Immune Modulation by Silencing IL-12 Production in
Dendritic Cells Using Small Interfering RNA1,2
Jonathan A. Hill,3*†Thomas E. Ichim,3‡§¶Kornel P. Kusznieruk,‡Mu Li,‡§?Xuyan Huang,§?
Xiaotao Yan,‡§Robert Zhong,†‡§¶?Ewa Cairns,*†¶David A. Bell,*†¶and Wei-Ping Min4§¶#
RNA interference is a mechanism of posttranscriptional gene silencing that functions in most eukaryotic cells, including human
and mouse. Specific gene silencing is mediated by short strands of duplex RNA of ?21 nt in length (termed small interfering RNA
or siRNA) that target the cognate mRNA sequence for degradation. We demonstrate here that RNAi can be used for immune
modulation by targeting dendritic cell (DC) gene expression. Transfection of DC with siRNA specific for the IL-12 p35 gene
resulted in potent suppression of gene expression and blockade of bioactive IL-12 p70 production without affecting unrelated genes
or cellular viability. Inhibition of IL-12 was associated with increased IL-10 production, which endowed the DC with the ability
to stimulate production of Th2 cytokines from allogenic T cells in vitro. Furthermore, siRNA-silenced DC lacking IL-12 produc-
tion were poor allostimulators in MLR. IL-12-silenced and KLH-pulsed DC polarized the immune response toward a Th2 cytokine
profile in an Ag-specific manner. These data are the first to demonstrate that RNA interference is a potent and specific tool for
modulating DC-mediated immune responses. The Journal of Immunology, 2003, 171: 691–696.
can evoke many cellular reactions, including the nonspecific inhi-
bition of protein synthesis seen in the IFN response of mammalian
cells (4). It has recently been discovered that short sequences of
RNA that are 21 nt in length (known as small interfering RNA or
siRNA5) can bypass the broad suppression of the IFN response and
can lead to the specific degradation of cognate mRNA (5, 6). This
process, known as RNA interference (RNAi), is immensely spe-
cific, since a single substitution in the 21-nt sequence can abrogate
its effects, and extremely efficient, since the siRNA is incorporated
into an enzymatic complex that conducts multiple rounds of target
mRNA degradation (7). As such, RNAi provides a powerful tool
ost-transcriptional gene silencing is a mechanism that
functions to inhibit viral replication in many eukaryotic
organisms (1–3). This process is mediated by dsRNA and
for inhibiting endogenous gene expression and could provide a
means to effectively modulate immune responses.
Dendritic cells (DC) serve as a junction between the innate and
adaptive immune system (8). Being the most potent APC, the DC
is endowed with the unique ability to stimulate and polarize naive
T cells to either Th1 or Th2 phenotypes (9). In addition, the DC
plays a critical role in the maintenance of self-tolerance by cur-
tailing T cell responses directly or indirectly through the genera-
tion of T regulatory cells (10–12). The difference between DC
subsets that stimulate and those that suppress immune responses
seems to reside in the expression of costimulatory molecules and
cytokines (13, 14). Generally, expression of IL-12 seems to stim-
ulate Th1 activation (15), whereas production of IL-10 by DC
stimulates Th2 activation (16) and in some cases regulatory T cell
generation (17, 18). Understanding this duality in function has led
to DC-based immunotherapies, which have been used to potentiate
T cell responses (in the case of cancer vaccines) or diminish them
(in autoimmune disorders and transplantation) (19–21). A poten-
tial barrier to DC immunotherapy is the plasticity of these cells in
an in vivo environment. Therefore, the ability to generate DC with
a specific phenotype and function would be advantageous. It is in
this context that RNAi would be a powerful tool for modulating
DC-initiated immune responses. However, to our knowledge, the
utility of RNAi for immune modulation has never been reported.
