original article
A Short Pulse of IL-4 Delivered by DCs
Electroporated With Modified mRNA
Can Both Prevent and Treat Autoimmune
Diabetes in NOD Mice
Rémi J Creusot1, Pearl Chang1, Don G Healey2, Irina Y Tcherepanova2, Charles A Nicolette2
and C Garrison Fathman1
1
Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, California, USA; 2Argos Therapeutics, Durham,
North Carolina, USA
Bone marrow-derived dendritic cells (DCs) are cells of the
immune system that have been used as a tool to boost,
modulate, or dampen immune responses. In the con-
text of autoimmunity, DCs can be modified to express
immuno­regulatory products encoded by transgenes, and
used therapeutically in adoptive cellular therapy. DCs
that were lentivirally transduced (lt) to express ­interleukin
4 (IL-4) can significantly delay or prevent the onset of
autoimmune diabetes in nonobese diabetic (NOD)
mice. However, modifying cells using viral vectors ­carries
the dual risk of oncogenicity or immuno­genicity. This
study demonstrates that NOD DCs, electroporated with
“translationally enhanced” IL-4 mRNA (eDC/IL-4), can
be equally efficient therapeutically, despite the reduced
amount and shorter duration of IL-4 secretion. Moreover,
a single injection of eDC/IL-4 in NOD mice shortly after
the onset of hyperglycemia was able to maintain stable
glycemia for up to several months in a significant ­fraction
of treated mice. Treatment with eDC/IL-4 boosted regu-
latory T (Tregs) cell functions and modulated T helper
responses to reduce pathogenicity. Thus, treatment with
DCs, electroporated with modified IL-4 mRNA to express
IL-4 for up to 24 hours, constitutes a viable cellular ther-
apy approach for the regulation of autoimmune diabetes,
as a preferred alternative to the use of viral vectors.
Received 24 May 2010; accepted 14 June 2010; published online
13 July 2010. doi:10.1038/mt.2010.146
Introduction
Type 1 diabetes (T1D) is a multifactorial autoimmune disease
characterized by the progressive T cell-mediated destruction of β
cells of the pancreatic islets. The nonobese diabetic (NOD) mouse
model of T1D shares many similarities with human T1D and has
been widely used to study the pathogenesis of disease and to test the
efficacy of therapeutic approaches aimed at maintaining or restor-
ing tolerance to and function of β cells.1,2 Among these approaches,
Correspondence: C Garrison Fathman, Division of Immunology and Rheumatology, 269 Campus Drive, CCSR Building Room 2225, Stanford, California
94305-5166, USA. E-mail: cfathman@stanford.edu
2112
© The American Society of Gene & Cell Therapy
dendritic cells (DCs) have received wide scrutiny because of their
inherent ability to regulate immune responses. Depending on their
type and maturation state, DCs can promote immune tolerance as
well as trigger immune responses. Tolerogenic properties of DCs
can be ascribed not only to their state of maturation (immature),
but also to certain molecules expressed on their surface (e.g., pro-
grammed death ligand-1), intracellularly (e.g., indoleamine 2,3-­
dioxygenase) or secreted [e.g., interleukin 10 (IL-10)].3,4 Some
other immunoregulatory products that are known to prevent
or ameliorate autoimmune disease are either not expressed by
DCs, or expressed in insufficient amounts. However, DCs remain
vehicles of choice to express these immunomodulatory products
because of their efficient migration to relevant lymphoid tissues
and interaction with T cells. In mice, the pancreatic lymph nodes
(PLNs) are among the few lymphoid tissues preferentially targeted
by adoptively transferred bone ­marrow-derived DCs, which makes
this strategy particularly relevant for the treatment of T1D.5
IL-4 is a pleiotropic cytokine whose contribution to immune
regulation is well established. One of its most important roles is to
support the differentiation and function of Th2 and B cells, while
antagonizing the development and function of Th1 cells, impli-
cated in numerous autoimmune diseases. IL-4 is not normally
produced by DCs, and viral vectors have so far been the method
of choice to express it ectopically. DCs modified to express IL-4
(DC/IL-4) have shown therapeutic efficacy in NOD mice,6,7 as well
as in collagen-induced arthritis, a mouse model of rheumatoid
arthritis.8,9 The approach of using viral vectors allows relatively
stable expression of the transgene, but carries certain risks and
disadvantages that are inherent to the type of viral vector used
(most typically adenoviral and lentiviral), ranging from immuno-
genicity to oncogenicity. Nonviral approaches for ectopic expres-
sion of immunoregulatory products are not only safer, but should
preserve the integrity of the DC, including its homing and T cell
clustering capacity.
Modification of DCs by mRNA electroporation has been
extensively studied for the treatment of cancer, where newly
introduced mRNAs typically encode tumor antigens and other
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© The American Society of Gene & Cell Therapy
NOD T1D Treatment With mRNA Electroporated DCs

proteins that boost the immunogenicity of the DCs.10–12 However, ltDCs were in contact with lentiviral particles for the final 2 days
this approach has not yet been successfully applied to autoimmune of a 7-day culture, eDCs were electroporated after the final harvest
diseases. Here, we report the use of an IL-4 mRNA optimized for on day 7. Using our extensively optimized conditions, electropo-
IL-4 protein expression to electroporate bone marrow-derived ration was carried out without significant loss of cells or change
DCs and then treat NOD mice before or immediately after the in viability (data not shown). Electroporation had the highest and
onset of hyperglycemia. DCs electroporated with translationally most consistent transfection efficiency (80–85%), whereas lenti-
enhanced IL-4 mRNA (eDC/IL-4), despite the short duration of viral transduction efficiency was more variable (40–70%), but in
IL-4 expression, had equivalent efficacy in preventing disease as general the transduced cells expressed higher levels of the trans-
did the previously described lentivirally transduced counterparts gene (Figure 1a and Supplementary Figure S1). Lentiviral trans-
(ltDC/IL-4).7 In addition, eDC/IL-4 cells were able to induce duction did not affect the differentiation of DCs (>90% CD11b+
a transient or sustained reversal of hyperglycemia in a signifi- CD11c+ at the end of the culture, Figure 1b), but increased their
cant fraction of mice treated after the onset of hyperglycemia. maturity, as seen by the expression of costimulatory molecules
Treatment with eDC/IL-4 reinforced tolerance through several and class II major histocompatibility complex (Figure 1c).
mechanisms, including induction of regulatory T cells (Tregs) and
skewing of helper T cell populations. This study represents a proof Function of modified DCs
of principle that electroporation of DCs with enhanced mRNA is The function of electroporated and transduced DCs was assessed by
a viable, safe, and effective substitute for viral vectors when using the amount of IL-4 secreted using enzyme-linked immunosorbent
cellular gene therapy to treat autoimmune diseases such as T1D. assay (ELISA). We observed that eDC/IL-4 consistently secreted
less than half the amount of IL-4 produced by ltDC/IL-4 within a
Results culture period of 24 hours in vitro at 37 °C, whereas unmodified
Characterization of electroporated DCs produced no detectable levels of IL-4 (Figure 2a). IL-4 intra-
and transduced DCs cellular staining showed that eDC/IL-4 no longer produced IL-4
We compared mRNA electroporation (eDCs) and lentiviral trans- by 24 hours whereas ltDC/IL-4 maintained IL-4 production, sug-
duction (ltDCs) for their efficiency of transgene transfer and gesting that, as expected, eDC/IL-4 use up their capacity to make
expression and for their effect on the phenotype of DCs. While IL-4 faster than ltDC/IL-4 (Figure 2b).

