Transcriptional re-programming of primary macrophages reveals distinct apoptotic and anti-tumoral...

Transcriptional re-programming of primarymacrophages reveals distinct apoptotic andanti-tumoral functions of IRF-3 and IRF-7

Delphine Goubau1,2, Raphaelle Romieu-Mourez1,2, Mayra Solis1,2,

Eduardo Hernandez1,2, Thibault Mesplede1,2, Rongtuan Lin1,3,

Douglas Leaman4 and John Hiscott1,2,3

1 Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, McGill

University, Montreal, Que., Canada2 Department of Microbiology and Immunology, McGill University, Montreal, Que., Canada3 Department of Medicine, McGill University, Montreal, Que., Canada4 Department of Biological Sciences, University of Toledo, Toledo, OH, USA

The immunoregulatory transcriptional modulators – IFN-regulatory factor (IRF)-3 and

IRF-7 – possess similar structural features but distinct gene-regulatory potentials. For

example, adenovirus-mediated transduction of the constitutively active form of IRF-3

triggered cell death in primary human MU, whereas expression of active IRF-7 induced a

strong anti-tumoral activity in vitro. To further characterize target genes involved in these

distinct cellular responses, transcriptional profiles of active IRF-3- or IRF-7-transduced

primary human MU were compared and used to direct further mechanistic studies. The

pro-apoptotic BH3-only protein Noxa was identified as a primary IRF-3 target gene and an

essential regulator of IRF-3, dsRNA and vesicular stomatitis virus-induced cell death. The

critical role of IRF-7 and type I IFN production in increasing the immunostimulatory

capacity of MU was also evaluated; IRF-7 increased the expression of a broad range of IFN-

stimulated genes including immunomodulatory cytokines and genes involved in antigen

processing and presentation. Furthermore, active IRF-7 augmented the cross-presentation

capacity and tumoricidal activity of MU and led to an anti-tumor response against the B16

melanoma model in vivo. Altogether, these data further highlight the respective functions

of IRF-3 and IRF-7 to program apoptotic, immune and anti-tumor responses.

Key words: Anti-tumor immunotherapy . Apoptosis . Cross-presentation .

IFN-regulatory factor . Microarray

Supporting Information available online

Introduction

Type I interferons (IFN-a/b) induce the expression of hundreds

of IFN-stimulated genes (ISG), which mediate a broad range of

anti-viral, growth-regulatory and immunomodulatory effects. In

virus-infected cells, these cytokines inhibit many RNA and DNA

viruses at various stages of their replication cycles [1, 2]. During

tumor-cell development, type I IFN exert direct cytotoxic or anti-

proliferative functions and negatively regulate angiogenesis

[3, 4]. Furthermore, direct anti-viral and anti-tumor propertiesCorrespondence: Dr. John Hiscotte-mail: [email protected]

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Eur. J. Immunol. 2009. 39: 527–540 DOI 10.1002/eji.200838832 Innate Immunity 527

of type I IFN are complemented by their role in immunosurveil-

lance [3]. Type I IFN are produced early during the innate

immune response and have important immunomodulatory effects

on T-lymphocyte and NK-cell activity [5–8], as well as on DC

differentiation and maturation [9–12].

Production of IFN-a/b following infection is dependent on the

activation of the IFN-regulatory factors (IRF)-3 and IRF-7 [13].

These IRF are phosphorylated by the kinases TANK-binding

kinase1 (TBK-1) and IkB kinase-e (IKK-e) following viral infection

and PRR signaling [14, 15] leading to their dimerization, nuclear

translocation and binding to the promoters of type I IFN genes

[16–19]. Although IRF-3 and IRF-7 share highly related structural

and functional characteristics [13, 20, 21], major biological and

functional distinctions can be identified between the two. While

IRF-3 is ubiquitously expressed [22], the basal expression of IRF-7

is low in most cells (with the notable exception of plasmacytoid DC

[23]) and is strongly increased by type I IFN-mediated signaling

[24]. IRF-3 and IRF-7 present both redundant and distinct tran-

scriptional profiles. Qualitative and quantitative differences exist in

type I IFN induction capacities of IRF-3 and IRF-7 upon activation:

IRF-7 can induce the efficient activation of IFNB genes and

multiple IFNA subtypes, whereas IRF-3 is a potent activator of IFNB

genes but not of IFNA genes with the exception of human IFNA1

and mouse Ifna4 genes [21, 24–26]. This observation may account

for the increased vulnerability to viral infections of Irf7�/� mice

compared with Irf3�/� mice [27]. The full range of IRF-3 or IRF-7

target genes has not been compared directly, although indepen-

dent studies in different cell types have suggested that the acti-

vation of IRF-3 or IRF-7 results in a broad perturbation in the

transcriptional profile of cellular genes involved in the anti-viral,

apoptosis and immune responses [28–30]. Generation of consti-

tutively active forms of IRF-3 and IRF-7, that translocate to the

nucleus, bind to the promoters of type I IFN genes and induce IFN-

a/b expression in the absence of viral infections [16–18], have

permitted further insight into the respective cellular functions of

these IRF. The constitutively active IRF-3 5D contains phospho-

mimetic aspartic acid substitutions at aa residues 396, 398, 402,

404 and 405, whereas IRF-7 D247–467 lacks the aa 247–467

region, which encompasses the inhibitory and nuclear export

domains [16–18]. Expression of IRF-3 5D triggered cell death in

numerous cell types, including primary human MF, monocyte-

derived DC, human lung epithelial carcinoma A549 cells, Jurkat

T cells and human embryonic kidney 293 cells [31, 32], whereas

expression of IRF-7 D247–467 in primary human MF induced the

expression of high levels of type I IFN and TRAIL as well as

strong cytostatic activity on different human cancer cells lines

in vitro [32].

In order to investigate the distinct mechanisms underlying

IRF-3 and IRF-7 function, as well as the possible use of these

transcription factors in viral and anti-tumor therapeutic strategies,

whole genome transcriptional profiles of primary human MFtransduced with adenoviral vectors expressing IRF-3 5D (Ad-F3) or

IRF-7 D247–467 (Ad-F7), were used to identify subsets of genes

triggered preferentially by active IRF-3 or IRF-7 expression. The

pro-apoptotic BH3-only protein Noxa was identified as a primary

IRF-3 target gene and an essential regulator of IRF-3, dsRNA and

vesicular stomatitis virus (VSV)-induced cell death. In contrast,

transduction of Ad-F7 was not pro-apoptotic, but rather induced

high levels of type I IFN and immunomodulatory cytokines, as well

as the expression of genes involved in antigen processing and

presentation. Murine MF transduced with Ad-F7 displayed an

increased capacity to cross-present antigens and activate

CD81 T lymphocytes in vitro. The potential immunotherapeutic

use of Ad-F7-transduced MF was further highlighted by their

enhanced tumor inhibitory effects in vivo using the B16 melanoma

model.

Results

Gene expression profiling in Ad-F3 or Ad-F7-transduced MU

To identify genes that may regulate distinct or overlapping

functions of IRF-3 and IRF-7, primary human MF preparations

were transduced with recombinant adenovirus-expressing GFP

(Ad-GFP), Ad-F3 or Ad-F7 vectors and evaluated by microarray

analysis. IRF-3 and IRF-7 transgene expression was readily

detected in transduced MF by immunoblot [32] and by tracking

the nuclear localization of GFP-tagged IRF-3 and IRF-7 proteins

following Ad-vector transduction (Fig. 1A). Seven of the 12

donors with the highest transduction efficiency (46–70% GFP

expression), as measured by flow cytometry (Fig. 1B) were

selected for genome-wide transcriptome analysis using Illumina

bead array. Hierarchical clustering of genes with an adjusted

p-value of o0.01 (1843 genes) showed that non-transduced (NT)

and control adenoviral vector (Ad-GFP) gene expression profiles

were nearly indistinguishable, whereas Ad-F3 and Ad-F7

profiles were distinct from these controls, as well as from each

other (Fig. 1C). Transduction of Ad-F7 in primary MF modulated

the expression of 292 genes with p-value o0.01 and fold-

change Z2.0 and r�2.0 (versus Ad-GFP values) compared

with 72 genes with Ad-F3. A subset of 49 genes was

commonly up-regulated by both active IRF-3 and active IRF-7

(Fig. 1D).

Ad-F3 and Ad-F7 versus Ad-GFP significant genes were

then manually refined and classified according to known

functions and pathways using manual searches in NCBI and the

ontology tools DAVID and BIORAG. Figure 2 provides a partial

list of significantly up-regulated genes including genes involved

in innate immunity, apoptosis, antigen processing and presenta-

tion, as well as cytokines and chemokines. Microarray data

were validated by performing real-time PCR on 12 genes (TAP1,

MCL1, RIGI, MDA5, G1P3, TLR8, NOXA, OAS1, RANTES, PELI1,

PSME2 and C5) for 4 donors included in the array (Supporting

Information Fig. 1A). Pearson correlations that averaged

0.88 for all 12 genes (minimum 0.68 and maximum 0.99)

revealed strong linear relationships between real-time PCR

validation and microarray data (Supporting Information

Fig. 1B).