We describe here the induction of RNAi in DC using siRNA
specific for IL-12 p35 (siRNA-IL12p35). We show that bioactive
IL-12 p70 production in bone marrow-derived DC is inhibited af-
ter stimulation with LPS plus TNF-? and is accompanied by an
increase in IL-10 production. Moreover, when siRNA-IL12p35-
treated DC were cultured with allogenic T cells, a Th2 polarization
was observed since T cell expression of IFN-? was reduced while
IL-4 was increased. Inhibiting IL-12 production using siRNA-
IL12p35 was associated with suppressed DC allostimulatory func-
tion. In vivo, initiation of Ag-specific Th2 responses was observed
when DC treated with siRNA-IL12p35 were pulsed with keyhole
limpet hemocyanin (KLH) and used for immunization experi-
ments. These results provide the first evidence that RNAi can be
*Department of Medicine, Division of Rheumatology, Departments of†Microbiology
and Immunology and‡Surgery, University of Western Ontario, London, Ontario,
Canada;§MultiOrgan Transplant Program, London Health Science Center,¶Immu-
nology and Transplantation, Lawson Health Research Institute, London, Ontario,
Canada;?John P. Robarts Research Institute, London, Ontario, Canada; and#Tolero-
Tech Inc., London, Ontario, Canada
Received for publication December 2, 2002. Accepted for publication May 6, 2003.
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 study was supported by grants from the MultiOrgan Transplant Program at
London Health Sciences Center and an Internal Research Fund from the Lawson
Health Research Institute. J.A.H. was supported by a K. M. Hunter/Canadian Insti-
tutes for Health Research doctoral research award. E.C. was supported by the Calder
2This study was partially presented at the 66th Annual Scientific Meeting of the
American College of Rheumatology, October 25–29, 2002, New Orleans, Louisiana;
the abstract was published in Ref. 9. Part of this work has won the Junior Faculty
Travel Award (W.-P.M.) by American Association of Immunologists 2003.
3J.A.H. and T.E.I. contributed equally to this work.
4Address correspondence and reprint requests to Dr. Wei-Ping Min, 339 Windermere
Road, LHSC-UC 9L9, London, Ontario, N6A 5A5, Canada. E-mail address:
5Abbreviations used in this paper: siRNA, small interference RNA; DC, dendritic
cells; RNAi, RNA interference; KLH, keyhole limpet hemocyanin.
The Journal of Immunology
Copyright © 2003 by The American Association of Immunologists, Inc.0022-1767/03/$02.00
induced in DC and that siRNA is a potent tool for modulating DC
function and subsequently T cell polarization.
Materials and Methods
Generation of bone marrow-derived DC
DC were generated from bone marrow progenitor cells as previously de-
scribed (22). Briefly, bone marrow cells were flushed from the femurs and
tibias of C57BL/6 mice (The Jackson Laboratory, Bar Harbor ME),
washed, and cultured in 24-well plates (2 ? 106cells/ml) in 2 ml of com-
plete medium (RPMI 1640 supplemented with 2 mM L-glutamine, 100
U/ml penicillin, 100 ?g of streptomycin, 50 ?M 2-ME, and 10% FCS (all
from Life Technologies, Ontario, Canada) supplemented with recombinant
GM-CSF (10 ng/ml; PeproTech, Rocky Hill, NJ) and recombinant mouse
IL-4 (10 ng/ml; PeproTech). All cultures were incubated at 37°C in 5%
humidified CO2. Nonadherent granulocytes were removed after 48 h of
culture and fresh medium was added. After 7 days of culture, ?90% of the
cells expressed characteristic DC-specific markers as determined by FACS.
DC were washed and plated in 24-well plates at a concentration of 2 ? 105
cells/well in 400 ?l of serum-free RPMI 1640.
siRNA synthesis and transfection
siRNA sequences were selected according to the method of Elbashir et al.