a GFP IL-4
100 100

80 80
Control DCs
% of Max

% of Max

60 60
Electroporated DCs (GFP mRNA on
40 40
left panel, IL-4 mRNA on right panel)
20 20

0 0 Transduced DCs (IL-4-IRES-GFP)

100 101 102 103 104 100 101 102 103 104
FL1-H: FITC FL4-H: APC

b Staining control Control DCs Electroporated DCs Transduced DCs

104 104 104 104

103 103 103 103

CD11c

102 102 102 102

101 101 101 101

100 100 100 100

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
CD11b

c 100 100 100 100

CD40
CD40 CD80 CD86 MHC-II
80 80 80 80

60 60 60 60

40 40 40 40

20 20 20 20

0 0 0 0
100 1
102 103 104 0 1
102 103 104 0 1
102 103 104 0 1
10 10 10 10 10 10 10 102 103 104

Figure 1  Phenotype of electroporated and transduced DCs. (a) GFP and IL-4 expression by unmodified DCs (harvested on day 7), mRNA elec-
troporated DCs (harvested and electroporated on day 7), and lentivirally transduced DCs (infected on day 5 and harvested on day 7). (b) CD11c and
CD11b expression by unmodified, mRNA electroporated, and lentivirally transduced DCs. (c) CD40, CD80, CD86, and class II MHC (I-Ag7) expression
by unmodified, mRNA electroporated and lentivirally transduced DCs. DC, dendritic cell; GFP, green fluorescent protein; IL, interleukin; MHC, major
histocompatibility complex.

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NOD T1D Treatment With mRNA Electroporated DCs

a 90 b GFP IL-4
100 100
eDC/GFP
80 80 80
eDC/IL-4

eDC/IL-4
60 60
70
ItDC/IL-4
40 40
IL-4 (pg per 100 modified DCs)

60
20 20

50 0 0
100 101 102 103 104 100 101 102 103 104

40 100 100

80 80
30

ItDC/IL-4
60 60
20
40 40

10 20 20

0 0
0
0 10 101 102 103 104 100 101 102 103 104
0 2 4 6 8 10 12 14 16 18 20 22 24
Time in culture (hours) 3 hours 6 hours 24 hours

Figure 2  Function of electroporated and transduced DCs. (a) Time course of IL-4 secretion by unmodified DCs, eDC/IL-4, and ltDC/IL-4 in the
first 24 hours after reculture at 37 °C in vitro. For better comparison between electroporation and lentiviral transduction, the values were normalized
based on the efficiency of transfection/transduction, and shown as mean ± SD from different numbers of cell plated (normalized to 100 cells). Data
from two independent experiments are shown. (b) GFP and intracellular IL-4 expression in eDC/IL-4 and ltDC/IL-4 after 3, 6, and 24 hours in culture.
DC, dendritic cell; eDC, enhanced DC; GFP, green fluorescent protein; ltDC, lentivirally transduced DC; IL, interleukin; MHC, major histocompatibility
complex.

Prevention of onset of hyperglycemia
We previously published that ltDC/IL-4, injected into 12-week-
old prediabetic NOD mice, can delay the time of disease onset and
prevent disease in about 50% of treated mice.7 We asked whether
eDC/IL-4 could work as a nonviral, potentially safer alternative to
ltDC/IL-4. Cohorts of nondiabetic 12-week-old female NOD mice
were treated with a single intravenous injection of phosphate-
buffered saline (PBS), eDC/green fluorescent protein (GFP), eDC/
IL-4, or ltDC/IL-4 (Figure 3). The control DCs (eDC/­GFP) slightly
delayed the onset of disease, but had no significant effect com-
pared to PBS (P = 0.692). However, both eDC/IL-4 and ltDC/IL-4
significantly reduced the incidence of disease (P = 0.013 and 0.026,
respectively). As previously shown with ltDC/IL-4,7 IL-4 expres-
sion by the DCs is required for the full therapeutic effect (eDC/
GFP versus eDC/IL-4, P = 0.023). Finally, despite the reduced
amount of IL-4 expressed by eDC/IL-4 compared to ltDC/IL-4,
these two treatments were equivalent therapeutically (P = 0.967).
It is noteworthy that some mice treated with eDC/IL-4 had mod-
est increases in glycemia (150–200 mg/dl) with transient episodes
>250 mg/dl. However, because 250 mg/dl was our cutoff thresh-
old for hyperglycemia in a prevention setting, some treated mice,
although initially considered diabetic, returned to <250 mg/dl and
did not develop full blown hyperglycemia (>500 mg/dl) during the
time they were monitored (Supplementary Figure S2). Thus, the
therapeutic efficacy as shown in Figure 3 was slightly underesti-
mated. Moreover, based on the amount of IL-4 produced in vitro,
we estimated that every million of the eDC/IL-4 and ltDC/IL4
injected produce respectively 260 ± 30 and 440 ± 100 ng of IL-4
within 24 hours in vivo (the variability is dependent on the effi-
ciency of transfection/transduction, which is more consistent
with electroporation) (Supplementary Figure S3). No IL-4 was
detected by ELISA in the serum of treated mice between 1 and
3 days after administration of eDC/IL-4 (data not shown).
Reversal of hyperglycemia
Next, we asked whether eDC/IL-4 had any beneficial effect when
administered after the onset of hyperglycemia. A cohort of female
NOD mice was monitored weekly for glycemia between the age of
11 and 30 weeks, and individual mice that became hyper­glycemic
(250–500 mg/dl) during the period of observation were treated
with a single injection of PBS, or freshly prepared eDC/GFP
or eDC/IL-4, and monitored twice weekly thereafter for blood
­glucose. Mice with a blood glucose >500 mg/dl over two consecu-
tive measurements were sacrificed. Data in Figure 4a,b show the
survival curve of control groups (PBS and eDC/GFP) and the
eDC/IL-4-treated group. Mice treated with either PBS or eDC/
GFP had to be sacrificed within 40 days, and there was no sig-
nificant difference between the two controls (P = 0.216). However,
one-third of mice treated with eDC-IL-4 survived well beyond 40
days, with a sustained period of reversal of hyperglycemia, com-
pared to all controls (P = 0.0014). Blood glucose measurements on
individual mice are reported in Figure 4c,d and demonstrate that
the responding mice did not return to a state of normoglycemia,
but instead to a state of controlled moderate hyperglycemia fluc-
tuating between 150 and 300 mg/dl. Two ­factors can contribute
to how well individual mice respond to the treatment: the sever-
ity of hyperglycemia and the age at the time of treatment; these
parameters are plotted for both responding and nonresponding
mice in Supplementary Figure S4. The average glycemia at time
of treatment with eDC/IL-4 was 307 ± 37 and 354 ± 53 mg/dl for
responding mice (n = 5) and nonresponding (n  = 10), respec-
tively. Although the data suggests better effectiveness in mice