Eur. J. Immunol. 2009. 39: 527–540Delphine Goubau et al.528


Involvement of Noxa in IRF-3-mediated apoptosis

The gene encoding the pro-apoptotic BH3-only protein Noxa

(Pmaip1) was more strongly induced in Ad-F3 as compared with

Ad-F7-transduced MF (6.6-fold change for Ad-F3 versus 2.7-fold

change for Ad-F7) (Fig. 2), a difference that was confirmed by

real-time PCR (Fig. 3A and Supporting Information Fig. 1A). The

increased expression of Noxa in Ad-F3-transduced MF was

independent of type I IFN, as demonstrated using a neutralizing

antibody for the type I IFN receptor (IFNAR) (Fig. 3A). Because

Noxa is known to induce mitochondrial membrane depolariza-

tion, we tested whether the intrinsic mitochondrial apoptotic

pathway was involved in Ad-F3-induced apoptosis. A 4-fold

increase in mitochondrial depolarization (as assessed by the loss

of MitoProbeTM DilC1(5) staining by flow cytometry) was

observed in MF transduced with Ad-F3 for 24 h, compared with

NT, Ad-GFP and Ad-F7 samples (Fig. 3B). Caspase-3 enzymatic

activity also increased 3.5-fold with Ad-F3 compared with NT

MF, whereas Ad-F7 transduction induced a 1.8-fold increase, as

measured by DEVD-AFC cleavage by fluorometric assay (Fig. 3C).

Disruption of mitochondrial membrane potential and increased

enzymatic activity of caspase-3 observed in Ad-F3-transduced MFcorrelated with increased cleavage of caspase-9 and caspase-3, as

well as the up-regulation of Noxa protein expression by

immunoblot (Fig. 3D). Ad-F7 and IFN-a-2b treatments did not

significantly increase caspase cleavage or Noxa protein levels.

Increased expression of Noxa at the RNA and protein levels, as

well as caspase-3 cleavage and enzymatic activity, was also

observed in Ad-F3-transduced human A549 epithelial carcinoma

cells relative to Ad-F7 and control samples (data not shown).

Importantly, use of pan-caspase inhibitor (Z-VAD-FMK) on these

cells following Ad-F3 transduction reduced caspase-3 enzymatic

activity to levels seen in controls without altering Noxa

expression levels (data not shown).

The direct contribution of Noxa in IRF-3-mediated apoptosis

was tested by assessing the effects of active IRF-3 in Noxa

knock-out and knock-down cells. In Noxa�/�, immortalized

epithelial baby mouse kidney (BMK) cells transduced with Ad-F3,

caspase-3 cleavage and mitochondrial depolarization were

reduced to levels seen in control Ad-GFP-transduced cells (Fig. 3E

and F). Using siRNA targeting, Noxa levels were reduced 60%

following transduction of Ad-F3 in primary human MF and a

corresponding 50% reduction in caspase-3 cleavage was also

observed (Fig. 3G).

Regulation of the NOXA promoter by IRF-3 was next evaluated,

using different promoter deletion constructs linked to a luciferase

reporter gene. The full-length (�1413 to 11) fragment contains

the previously identified IRF-E, CRE and p53 binding sites of the

NOXA promoter [33, 34]. Co-transfection of the plasmid-expres-

sing IRF-3 5D with this 1.4 kb fragment of the NOXA promoter in

human embryonic kidney 293 cells led to a 9-fold increase in

luciferase activity compared with control GFP plasmid or IRF-3 WT

transfected cells (Fig. 3H). Deletion to �677 reduced activation to

3.5-fold following active IRF-3 co-expression, whereas constructs

(�642 to 11) and (�360 to 11) displayed no increase in luci-

Figure 1. Distinct gene expression profiles of Ad-F3 and Ad-F7-transduced MF. Primary human MF from healthy donors weretransduced or not with Ad-GFP, Ad-F3 or Ad-F7. (A) Nuclear transloca-tion of GFP-tagged IRF proteins was monitored 24 h post-transductionusing the nuclear marker To-Pros-3. Confocal fluorescent images shownuclear staining (left), Ad-vector GFP expression (middle) and merge(right). Images are representative of cells for two donors. (B) Cells wereanalyzed by flow cytometry 24 h post-transduction to determine thepercentage of GFP positive cells. MF with the highest transductionefficiency (donors 1–7) were selected for microarray analysis. (C) RNAfrom MF (donors 1–7) was collected 24 h post-transduction, purified,amplified and labeled before Illuminas bead array and data analysiswas carried out as described in Materials and methods. Hierarchical geneaverage linkage clustering was performed using genes with po0.01.Each row represents the relative rank based levels of expression of asingle gene (low expression in light blue and high expression in darkblue) and each column represents a sample (28 samples total). Thesamples include seven donors and four conditions (NT, Ad-GFP, Ad-F3and Ad-F7). (D) Venn diagram represents the number of genes fromarray data with po0.01 and fold-change Z2.0 (up) or r2.0 (down) in Ad-F7 versus Ad-GFP and Ad-F3 versus Ad-GFP-transduced MF.