(23). The siRNA sequences specific for IL-12p35 (AACCUGCUGAAG
GAUGGUGAC), IL-12p40 (AAGAUG ACAUCACCUGGACCU), and
IFN-? (AACTGGCAAAAGGATGGTGAC) were synthesized and an-
nealed by the manufacturer (Dharmacon, Lafayette, CO). siRNA for IFN-?
was used as a control since bone marrow-derived DC generated by the
conditions described above did not produce IFN-? after stimulation. Trans-
fection efficiencies were determined using unlabeled and fluorescein-la-
beled siRNA luciferase GL2 duplex (Dharmacon). Transfection was con-
ducted as described previously (23). Briefly, 3 ?l of 20 ?M annealed
siRNA were incubated with 3 ?l of GenePorter (Gene Therapy Systems,
San Diego, CA) in a volume of 100 ?l of RPMI 1640 (serum free) at room
temperature for 30 min. This was then added to 400 ?l of DC cell culture
as described above. Mock controls were transfected with 3 ?l of Gene-
Porter alone. After 4 h of incubation, an equal volume of RPMI 1640
supplemented with 20% FCS was added to the cells. Twenty-four to 48 h
later, transfected DC were washed and used for subsequent experiments.
In the transfection by phagocytosis, bone marrow DC progenitors at day
4 of culture were incubated in a final concentration of 60 pM FL-siRNA-
Luc. Cells remained in culture with GM-CSF and IL-4 as described above.
At day 8 of culture, cells were activated with LPS/TNF-? and incorporated
FL-siRNA-Luc was assessed by flow cytometry on day 9.
DC activation and MLR
Transfected DC (1 ? 106cells) were plated in 24-well plates and stimu-
lated with LPS (10 ng/ml; Sigma-Aldrich, St. Louis, MO) plus TNF-? (10
ng/ml; PeproTech) for 48 h, at which point supernatants were used for
ELISA and RNA was extracted from the cells for RT-PCR. For MLR, T
cells were purified from BALB/c splenocytes using nylon wool columns
and were used as responders (1 ? 106/well). siRNA-treated DC (5–40 ?
103from C57BL/6 mice) were used as stimulators. Seventy-two-hour MLR
was performed and the cells were pulsed with 1 ?Ci [3H]thymidine for the
last 18 h. The cultures were harvested onto glass fiber filters (Wallac,
Turku, Finland). Radioactivity was counted using a Wallac 1450 Microbeta
liquid scintillation counter and the data were analyzed with UltraTerm 3
software (Microsoft, Seattle, WA).
Phenotypic analysis of siRNA-treated DC was performed on a FACScan
(BD Biosciences, San Jose, CA) and analyzed using CellQuest software
(BD Biosciences). The following FITC-conjugated anti-mouse mAbs were
used: anti-I-Ab, anti-CD11c, anti-CD40, and anti-CD86 (BD PharMingen,
San Diego, CA). The annexin V-propidium iodide method of determining
apoptosis/necrosis was used as previously described (22). All flow cyto-
metric analyses were performed using appropriate isotype controls (Cedar-
lane Laboratories, Hornby Ontario, Canada).
Total RNA from siRNA-treated DC (106cells) or from T cells purified
from MLR (106cells) was isolated by TRIzol reagent (Life Technologies,
Grand Island, NY) according to the manufacturer’s instructions. First-
strand cDNA was synthesized using a RNA PCR kit (Life Technologies)
with the supplied oligo(dT)16 primer. One micromole of reverse transcrip-
tion reaction product was used for the subsequent PCR. The primers used
for IL-12p35 and IL-12p40 flanked the sequences targeted by siRNA (IL-
12p35, forward primer 5?-GCCAGGTGTCTTAGCCAGTC-3?, reverse
primer 5?-GCTCCCTCTTGTTGTGGAAG-3?; IL-12p40, forward primer
GCAGGTGTACTGG-3?). In addition, IL-10, IFN-?, IL-4, and GAPDH
(internal control) primers were used as previously described (24). The PCR
conditions were: 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min, and
PCR was done for 35 cycles. PCR products were visualized with ethidium
bromide on 1.5% agarose gel.
siRNA-treated DC (105, C57BL/6 origin) were cultured with the allogenic
T cells (1 ? 106) for 48 h. The supernatants were harvested and assessed
for DC cytokines (IL-12p70, IL-10) and T cell cytokines (IFN-?, IL-4) by
ELISA. Cytokine-specific ELISA (Endogen, Rockford, IL) was used for
detecting cytokine concentrations in culture supernatants according to the
manufacturer’s instructions using a Benchmark Microplate Reader (Bio-
Rad, Hercules, CA).