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© The American Society of Gene & Cell Therapy
NOD T1D Treatment With mRNA Electroporated DCs

100 with less severe hyperglycemia, statistical significance was not

PBS iv (n = 27)
90
reached (P = 0.071). The median age of treatment of controls and
eDC/GFP iv (n = 24) eDC/IL-4 treated mice (in weeks) was similar (18 and 19, respec-
80
eDC/IL-4 iv (n = 25) tively), but within eDC/IL-4 treated mice, the median age at the
70 ItDC/IL-4 iv (n = 22) time of treatment was greater for responding mice (24) than for
­nonresponding mice (19).
Percentage diabetic

60

50 Changes in immune cell populations and cytokine

40
30
20
10
0
10
Figure 3  Incidence of diabetes after a single intravenous injection
of PBS, eDC/GFP, eDC/IL-4, or ltDC/IL-4 in 12-week-old prediabetic
NOD mice. The arrow indicates the time of treatment. Data are pooled
from two independent experiments. Log rank test was used for statisti-
cal analysis. eDC, enhanced dendritic cell; GFP, green fluorescent pro-
tein; ltDC, lentivirally transduced DC; IL, interleukin; NOD, nonobese
diabetic; PBS, phosphate-buffered saline.
profiles in targeted tissues
Targeted tissues (PLNs and spleen) and nontargeted tissue (­cervical
lymph nodes) were collected 1 and 3 days after intravenous injec-
tion of PBS, eDC/GFP, or eDC/IL-4. No major change in the per-
centage of the overall population of CD25+ Foxp3+ cells among
CD4+ T cells was observed, except for a modest but significant
15 20 25 30
increase in the spleen with eDC/IL-4 treatment (Figure  5a and
Age of mice (weeks) Supplementary S5a). However, a small population (up to 1% of
CD4+ T cells) emerged with higher levels of CD25 and CD4 than
the general CD4+ CD25+ population (Supplementary Figure
S5b). This small population significantly increased between day 1
and 3 following treatment with both eDC/GFP and eDC/IL-4, but
was further increased with eDC/IL-4 (Figure 5b). Moreover, these
CD25hi cells were made up of two populations of Foxp3+ cells, one
expressing higher levels of Foxp3 than the general CD25+ Foxp3+

a 100 b 100

90 90

80 Controls (n = 20)
80 PBS (n = 15)
eDC/IL-4 (n = 15)
eDC/GFP (n = 5)
70
70
60
60
Survival rate

50
50
40
40
P = 0.216 30
30
20 P = 0.0014
20
10
10
0
0 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320
0 20 40 60
c 600 d 600

500 500
Blood glucose (mg/dl)

400 400

300 300

200 200

100 100
Normoglycemia

0 0
0 20 40 60 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320
Days after treatment

Figure 4 Reversal of hyperglycemia after treatment with eDC/IL-4. (a,b) Survival curve of mice treated with (a) PBS or eDC/GFP, and (b) of all
control mice (PBS and eDC/GFP) or eDC/IL-4-treated mice. Mice were sacrificed when their blood glucose reached over 500 mg/dl after two consecu-
tive measurements. Log rank test was applied for statistical analysis. (c,d) Blood glucose measurements in mice treated with (c) PBS (dashed line,
n = 15) or eDC/GFP (solid lines, n = 5), and in mice treated with (d) eDC/IL-4 (n = 15). In d, responders were eDC/IL-4-treated mice that survived
longer than control mice (n = 5, solid lines); nonresponders (n = 10) are represented in dashed lines. Initial blood glucose value (day 0) is the mean
of two consecutive measurements (between 250 and 500 mg/dl) before treatment. eDC, enhanced dendritic cell; GFP, green fluorescent protein;
IL, interleukin; NOD, nonobese diabetic; PBS, phosphate-buffered saline.

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NOD T1D Treatment With mRNA Electroporated DCs

a 12 Day 1 Day 3 P = 0.037 d 700 Splenocytes from

12
PBS-treated
600
10 10
500
% CD25 + Foxp3+

8 8 400
6 300
6
200
4 4
100

Blood glucose (mg/dl)

2
2 0
10 12 14 16 18 20 22 24 26
0 0
PBS eDS/GFP eDC/IL-4 eDC/IL-4 PBS eDS/GFP eDC/IL-4
700 Splenocytes from
PLNs CLNs Spleen eDC/IL-4-treated
600
b 1.2 P = 0.009 P = 0.004
1 500
1 400
0.8
P = 0.004
0.8 300
hi

0.6
% CD25

200
0.6
0.4 100
0.4 P = 0.0004
0
0.2 0.2 10 12 14 16 18 20 22 24 26

0 0
PBS eDC/GFP eDC/IL-4 eDC/IL-4 PBS eDC/GFP eDC/IL-4 100 Splenocytes from
PBS-treated ( ---- )
PLNs CLNs Spleen 80 eDC/IL-4-treated ( ___ )

% diabetic
eDC/GFP eDC/IL-4 eDC/GFP eDC/IL-4 60 P = 0.041
100 100 100 100
40
80 80 80 80
Spleen

