
reports directly implicating these cells in preventing diabetes onset by
regulating immune cell function compel us to further study these spe-
cific cell types and their mechanism of action in well-defined in vivo
studies (16, 17, 74–76, 81–84).
A respectable body of evidence supports the existence of en-
dogenous immunosuppressive DC in vivo and suggests molecular
pathways which can be exogenously manipulated to make their
immunosuppressive activity persistent in vivo (85, 86). A number
of factors, which at this time remain poorly understood or unex-
plored, could influence the effectiveness of the DC in maintaining
such a regulatory DC network along with a regulatory T cell pop-
ulation: 1) the phenotypic nature of the DC; 2) their maturational
status at the time of administration, the route of administration; 3)
the anatomical site of action; 4) the precise number of cells ad-
ministered, and the effects of multiple dosings. Addressing the
mechanisms of immunoregulation by DC as well as the precise
phenotype of the “active” DC and/or regulatory immune cells in
appropriate in vivo models, like the NOD mouse, is important
because a DC-based approach can be potentially translated to the
clinic, for prophylaxis in high-risk individuals or in newly onset
cases of T1DM to save residual ? cell mass.
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4341The Journal of Immunology