Immunization of mice with peptide-pulsed DC
Day 7 bone marrow-derived DC were transfected with siRNA-IL12p35, or
transfection reagent alone as described above, and pulsed with 10 ?g/ml
KLH (Sigma-Aldrich) for 24 h. DC were then activated with LPS plus
TNF-? for 24 h, washed extensively, and used for subsequent transfer
experiments. Ag-pulsed DC (5 ? 105cells/mouse) were injected s.c. into
syngeneic mice. Mice were sacrificed after 10 days and cell suspensions
were prepared from the draining lymph nodes. These cells were cultured in
96-well plates at a concentration of 4 ? 105cells/well in the presence or
absence of Ag for 48 h, at which point culture supernatants were used for
analyzing cytokine production by ELISA.
One-way ANOVA followed by the Newman-Keuls test was used to de-
termine the significance between groups for cytokine production and MLR.
Differences with p ? 0.05 were considered to be significant.
DC are efficiently transfected with siRNA
To establish a protocol for RNAi in DC, we first assessed the
siRNA transfection efficacy. Although many studies have shown a
limited ability of DC to be transfected with DNA, RNA transfec-
tion has proven to be efficient. To determine the transfection effi-
cacy, we synthesized fluorescein-labeled siRNA specific for lucif-
erase (FL-siRNA-Luc), a gene that does not exist in mammalian
cells and thus does not affect cellular function. siRNA lacking
fluorescein (siRNA-Luc) was used as a nonlabeled control. FL-
siRNA-Luc and siRNA-Luc were transfected by GenePorter into
bone marrow-derived and cultured DC. After 24-h siRNA trans-
fection, the percentages of DC that had incorporated FL-siRNA-
Luc were quantified by flow cytometry. As seen in Fig. 1, FL-
row-derived DC (1 ? 106) were transfected with unlabeled control siRNA
(Ctrl-siRNA, left) or fluorescein-labeled siRNA specific for luciferase GL2
duplex (Fl-siRNA, middle) at 60 pM concentration. Alternatively, Fl-
siRNA was added to day 4-cultured DC without transfection reagents
(Phagocytosis, right). DC were activated with LPS/TNF-? on day 8 and the
transfection efficacy was assessed by flow cytometry on day 9. Data are
representative of three independent experiments. Ctrl, Control; Fl, fluores-
Efficacy of siRNA transfection into DC. Day 7 bone mar-
692 IMMUNE MODULATION BY siRNA
siRNA-Luc had been successfully incorporated into 88% of the
cells, as analyzed by flow cytometry.
It is known that immature DC are highly phagocytic and can
internalize a variety of molecules, including nucleic acids. Thus,
we asked whether immature DC are able to internalize naked
siRNA. To assess this, immature DC on day 4 were cultured with
FL-siRNA-Luc in the absence of transfection reagent and assessed
for siRNA internalization by flow cytometry on day 9 of culture.
Despite the long incubation period, 19% of DC still contained
incorporated siRNA (Fig. 1), suggesting that naked siRNA may be
used for transfection of DC.
siRNA transfection does not alter DC viability, maturation or
One of the major concerns for gene transfection is that transfection
reagents may affect cellular function or viability. Although we
have demonstrated a high level of transfection efficiency using the
GenePorter method, we needed to establish whether siRNA or the
transfection procedure itself altered the viability of the DC. Thus,
day 7 bone marrow-derived DC were treated with transfection re-
agent (GenePorter) alone, siRNA-IL12p35 alone, or the combina-
tion of transfection reagent and siRNA-IL12p35. After 24 h of
transfection, apoptosis and necrosis were assessed using annexin V
and propidium iodine staining, respectively. Compared with un-
treated DC, neither the transfection protocol alone nor the siRNA
affected cell viability (Fig. 2).