60 60 60 60 20
PLNs

40 40 40 40
20 0
20 20 20
10 12 14 16 18 20 22 24 26
0 0 0 0
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Age (weeks)

Figure 5  Induction of functional Tregs. (a) Frequency of CD25+ Foxp3+ cells gated on CD4+ T cells in select lymphoid tissues 1 and 3 days after
treatment. (b) Frequency of induced CD25hi cells among CD4+ T cells (gating shown in Supplementary Figure S5b). Mean ± SD is from n = 5 mice
per group per time point. t-Test was performed for statistical analysis. P values shown on graph are between day 1 and day 3. Difference between
eDC/GFP and eDC/IL-4 in spleen on day 3 was also significant (P = 0.035 on a; P = 0.01 on b). (c) Expression level of Foxp3 on CD4+ CD25−
cells (shaded histogram, no line), CD4+ CD25+ cells (shaded histogram with line) and induced CD4+ CD25hi cells (thick line). Gating is shown in
Supplementary Figure S5b. (d) Development of hyperglycemia in NOD.SCID mice injected at 10 weeks of age with 3 × 106 splenocytes from PBS-
treated (upper panel) or eDC/IL-4-treated (middle panel) NOD mice (n = 6 mice per group). The incidence of diabetes is reported in the lower panel
(mice were considered diabetic after two consecutive measurement of blood glucose over 250 mg/dl). Log rank test was used in statistical analysis.
eDC, enhanced dendritic cell; GFP, green fluorescent protein; IL, interleukin; NOD, nonobese diabetic; PBS, phosphate-buffered saline.

population, and one expressing lower levels (Figure 5c). Adoptive
transfer of splenocytes from eDC/IL-4-treated mice into NOD.
SCID mice did not efficiently transfer disease when compared to
splenocytes from PBS-treated mice (Figure 5d). Treatment with
modified DCs did not significantly alter the population of acti-
vated (CD44+ CD69+) T cells (Supplementary Figure S6), nor
did it lead to dramatic changes in different populations of splenic
DCs (Supplementary Figure S7). Cells isolated from selected tis-
sues were also cultured in vitro without stimulation to assess the
relative levels of interferon γ (IFN-γ), IL-4, and IL-10 induced
(Figure 6). No expression of these cytokines was seen from any
tissue obtained at 1 day after treatment, from the tissues from PBS-
treated mice or from control cervical lymph nodes from eDC/
IL-4-treated mice. The cytokine profile on day 3 following treat-
ment with control DCs (eDC/GFP) differed substantially between
PLNs and spleen. eDC/GFP induced similar amounts of IFN-γ
and IL-4 in the PLNs (IL-4/IFN-γ ratio = 1.26) but mostly IFN-γ
in the spleen (IL-4/IFN-γ ratio = 0.007). The additional effects of
IL-4 delivered by eDC/IL-4 led to different changes depending on
the target tissue. In the PLNs, eDC/IL-4 reduced the expression
of IFN-γ significantly, resulting in a more than sixfold increase of
the IL-4/IFN-γ ratio to 7.8. In the spleen, eDC/IL-4 increased IL-4
and IL-10 more than it increased IFN-γ, resulting in a >14-fold
increase of the IL-4/IFN-γ ratio to 0.1. The expression profile of
IL-10 mimicked that of IL-4.
Discussion
DCs play a crucial role in regulating immune responses, and
have attracted much attention as tools to manipulate the immune
response toward a desired outcome (stronger immune responses
against infections and tumors, versus dampened autoimmune
or allergic responses). The use of DCs generated ex vivo to treat
T1D has been proposed and tested by several groups.13–17 These
approaches consist, for example, of rendering the DCs more
tolerogenic by the use of particular cytokines in the culture, or
introducing antisense oligonucleotides to downregulate costimu-
latory molecules. DCs can also be used as vehicles to express and
deliver immunoregulatory products to sites of relevance in vivo.

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NOD T1D Treatment With mRNA Electroporated DCs

a 1,800
1,600
1,400
1,200 P = 0.77 P = 0.036

IL-4 (pg/ml)
1,000
800
600
P = 0.014
400 P = 0.39
200
0
−200 PBS eDC/GFP eDC/IL-4 eDC/IL-4 PBS eDC/GFP eDC/IL-4
b 8,000
7,000 P = 0.00068
36 hours 72 hours
6,000
5,000
IFN-γ (pg/ml)

4,000
P = 0.024
3,000
2,000 P = 0.036

1,000
0

−1000 PBS eDC/GFP eDC/IL-4 eDC/IL-4 PBS eDC/GFP eDC/IL-4

c 5,000
P = 0.81 P = 0.0039
4,000
IL-10 (pg/ml)

3,000

2,000
P = 0.027
P = 0.56
1,000

0
PBS eDC/GFP eDC/IL-4 eDC/IL-4 PBS eDC/GFP eDC/IL-4

PLN CLN Spleen

Figure 6 Local cytokine profile in targeted tissues. Cells from PLNs, CLNs, and spleen obtained 3 days after different treatments were cultured
in vitro. (a) IL-4, (b) IFN-γ, and (c) IL-10 were measured by ELISA on supernatant collected 36 hours (white bars) or 72 hours (gray bars) after replat-
ing. Mean ± SD is from n = 5 mice per group. t-Test was performed for statistical analysis. P values shown on graph are between eDC/GFP and eDC/
IL-4 for each time point. CLN, cervical lymph node; eDC, enhanced dendritic cell; GFP, green fluorescent protein; IFN-γ, interferon-γ; IL, interleukin;
NOD, nonobese diabetic; PBS, phosphate-buffered saline.