Eur. J. Immunol. 2009. 39: 527–540 Innate Immunity 529


ferase activity. As expected, transfection with plasmids coding IRF-

7 WT or constitutively active IRF-7 (D247–467) did not increase

NOXA promoter activity (Fig. 3H). Importantly, the differential

effects of IRF-3 and IRF-7 on the NOXA promoter contrasted with

their redundant capacity to induce type I IFN as demonstrated

using the luciferase reporter plasmid-encoding tandem repeats of

the IFN-stimulated response element (ISRE) (Fig. 3I). These data

thus identify NOXA as a primary IRF-3, but not IRF-7, target gene –

involved in the pro-apoptotic response in primary MF, and further

suggest that the region between �642 and �1413 of the NOXA

promoter which includes an IRF-E element (region (�642 to

�656)) [33] is exclusively required for IRF-3 transcriptional

activity.

Involvement of IRF-3 and Noxa in dsRNA andviral-mediated apoptosis

To further address the role of Noxa in dsRNA and virus-induced

apoptosis, WT or Noxa�/� BMK cells were treated with

synthetic dsRNA (poly(I:C)) or infected with VSV for 16 h.

Whereas WT BMK cells exhibited an acute cytopathic response

to VSV, Noxa�/� BMK cells remained unaffected (Fig. 4A).

Noxa�/� BMK cells reconstituted with empty vector (1Vec)

were similarly resistant to the cytopathic effects of VSV,

whereas Noxa-reconstituted BMK cells (1Noxa) restored the

cytopathic response (Fig. 4A). VSV-induced cytopathicity

was also associated with cleavage of caspase-3 (Fig. 4B).

Figure 2. List of selected Ad-F3 and/or Ad-F7-induced genes following transduction in primary human MF. Data depict average mean fold changesof Ad-F3 versus Ad-GFP and Ad-F7 versus Ad-GFP samples from Illumina bead array analysis presented in Fig. 1. p-Values for all genes are o 0.01with the exception of values in italic (t.v.: transcript variant).



Figure 3. Involvement of Noxa in IRF-3-mediated apoptosis. (A–D) Primary human MF were transduced or not with Ad-GFP, Ad-F3, Ad-F7 ortreated with IFN-a-2b for (D). (A) Transduced MF were plated with an isotype control (IgG) or a neutralizing antibody for the human IFNAR (anti-IFNAR2). Expression of human NOXA was analyzed 24 h later by real-time PCR. RQ was calculated by normalizing to BETA-ACTIN and Ad-GFP MF.Data are representative of four donors. (B) Cells were harvested at 24 h, incubated with the MitoProbeTM DilC1(5) dye, which accumulates inmitochondria with active membranes, and analyzed by flow cytometry. Data represent mitochondrial depolarization as the percentage of cell thathave lost MitoProbeTM DilC1(5) staining. Data are representative of two donors. (C) Whole-cell extracts (WCE) were prepared at 24 h and incubatedwith the fluorogenic substrate for caspase-3 (Ac-DEVD-AFC). Fold change (7SD; n 5 3) in caspase-3 enzymatic activity is represented by thefluorescence ratio between NT and transduced cells. Data are representative of two donors. (D) WCE were prepared at 24, 48 and 72 h, andsubjected to immunoblot analysis for caspase-3 and caspase-9 cleavage, Noxa expression and tubulin as loading control. Data are representativeof three donors. (E and F) WT and Noxa�/� (noted: �/�) BMK cells were transduced with Ad-GFP, Ad-F3 or Ad-F7. Cells were harvested at 24 h andanalyzed for (E) caspase-3 cleavage by immunoblot and tubulin as loading control and (F) mitochondrial depolarization as described in (B). Data arerepresentative of three independent experiments. (G) Primary human MF were electroporated with control siRNA (lane 1) or Noxa siRNA (lane 2)for 48 h before being transduced with Ad-GFP or Ad-F3. Cells were harvested 24 h post-transduction and subjected to immunoblot analysis as in (D)for caspase-3 cleavage, Noxa and tubulin as loading control. Noxa and cleaved-caspase-3 expression levels were quantified and normalized totubulin levels using the Scion Image 4.0 software. (H and I) 293 cells were transfected with plasmids harboring the luciferase reporter gene underthe control of (H) full length and deletion constructs of the NOXA promoter or of (I) the luciferase reporter plasmid-encoding tandem repeats of theIFN-stimulated response element (ISRE) and indicated IRF-3 or IRF-7-expressing plasmids. Relative luciferase activity (7SD; n 5 3) was analyzed24 h later after normalization with co-transfected Renilla luciferase encoding plasmid. Data are representative of three independent experiments.Casp: caspase.