Next, we addressed whether the siRNA or the transfection pro-
cedure affected DC maturation. DC were transfected with siRNA
following activation with LPS and TNF-?. DC maturation was
assessed by flow cytometry to analyze expression of MHC II,
CD40, and CD86 or the DC-specific marker CD11c. It can be seen
that neither treatment with siRNA nor mock transfection altered
DC maturation in response to LPS and TNF-? (Fig. 3A).
An additional concern associated with transfecting DC with nu-
cleic acids is induction of maturation. Since long dsRNA (poly(I):
poly(C)) has previously been shown to induce maturation and ac-
tivation of immature DC (25), we wished to determine whether or
not siRNA had the same effect. Thus, immature DC were treated
with siRNA-IL12p35 for 24 h and cell surface maturation markers
were assessed by FACS. Fig. 3B illustrates that siRNA treatment
alone failed to up-regulate MHC II, CD40, or CD86 on immature
DC. Although these experiments used a concentration of 60 pM
siRNA-IL12p35, higher concentrations of siRNA-IL12p35 (up to
10-fold) were also assessed, with no alteration in viability or dif-
ferentiation (data not shown). These data indicate that transfection
of DC with siRNA-IL12p35 affects neither the viability nor
siRNA induces specific gene silencing in DC
The gene specificity of siRNA targeting of immune-associated
molecules in T cells has been previously demonstrated (26). How-
ever, since DC exhibit distinct cellular features from T cells, we
first chose to confirm the specificity of siRNA-induced gene si-
lencing in DC. The specificity of siRNA gene inhibition was in-
vestigated by transfecting DC with siRNA-IL12p35 and siRNA
cultured from bone marrow progenitors as indicated in Materials and
Methods. Day 7 (1 ? 106) immature DC were left untreated or were trans-
fected with GenePorter alone, siRNA-IL12p35 alone, or the combination
of both for 48 h. Percentage apoptosis and necrosis was assessed using
annexin V and propidium iodine, respectively, by flow cytometry. Data are
representative of three independent experiments.
DC viability is not affected by siRNA transfection. DC were
induces DC maturation. A, Immature DC (1 ? 106)
were cultured alone (untransfected), pretreated for
24 h with GenePorter (mock transfected), or trans-
fected with 60 pM siRNA-IL12p35. The transfected
DC were subsequently activated for 24 h with 10
ng/ml LPS and 10 ng/ml TNF-?. Maturation was
assessed by expression of CD11c, MHC II, CD40,
and CD86 by flow cytometry using FITC-conju-
gated Abs (solid line) and isotype controls (dashed
line). B, Immature DC (1 ? 106) were untreated (un-
transfected), treated with GenePorter alone (mock
transfected), or transfected with 60 pM siRNA-
IL12p35 for 24 h, at which time maturation was as-
sessed by expression of CD11c, MHC II, CD40, and
CD86 by flow cytometry using FITC-conjugated
Abs (solid line) and isotype controls (dashed line).
Data are representative of three independently per-
siRNA transfection neither alters nor
693 The Journal of Immunology
targeted to the p40 component of IL-12 (siRNA-IL12p40). Tran-
scripts of IL-12 p35 and IL-12 p40 were detected by RT-PCR
using primers flanking the siRNA-targeted sequence. Specific in-
hibition was demonstrated at the transcript level: siRNA-IL12p35
exclusively suppressed p35 transcripts while siRNA-IL12p40 sup-
pressed only p40 transcripts (Fig. 4). In addition, both siRNA-
IL12p35 and siRNA-IL12p40 failed to affect transcripts of the
housekeeping gene GAPDH. These data suggested that siRNA-
mediated gene silencing is specific in DC.