If  such immunoregulatory products are not normally expressed
by DCs, their respective genes must be introduced and adequately
expressed. To date, only viral means of DC modification have
been successfully applied to achieve prevention or treatment of
autoimmune diseases in mouse models, but safety concerns have
impeded the use of this approach in attempts at human therapy.
In the field of tumor immunology, researchers have achieved
expression of certain tumor antigens using electroporation of auto­
logous DCs with mRNA or translationally enhanced mRNA for
vaccination strategies.10–12 This has led to several trials in humans,
demonstrating the safety of mRNA electroporated DCs.18–21 In
this present study, we explore and report for the first time the use
of translationally enhanced mRNA electroporation as an alterna-
tive to viral means of modifying DCs to express immunomodula-
tory cytokines for adoptive cellular gene therapy of autoimmune
diseases. Electroporation with mRNA was more efficient than
lentiviral transduction in modifying a greater number of DCs,
and did so with no apparent effect on their phenotype. Dullaers
et al.22 have conducted a similar comparison and also noted that
­electroporation was more efficient, but that transduction led
to higher levels of transgene expression, especially at later time
points (beyond 24 hours). In agreement with this study, we found
that certain proteins, like GFP, are still present in high amount in
cells up to 3 days after electroporation. Expression of such pro-
teins does not necessarily reflect the duration of mRNA expres-
sion if the expressed protein is very stable (slowly degraded).
In the case of a secreted protein like IL-4, however, we did not
detect expression beyond 24 hours, whereas ltDCs were still
making IL-4 at this time. Unlike transduced cells, electroporated
DCs bear a finite quantity of mRNA, most of which will in turn
be translated into protein as long as the mRNA persists. In the
case of cytokines like IL-4, most of the mature protein presum-
ably gets secreted within 12 hours. Although eDC/IL-4 produced
less IL-4 in a 24-hour period than ltDC/IL-4, they expressed
higher levels on per cell basis within the first 6 hours after elec-
troporation. This elevated initial level of expression, at the time