siRNA-IL12p35 inhibits IL-12 expression in DC
We next wished to verify whether siRNA-IL12p35 can block pro-
duction of IL-12 protein. Since IL-12p35 is critical for the forma-
tion of the IL-12 p70 heterodimer, we assessed production of this
cytokine in the supernatant of LPS/TNF-?-activated DC using
ELISA. DC transfected with siRNA-IL12p35 were stimulated with
LPS plus TNF-? for 48 h to induce maturation and cytokine ex-
pression. To confirm specificity of gene silencing, siRNA specific
for IFN-? (siRNA-control) was used since this cytokine is not
expressed in bone marrow-derived DC. Additionally, negative
controls included DC transfected with GenePorter alone (mock-
transfected DC) and unmanipulated DC (untreated control). As
shown in Fig. 5A, siRNA-IL12p35 reduced IL-12p70 heterodimer
production (as determined by ELISA) by 85–90% compared with
untreated or mock-transfected DC. More important, this effect was
specific since no significant difference in IL-12p70 production was
seen in DC treated with the IFN-? siRNA-control. In addition, we
tested for levels of IL-10 production since a reciprocal relationship
with IL-12 production has been previously reported (27). IL-10
production in DC treated with siRNA-IL12p35 was significantly
and specifically up-regulated compared with controls (Fig. 5B).
siRNA-IL12p35 suppresses DC allostimulatory activity
DC function can be characterized in part by their ability to stim-
ulate alloreactive T cells in the MLR (8). To determine whether
siRNA-IL12p35 affected DC allostimulatory activity, MLR was
performed using DC transfected with siRNA-IL12p35, siRNA-
control, mock transfected, or untreated controls. Allogenic T cells
were cultured with siRNA-transfected DC for 48 h, at which point
allostimulation was determined by proliferation. Although the con-
trol DC groups all showed similar allostimulatory activity, DC
transfected with siRNA-IL12p35 significantly suppressed this re-
sponse (Fig. 6).
siRNA-IL12p35-treated DC promote Th2 differentiation
Since IL-12p70 is a key cytokine responsible for polarizing T cells
toward an IFN-?-producing or Th1 phenotype (28), we assessed
whether allostimulation with DC that were transfected with
siRNA-IL12p35 could alter cytokine production from responding
ability. C57BL/6-derived DC (1 ? 106) were untreated (untransfected, E),
transfected with GenePorter alone (mock transfected, ?), transfected with
60 pM siRNA-IFN-? (Control siRNA, ‚), or transfected with 60 pM
siRNA-IL12p35 (F) for 24 h. Allogenic (BALB/c) T cells (2 ? 105/well)
were incubated with siRNA-treated DC at the indicated numbers for 72 h.
Proliferation was determined using [3H]thymidine incorporation. Data are
representative of three independent experiments (?, p ? 0.01, by one-way
ANOVA and Newman-Keuls test).
siRNA-IL12p35 transfection inhibits DC allostimulatory
transfected with 60 pM siRNA-IL12p35, siRNA-IL12p40, or Geneporter
alone (mock transfected). The transfected DC were activated with 10 ng/ml
LPS and 10 ng/ml TNF-? for 24 h. RNA from the treated DC was extracted
using the TRIzol method. RT-PCR was performed to assess expression of
IL-12p35, IL-12p40, and GAPDH using primers described in Materials
and Methods. Data are representative of three independent experiments.
Specificity of gene inhibition by siRNA. DC (1 ? 106) were
IL-10. DC (1 ? 106) were unmanipulated (control), transfected with Gene-
porter alone (mock transfected), transfected with 60 pM siRNA-IL12p35,
or 60 pM siRNA-IFN-? (siRNA control). The transfected DC were acti-
vated with 10 ng/ml LPS and 10 ng/ml TNF-? for 24 h. A, Supernatants
were harvested from cultures and analyzed for IL12p70 production using
ELISA. B, Supernatants were harvested from cultures and analyzed for
IL-10 production using ELISA. Data represent mean ? SD and are rep-
resentative of three experiments (?, p ? 0.01, by one-way ANOVA and
siRNA-IL12p35 specifically blocks IL-12 and up-regulates
694IMMUNE MODULATION BY siRNA
T cells. Mock-transfected DC stimulated high IFN-? and low IL-4
mRNA transcripts from responding T cells; however, stimulation
with siRNA-IL12p35-treated DC resulted in low IFN-? and high
IL-4 transcripts (Fig. 7A). To confirm these results at the protein
level, we assayed for IFN-? and IL-4 from MLR culture superna-
tants using ELISA. The T cells incubated with siRNA-IL12p35-
treated DC produced low levels of IFN-? (Fig. 7B) and high levels
of IL-4 (Fig. 7C). In contrast, T cells incubated with untransfected
DC, GenePorter-transfected DC, or DC transfected with control
siRNA showed a cytokine profile of high IFN-? and low IL-4.
These data suggest that siRNA-IL12p35-treated DC have the abil-
ity to polarize naive T cells along the Th2 pathway.
Modulation of Ag-specific response in vivo using
Although a shift from Th1 cytokine production to Th2 is seen
when naive T cells are incubated with siRNA-IL12p35-treated DC,
we wondered whether this effect could also be obtained in vivo. To
accomplish this, we pulsed siRNA-IL12p35-treated or mock-trans-
fected DC with KLH and used these modified DC as immunogens
in vivo by injecting into syngeneic hosts. Ten days after immuni-
zation with KLH-pulsed control DC, a Th1 recall response was
evident when draining lymph node cells from recipient mice were
challenged with KLH in vitro, as determined by up-regulated
IFN-? and down-regulated IL-4 production (Fig. 8). Under the
same conditions, the siRNA-IL12p35-treated DC promoted a Th2
shift in the recall cytokine response, showing increased IL-4 pro-
duction and suppressed IFN-?. These results suggest that Ag-
pulsed and siRNA-modified DC can be used to modulate the Th1
vs Th2 balance in vivo during a primary immune response.
We have demonstrated that RNAi can be successfully used for
immune modulation of DC by targeting expression of the Th1-
polarizing cytokine IL-12. Using a siRNA sequence specific for
the p35 subunit of IL-12, we have successfully blocked the ex-
pression of bioactive IL-12p70. Suppression was noted at the tran-
script and protein level and endowed the DC with higher IL-10
production, inhibited DC allostimulatory activity but allowed pref-
erential Th2 differentiation in vitro and in vivo. This study is, to
our knowledge, the first to report that RNAi can be successfully
used for the purpose of DC immune modulation.
Although many techniques have been used to block specific
molecules in vitro and in vivo, such as antisense oligonucleotides
(29) and mAbs (30), RNAi provides several distinct advantages.
First, mRNA degradation by siRNA is extremely efficient as only
a few copies of dsRNA are necessary to activate the RNA-induced
silencing complex (31). Once the RNA-induced silencing complex
is activated. it can conduct multiple rounds of gene-specific mRNA
cleavage. Second, RNAi is specific, in that only sequences with
identity to one of the strands of dsRNA will be cleaved (1). Third,
the RNAi effect is long lasting and can be spread to progeny cells
after replication, although a dilution effect is evident in mamma-
lian cells (32). Finally, this technique is relatively simple, giving
rise to an in vitro knock-down phenotype within days that can be
confirmed with many Ab-based detection systems (such as ELISA
or Western blotting), or if an Ab is not available, by RT-PCR or
functional assays. RNAi can also be used to study genes of un-
known function since siRNA sequences from predicted exon re-
gions should be effective in inducing a knock-down phenotype.