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NOD T1D Treatment With mRNA Electroporated DCs
of administration, may account for the biological effect of delayed
disease ­progression  which was ­comparable to that of ltDCs.
Interestingly, electroporated DCs, in contrast to unmodified
and transduced DCs, may become more refractory to producing
IL-12 under certain maturation conditions.12,22 This phenomenon
is worth further exploration, as impaired induction of IL-12 can
enhance the effect of IL-4 expressed by therapeutic DCs.
Although various studies have reported an effect of DCs alone
in preventing diabetes when injected into young NOD mice, DCs
transduced by adenovirus or lentivirus to express IL-4, but not
untransduced DCs, were able to significantly delay and/or prevent
the onset of disease when injected in older prediabetic mice.6,7 One
major concern with the electroporation method was the transient
nature of IL-4 expression (limited to 24 hours). However, we have
also reported that DCs are detected in the spleen within 4 hours
and in the PLNs between 7 and 10 hours after injection,5 thus
sufficiently early to expect IL-4 to still be produced in the ­tissue
following transfer. We showed that eDC/IL-4 had a significant
effect in preventing onset of disease, whereas control eDC/GFP
did not, suggesting that IL-4, even transiently expressed, played
an important role. Thanks to recent developments, T1D can be
diagnosed earlier, allowing prevention strategies to be considered
for patients with a high risk of disease (family history, permissive
major histocompatibility complex, and the presence of relevant
autoantibodies in the serum),23 and treatments that are still effec-
tive at a late stage before the onset of overt hyperglycemia deserve
more attention. Currently, most efforts are still focused on treating
recently diagnosed patients, and most successful therapies in the
NOD mouse are only seriously considered if they are also able to
reverse overt hyperglycemia. We showed that a single injection
of eDC/IL-4 after onset of hyperglycemia had a significant effect,
decreasing the severity of disease in a third of the mice for pro-
longed periods of time. The treatment was most effective in mice
that had less severe hyperglycemia. In addition, older mice were
more likely to respond to the treatment, possibly a reflection of
late onset being the result of a more slowly progressing and less
aggressive disease.
We have previously shown that the homing of DCs to target
tissues results in increased expression of CD25 and CD69 on
CD4+ T cells.5 In this study, with a fivefold lower number of DCs
used for therapy, we continued to observe induction of CD25hi
cells that express Foxp3. This, combined with the lack of CD69
upregulation, suggests that induction of Tregs may contribute
to the therapeutic effect of the DCs. The fact that splenocytes
from eDC/IL-4-treated mice lose their ability to transfer disease
in NOD.SCID mice is further suggestive that Tregs have been
induced, but an enhanced deletion of autoreactive T cell within
3  days of treatment cannot be ruled out. Although CD25hi cells
were induced by all electroporated DCs, the presence of IL-4
boosted their frequency, particularly in the spleen. Such beneficial
effect of IL-4 on Tregs has been reported before.24,25 Another aspect
of the therapeutic effect of eDC/IL-4 is a significant shift from
Th1 to Th2 compared to eDC/GFP, characterized by a reduced
Th1 response and/or an increased Th2 response, depending on
the tissue ­analyzed, as well as significantly greater levels of IL-10
produced. The lack of response in the cervical lymph nodes of
eDC/IL-4-treated mice confirms that therapeutic DCs only target
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© The American Society of Gene & Cell Therapy
limited and specific lymphoid tissues.5 Overall, it appears that the
effect of eDC/IL-4 was more pronounced in the spleen than in the
PLNs, possibly because DCs migrate faster to the spleen than to
the PLNs5 and can therefore secrete more IL-4 before the mRNA
get depleted and the expression shut down. In other published
studies,7 we have determined that the efficacy of ltDC/IL-4 was
more consistent using the intravenous route (which allows spleen
homing) compared to the intraperitoneal route (which improves
PLN homing, but prevents spleen homing), suggesting that hom-
ing to the spleen may be important in addition to homing to the
PLNs. We have previously reported that certain changes in gene
expression affect the PLNs very early in young NOD mice, but
appear in the spleen only around 12 weeks of age.26 Because, we
treated mice at 12 weeks of age, it is possible that homing to both
the spleen and the PLNs is desired.
Although the therapeutic effect of eDC/IL-4 may not appear
as effective as other therapies being investigated in NOD mice,
including treatment with anti-CD3 antibodies or ex vivo amplified
and stimulated Tregs, mRNA electroporation of DCs offers poten-
tial flexibility and room for improvement. First, different types of
mRNA may be combined. For cancer immunotherapy, a combina-
tion of mRNA encoding both tumor antigens and factors enhanc-
ing DC maturity and stimulatory function has been used.13,27,28 In
the case of autoimmune diseases, retaining DC immaturity and/or
inducing tolerogenic properties is often preferable. In that respect,
electroporation is a safer choice over transduction with viral vec-
tors, which tends to increase DC maturity. Although IL-4 is an
effective cytokine in the treatment of autoimmune diabetes, other
molecules may be coexpressed to improve the effects of the DCs
by, for example, preventing the maturation of the DCs, express-
ing inhibitory molecules (e.g., programmed death ligand-1,
indoleamine 2,3-dioxygenase), enhancing trafficking to lymphoid
or inflamed tissues (via certain chemokine receptors and adhesion
molecules) or inducing more antigen-specific responses (expres-
sion of peptides recognized by self-reactive T cells). Secondly,
the effect of both prophylactic and therapeutic treatments was
achieved with a single injection of eDC/IL-4, but it is possible
that several treatments with eDC/IL-4 at different intervals may
increase the duration of the effect. To this end, cryopreservation
of DCs will be required as adoptive cellular therapy with DCs
­represents a personalized medicine in which the cells cannot be
continuously prepared. We have started to test cryopreserved
eDC/IL-4 in therapy settings, and found that a similar proportion
of mice responded to treatment, but with a more transient rever-
sal of hyperglycemia. Although optimization will be required to
improve recovery, viability, and function of cryopreserved murine
bone marrow-derived eDC/IL-4, there is evidence that human
(monocyte-derived) DCs retain excellent viability and function
following recovery from cryopreservation.11,29
In conclusion, we have demonstrated that electroporation
with translationally enhanced mRNA can be used to modify DCs
to express immunoregulatory products for adoptive cellular gene
therapy of autoimmune diseases. In the case of IL-4, which is
secreted and not retained in the cells, the timeline of expression is
short and lasts no longer than 24 hours. Nevertheless, this short
“pulse” of IL-4 delivered in vivo was sufficient to induce thera-
peutic effects in NOD mice, both in prevention and treatment of
www.moleculartherapy.org vol. 18 no. 12 dec. 2010
© The American Society of Gene & Cell Therapy
disease, which were in part ascribed to Treg induction and helper
T cell skewing. Modification by mRNA electroporation is a flex-
ible method that allows for coexpression of multiple products that
could be used to expand current therapeutic options.
Materials and Methods