Thus, RNAi may help to elucidate much of the genomic data that
is presently being accumulated.
inhibit Th1 responses in vivo. Day 7 bone marrow-derived DC cultured in
GM-CSF and IL-4 were transfected with IL12p35-siRNA or mock trans-
fected. Subsequently, cells were pulsed with 10 ?g/ml KLH for 24 h and
injected s.c. (5 ? 105cells/mouse) into syngeneic C57BL/6 mice. After 10
days, T cells from lymph nodes were isolated from recipient mice. A KLH-
specific recall response was performed as described in Materials and Meth-
ods. IFN-? and IL-4 responses to KLH were assessed by ELISA. Data
shown are pooled from three independent experiments.
siRNA-IL12p35-treated DC stimulate Ag-specific Th2 and
C57BL/6 bone marrow-derived DC were pretreated with GenePorter alone
(mock transfected) or transfected with 60 pM siRNA-IL12p35 for 24 h. Sub-
sequently, siRNA-treated DC (106) were cultured with allogenic (BALB/c) T
cells (10 ? 106) for 48 h. T cells were purified from coculture using a T cell
column and RT-PCR was performed for IL-4, IFN-?, and GAPDH. B,
C57BL/6 bone marrow-derived DC were unmanipulated (control), pretreated
with GenePorter alone (mock transfected), or transfected with 60 pM siRNA-
IL12p35 or 60 pM siRNA-IFN-? (siRNA control) for 24 h. siRNA-treated DC
(106) were subsequently cultured with allogenic (BALB/c) T cells (10 ? 106)
for 48 h. Supernatants were collected from the cultures and IFN-? (Th1 cy-
tokine) and IL-4 (Th2 cytokine) production was assessed by ELISA (?, p ?
0.01, by one-way ANOVA and Newman-Keuls test).
siRNA-IL12p35-transfected DC promote Th2 polarization. A,
695 The Journal of Immunology
Interestingly, DC silenced by siRNA-IL12p35 showed de- Download full-text
creased allostimulatory capacity in this study. This is in contrast to
results reported using DC generated from IL-12 knockout mice
that possess normal allostimulatory activity (33, 34). We attribute
this discrepancy to compensatory immunological mechanisms that
may have arisen in the lifetime of the IL-12 knockout mice. This
is suggested by studies that have demonstrated the importance of
IL-12 in MLR. First, IL-12 production by APC was demonstrated
to be critical for MLR proliferative response since addition of anti-
IL-12 Abs resulted in suppression of proliferation (35). Second,
overexpression of IL-12 in DC results in increased allostimulatory
function (36). Another possible explanation for suppressed MLR
in siRNA-IL12p35-transfected DC is that the increased IL-10 pro-
duction may act as an inhibitor of T cell proliferation (37, 38).
Other studies examining naturally occurring Th2-promoting DC
have shown that these cells have a reduced allostimulatory func-
tion and reduced IL-12 production (39, 40). The combination of
Th2-promoting properties as well as poor allostimulation suggests
that siRNA-IL12p35-transfected DC may possess the phenotype of
a “tolerogenic” DC and thus may be useful for treatment of Th1-
mediated autoimmune diseases and transplant rejection.
Manipulating immune response at the level of DC is attractive
since the DC plays a critical role in both the initiation and sup-
pression of immune responses. We and others have used DC-mod-
ifying approaches for induction of tolerance or treatment of auto-
immunity (22, 41). Although antisense manipulation of DC has
previously been reported (42), low transfection efficacy of DNA
into DC presents a problem for widespread use of this approach
(43). In contrast, transfection of DC with siRNA is quite efficient and
therefore may offer the possibility of treating immune-based diseases
in a specific and effective manner. The successful utility of Ag-pulsed
siRNA-modified DC to shift the Ag-specific immune response from
Th1 to Th2 (Fig. 8) suggests that this approach could be used for
Ag-specific immune modulation. Future work will focus on these av-
enues of in vivo immune modulation and may expand the armamen-
tarium of strategies available for immunotherapy.
We thank Dr. Gill Strejan (University of Western Ontario, London, On-
tario, Canada) for helpful advice and review of the manuscript and Dionne
White for technical assistance.
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696 IMMUNE MODULATION BY siRNA