Mice. Female NOD and NOD.SCID mice were purchased from the Jackson
Laboratories (Bar Harbor, ME), and kept in our animal facility under spe-
cific pathogen free conditions. Bone marrow donors were 8–10 weeks old,
while recipient mice were used as nondiabetic at 12 weeks of age (pre-
vention experiments), or as recent onset hyperglycemic between 11 and
30 weeks of age. NOD.SCID mice were adoptively transferred at 10 weeks
of age. All manipulations were approved by the Stanford Administrative
Panel on Laboratory Animal Care.
Subcloning of mIL4Rot6 for RNA generation. mIL4 coding sequence was
subcloned from pHR-IL4IG plasmid7 into pGem 4Z 64T ­plasmid  using
SmaI restriction sites. Translation enhancer element from the 3′ untrans-
lated region of rotavirus gene 6 mRNA (rot 6 element)30 was PCR ampli-
fied from synthetically generated construct (Blue Heron Biotechnology,
Bothell, WA) encoding the rot6 sequence using primers forward 5′-ACT
GCTCGAGGACCAAGCTAACAACTTGG-3′ and reverse 5′-ACTGTTA
ATTAAGGTCACATCCTCTCACTATACC-3′ containing XhoI and PacI
recognition sites indicated in italics, respectively. The amplified PCR frag-
ment containing rot 6 element was subcloned downstream of stop codon
of mIL4 in pGem4Z64T using XhoI and PacI restriction sites. The final
construct was verified by sequencing through the coding region of mIL4
and junction to pGem4Z64T plasmid.
Generation of the GFP and mIL4Rot6 type I cap RNAs. Plasmid
pGem4Z64TmIL4Rot6 was purified using QiaFilter maxiprep kit (Qiagen,
Valencia, CA) and linearized with SpeI. Twenty-five microgram of puri-
fied linearized template was used to establish 1-ml transcription reaction
using T7 Flash kit according to manufacturing instruction (Epicentre
Biotechnologies, Madison, WI). The in vitro transcription was conducted
for 2 hours at 37 °C. After digestion with added 50 μl of DNAseI for addi-
tional 30 minutes at 37 °C, the uncapped RNA was purified using RNeasy
maxi columns (Qiagen). The purified uncapped RNA was capped in the
post-transcriptional capping reaction for 1 hour at 37 °C using Script
Cap Type I Capping kit scaled up to 3,340 μl reaction volume (Epicentre
Biotechnologies). The polyadenylation reaction was carried out using
A-plus PolyA Tailing kit (Epicentre Biotechnologies). The unpurified cap-
ping reaction was adjusted to 6,664 μl with water, 10× A-plus reaction buffer
and ATP substrate according to manufacturer’s instructions and incubated
for 45 minutes at 37 °C swirling the reaction every 15 minutes. The final
polyadenylated RNA modified with type I cap and ~200 polyA tail was
purified using RNeasy maxi columns (Qiagen). The length of polyA tail
was measured by comparing relative migration of unpolyadenylated and
polyadenylated mIL4 RNAs on denaturing agarose gel followed by collect-
ing image with Alphaimager and size analysis using ImageQuant Software
(Alpha Innotech, San Leandro, CA). 2.2 mg of RNA eluted in water was
adjusted to final concentration of 1 mg/ml and stored frozen in single-
size aliquots in pyrogen-free RNAse-free Safe Lock tubes (Eppendorf,
Hamburg, Germany). GFP RNA was generated from pGem4Z/GFP/A64
plasmid as described previously.31
Lentivirus generation, DC culture, transduction, and electroporation.
Lentiviral particles expressing IL-4 and GFP were generated using the
pHR-IL4IG vector as previously described.7 Bone marrow was harvested
from the femurs, tibias, and pelvis of female mice. The bone marrow cells
were depleted of CD3+, B220+, and Gr-1+ cells on AutoMACS (Miltenyi
Biotec, Bergisch Gladbach, Germany) using biotinylated antibodies (eBio-
science, San Diego, CA) and antibiotin beads (Miltenyi Biotec), and then
­cultured at 37 °C in complete RPMI medium (10% fetal calf serum, 2 mmol/l
Molecular Therapy vol. 18 no. 12 dec. 2010
NOD T1D Treatment With mRNA Electroporated DCs
l-glutamine, 100 U/ml penicillin, 100 μg/ml ­streptomycin, 0.1 mmol/l
nonessential amino acids, 1 mmol/l sodium pyruvate, 14.3 μmol/l
β-mercaptoethanol) in presence of recombinant mouse granulocyte-
macrophage colony-stimulating factor and IL-4 (Peprotech, Rocky Hill,
NJ) at 10 ng/ml each. On day 5, cells were infected with lentiviral ­particles
(multiplicity of infection = 15–20) in presence of 10 μg/ml protamine sul-
fate (Sigma, St Louis, MO). After 16–24 hours incubation with virus, the
medium was changed. Infected and noninfected DCs were collected on
day 7. Noninfected DCs were washed three times in OptiMEM and elec-
troporated in a 4 mm cuvette (5 × 106 DCs in 200 μl OptiMEM with 30 μg
mRNA) on a Gene Pulser Xcell II electroporator (Bio-rad, Hercules, CA)
using optimized parameters (300 V, 150 μF, 100 Ω).
Treatments and monitoring. 1 × 106 DCs (in 200 μl PBS) were injected
intravenously into 12-week-old nondiabetic NOD mice (prevention) or
11–30-week-old NOD mice with recent onset hyperglycemia. In preven-
tion studies, blood glucose was measured weekly and animals with levels
>250 mg/dl over two consecutive bleedings were considered diabetic. In
therapy studies, blood glucose was monitored weekly. Mice with high-
blood glucose (250–500 mg/dl) were bled again 1 or 2 days later to confirm
hyperglycemia and immediately treated. Treated mice were monitored
twice weekly and animals with levels >500 mg/dl over two consecutive
bleedings were sacrificed. In transfer experiments, splenocytes obtained
3 days after treatment were injected intravenously into 10-week-old NOD.
SCID mice (3 × 106 cells per recipient). Recipient mice were followed
every other week until the first onset of disease, and weekly thereafter.
Mice were considered diabetic using the same criteria as in prevention
studies.
Flow cytometric analysis. DCs were analyzed for GFP and surface mark-
ers expression (anti-CD11b, CD11c and I-Ak (also stains I-Ag7) were from
BD Biosciences (San Jose, CA); anti-CD40, CD80, CD86, CD8a and SIRPα
were from eBioscience). For intracellular staining of IL-4, transduced and
electroporated DCs were recultured for 3–24 hours in complete medium
without cytokines and with GolgiStop (BD Biosciences) added in the last
3–4 hours, processed with BD Fix/Perm kit (BD Biosciences) and stained
using allophycocyanin-conjugated anti-IL-4 (eBioscience). T cells were
stained for CD4, CD8a, CD25, CD44, CD69, and Foxp3 using antibodies
from BD Biosciences and eBioscience. Fix/Perm kit from eBioscience was
used for Foxp3 intracellular staining. All cells were analyzed on LSR1 (BD
Biosciences) and data were computed using the FlowJo software (Tree Star,
Ashland, OR).
ELISA. DCs were washed and replated at different numbers and several
replicates in complete RPMI without granulocyte-macrophage colony-
stimulating factor or IL-4. Supernatant was collected at various time points
ranging from 30 minutes to 24 hours. ELISA was performed using stan-
dard protocol and reagents from BD Biosciences (anti-IL-4 antibody pair),
Peprotech (IL-4 standard) and Sigma (ExtrAvidin peroxidase and TMB
substrate). Data shown are mean ± SD from different number of live cells
plated (5 × 103, 2.5 × 104, 1 × 105). The amount of IL-4 secreted was lin-
early correlated with the number of DCs (R2 > 0.98). For cytokine profile,
splenocytes (1 × 106/well), PLN, or cervical lymph node cells (0.8 × 106/
well) were replated in complete c-RPMI in four replicates. Supernatant
was ­collected after 36 and 72 hours (one duplicate per time point). ELISA
was performed as described above for IL-4, or using ELISA Max kits
(Biolegend, San Diego, CA) for IFN-γ and IL-10.
SUPPLEMENTARY MATERIAL
Figure S1.  Efficiency of electroporation or transduction of DCs.
Figure S2.  Evolution of blood glucose on a selection of mice treated
with eDC/IL-4, showing occasional transient increase of glycemia over
250 mg/dl.
Figure S3.  Consistency in the amount of IL-4 secreted by modified
DCs in a 24 hours culture.
2119

NOD T1D Treatment With mRNA Electroporated DCs
Figure S4.  Factors influencing effectiveness of eDC/IL-4 treatment.
Figure S5.  CD25+ Foxp3+ cells in the PLNs and spleen of treated
mice.
Figure S6.  Activated CD4+ and CD8+ T cells in the PLNs and spleen
of treated mice.
Figure S7.  Frequency of several DC subsets in the spleen of treated
mice.
ACKNOWLEDGMENTS
D.G.H., I.Y.T., and C.A.N. are employed by Argos Therapeutics. The
authors also thank Melissa Adam and Aijing Starr, also employed by
Argos Therapeutics, for preparation of optimized IL-4 cDNA template
and for mRNA generation, respectively. This work was supported by
grants from the National Institutes of Health (DK078123, to C.G.F.),
the Juvenile Diabetes Research Foundation (3-2005-1019, to R.J.C.)
and by a gift from Argos Therapeutics (to C.G.F.).
REFERENCES
1. Anderson, MS and Bluestone, JA (2005). The NOD mouse: a model of immune
dysregulation. Annu Rev Immunol 23: 447–485.
2. Shoda, LK, Young, DL, Ramanujan, S, Whiting, CC, Atkinson, MA, Bluestone, JA et al.
(2005). A comprehensive review of interventions in the NOD mouse and implications
for translation. Immunity 23: 115–126.
3. Smits, HH, de Jong, EC, Wierenga, EA and Kapsenberg, ML (2005). Different faces of
regulatory DCs in homeostasis and immunity. Trends Immunol 26: 123–129.
4. Morelli, AE and Thomson, AW (2007). Tolerogenic dendritic cells and the quest for
transplant tolerance. Nat Rev Immunol 7: 610–621.
5. Creusot, RJ, Yaghoubi, SS, Chang, P, Chia, J, Contag, CH, Gambhir, SS et al. (2009).
Lymphoid-tissue-specific homing of bone-marrow-derived dendritic cells. Blood 113:
6638–6647.
6. Feili-Hariri, M, Falkner, DH, Gambotto, A, Papworth, GD, Watkins, SC, Robbins, PD
et al. (2003). Dendritic cells transduced to express interleukin-4 prevent diabetes in
nonobese diabetic mice with advanced insulitis. Hum Gene Ther 14: 13–23.
7. Creusot, RJ, Yaghoubi, SS, Kodama, K, Dang, DN, Dang, VH, Breckpot, K et al. (2008).
Tissue-targeted therapy of autoimmune diabetes using dendritic cells transduced to
express IL-4 in NOD mice. Clin Immunol 127: 176–187.
8. Kim, SH, Kim, S, Evans, CH, Ghivizzani, SC, Oligino, T and Robbins, PD (2001).
Effective treatment of established murine collagen-induced arthritis by systemic
administration of dendritic cells genetically modified to express IL-4. J Immunol 166:
3499–3505.
9. Morita, Y, Yang, J, Gupta, R, Shimizu, K, Shelden, EA, Endres, J et al. (2001). Dendritic
cells genetically engineered to express IL-4 inhibit murine collagen-induced arthritis. J
Clin Invest 107: 1275–1284.
10. Michiels, A, Tuyaerts, S, Bonehill, A, Corthals, J, Breckpot, K, Heirman, C et al. (2005).
Electroporation of immature and mature dendritic cells: implications for dendritic cell-
based vaccines. Gene Ther 12: 772–782.
11. Van Tendeloo, VF, Ponsaerts, P and Berneman, ZN (2007). mRNA-based gene transfer
as a tool for gene and cell therapy. Curr Opin Mol Ther 9: 423–431.
12. Calderhead, DM, DeBenedette, MA, Ketteringham, H, Gamble, AH, Horvatinovich, JM,
Tcherepanova, IY et al. (2008). Cytokine maturation followed by CD40L mRNA
electroporation results in a clinically relevant dendritic cell product capable of inducing
a potent proinflammatory CTL response. J Immunother 31: 731–741.
13. Feili-Hariri, M, Flores, RR, Vasquez, AC and Morel, PA (2006). Dendritic cell
immunotherapy for autoimmune diabetes. Immunol Res 36: 167–173.
2120
© The American Society of Gene & Cell Therapy
14. Lo, J and Clare-Salzler, MJ (2006). Dendritic cell subsets and type I diabetes: focus
upon DC-based therapy. Autoimmun Rev 5: 419–423.
15. Trucco, M and Giannoukakis, N (2007). Immunoregulatory dendritic cells to
prevent and reverse new-onset Type 1 diabetes mellitus. Expert Opin Biol Ther 7:
951–963.
16. Besin, G, Gaudreau, S, Ménard, M, Guindi, C, Dupuis, G and Amrani, A (2008).
Thymic stromal lymphopoietin and thymic stromal lymphopoietin-conditioned
dendritic cells induce regulatory T-cell differentiation and protection of NOD mice
against diabetes. Diabetes 57: 2107–2117.
17. Giannoukakis, N, Phillips, B and Trucco, M (2008). Toward a cure for type 1 diabetes
mellitus: diabetes-suppressive dendritic cells and beyond. Pediatr Diabetes 9(3 Pt 2):
4–13.
18. Kyte, JA, Mu, L, Aamdal, S, Kvalheim, G, Dueland, S, Hauser, M et al. (2006).
Phase I/II trial of melanoma therapy with dendritic cells transfected with autologous
tumor‑mRNA. Cancer Gene Ther 13: 905–918.
19. Van Driessche, A, Van de Velde, AL, Nijs, G, Braeckman, T, Stein, B, De Vries, JM et al.
(2009). Clinical-grade manufacturing of autologous mature mRNA-electroporated
dendritic cells and safety testing in acute myeloid leukemia patients in a phase I
dose‑escalation clinical trial. Cytotherapy 11: 653–668.
20. Schuurhuis, DH, Verdijk, P, Schreibelt, G, Aarntzen, EH, Scharenborg, N, de Boer, A
et al. (2009). In situ expression of tumor antigens by messenger RNA-electroporated
dendritic cells in lymph nodes of melanoma patients. Cancer Res 69: 2927–2934.
21. Routy, JP, Boulassel, MR, Yassine-Diab, B, Nicolette, C, Healey, D, Jain, R et al. (2010).
Immunologic activity and safety of autologous HIV RNA-electroporated dendritic
cells in HIV-1 infected patients receiving antiretroviral therapy. Clin Immunol 134:
140–147.
22. Dullaers, M, Breckpot, K, Van Meirvenne, S, Bonehill, A, Tuyaerts, S, Michiels, A et al.
(2004). Side-by-side comparison of lentivirally transduced and mRNA-electroporated
dendritic cells: implications for cancer immunotherapy protocols. Mol Ther 10:
768–779.
23. Sherr, J, Sosenko, J, Skyler, JS and Herold, KC (2008). Prevention of type 1 diabetes:
the time has come. Nat Clin Pract Endocrinol Metab 4: 334–343.
24. Skapenko, A, Kalden, JR, Lipsky, PE and Schulze-Koops, H (2005). The IL-4 receptor
alpha-chain-binding cytokines, IL-4 and IL-13, induce forkhead box P3-expressing
CD25+CD4+ regulatory T cells from CD25-CD4+ precursors. J Immunol 175:
6107–6116.
25. Pillai, AB, George, TI, Dutt, S and Strober, S (2009). Host natural killer T cells induce
an interleukin-4-dependent expansion of donor CD4+CD25+Foxp3+ T regulatory cells
that protects against graft-versus-host disease. Blood 113: 4458–4467.
26. Kodama, K, Butte, AJ, Creusot, RJ, Su, L, Sheng, D, Hartnett, M et al. (2008). Tissue-
and age-specific changes in gene expression during disease induction and progression
in NOD mice. Clin Immunol 129: 195–201.
27. Michiels, A, Breckpot, K, Corthals, J, Tuyaerts, S, Bonehill, A, Heirman, C et al.
(2006). Induction of antigen-specific CD8+ cytotoxic T cells by dendritic cells
co‑electroporated with a dsRNA analogue and tumor antigen mRNA. Gene Ther 13:
1027–1036.
28. Bonehill, A, Tuyaerts, S, Van Nuffel, AM, Heirman, C, Bos, TJ, Fostier, K et al.
(2008). Enhancing the T-cell stimulatory capacity of human dendritic cells by co-
electroporation with CD40L, CD70 and constitutively active TLR4 encoding mRNA.
Mol Ther 16: 1170–1180.
29. Ponsaerts, P, Van Tendeloo, VF, Cools, N, Van Driessche, A, Lardon, F, Nijs, G et al.
(2002). mRNA-electroporated mature dendritic cells retain transgene expression,
phenotypical properties and stimulatory capacity after cryopreservation. Leukemia 16:
1324–1330.
30. Yang, AD, Barro, M, Gorziglia, MI and Patton, JT (2004). Translation enhancer in the
3’-untranslated region of rotavirus gene 6 mRNA promotes expression of the major
capsid protein VP6. Arch Virol 149: 303–321.
31. Boczkowski, D, Nair, SK, Nam, JH, Lyerly, HK and Gilboa, E (2000). Induction of tumor
immunity and cytotoxic T lymphocyte responses using dendritic cells transfected with
messenger RNA amplified from tumor cells. Cancer Res 60: 1028–1034.
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