Although poly(I:C) treatment did not induce a change in

BMK-cell morphology at this timepoint (Fig. 4A), caspase-3

cleavage was evident in WT cells and Noxa�/�cells reconstituted

with Noxa (1Noxa) (Fig. 4B). We next evaluated if the

ablation of Noxa in BMK cells had an effect on the capacity

of VSV to infect these cells. As demonstrated by RT-PCR

examining the expression of VSV-G mRNA, viral gene expression

occurred regardless of Noxa expression in these cells (Fig. 4C).

Futhermore, VSV-infected Noxa�/� cells continued to shed

virus for many days following infection (D.L., unpublished

observations).

To investigate the role of IRF-3 on cell death, human MF were

treated with the IRF-3 activator, poly(I:C). Increased expression of

ISG56 and Noxa, as well as caspase-3 cleavage, was observed by

immunoblot as early as 2 h post-treatment (data not shown), thus

suggesting a role for IRF-3 in the apoptotic response triggered in

the course of anti-viral defense. To further address this, WT and

Irf3�/� mouse embryonic fibroblasts (MEF) were transfected or

not with poly(I:C) for 20 h. While dsRNA treatment induced

massive cell death in WT MEF, the morphology of treated and non-

treated Irf3�/� MEF was nearly indistinguishable (Fig. 5A).

Observed differences in cytopathology correlated with the 75%

reduction in Noxa mRNA levels following 12 h of poly(I:C) treat-

ment in Irf3�/� MEF compared with WT MEF (Fig. 5B).

ChIP studies were performed to establish a direct association

of IRF-3 with the NOXA promoter in human HT1080 fibro-

sarcoma cells transfected with poly(I:C). A 4-fold increase in IRF-

3 binding to the NOXA promoter was observed within 2 h after

poly(I:C) treatment as compared with the 0 h control

(Fig. 5C). The IFNB promoter used as a positive control also

exhibited an increased IRF-3 association (Fig. 5C). Altogether,

these data support a direct involvement for IRF-3 in Noxa-

mediated apoptosis following viral infection.

Antigen cross-presentation in Ad-F7-transduced MU

From the microarray analysis, Ad-F7 but not Ad-F3 transduction

into primary human MF up-regulated the expression of genes

involved in antigen processing and presentation in the immuno-

logical synapse (Fig. 2). Transduction of active IRF-7 up-regulated

the expression of all three subunits of the immunoproteasome that

replace the catalytic 20S proteasomal-core: LMP2, LMP10 and

LMP7 as well as PA28A and -B subunits of the proteasome activator

(Fig. 2 and Fig. 6A). Furthermore, active IRF-7 induced the

expression of the TAP1 and TAP2 subunits of TAP responsible for

transporting peptides into the lumen of the ER (Fig. 2 and Fig.

6A). Co-stimulatory molecules of the MHC–peptide complex and

MF activation markers CD40, CD80 and CD86 were also up-

regulated in Ad-F7-transduced MF (Fig. 2 and Fig. 6C). The use of

a neutralizing antibody for the IFNAR2 in a second microarray

data set and in real-time PCR analysis following Ad-F7 transduc-

tion demonstrated that type I IFN mediated the increased

expression of genes involved in antigen processing and presenta-

tion (Fig. 6A, Supporting Information Figs. 2 and 3).

Given the increased expression of genes involved in antigen

processing and presentation following Ad-F7 transduction, we

sought to determine if Ad-F7 MF possessed an increased capacity

to cross-present exogenous antigens in the context of MHC class I

and to activate CD81 T cells in vitro. Murine peritoneal MF were

transduced with Ad-vectors, exposed to 0.125–2.0 mg/mL of

soluble chicken egg OVA and co-cultured with syngeneic naıve

OVA-specific MHC class I-restricted CD81 T lymphocytes isolated

from the spleen of transgenic mice that bear an MHC-class-I

Figure 4. Involvement of Noxa in dsRNA and VSV-induced apoptosis.WT and Noxa�/� BMK cells, or Noxa�/� BMK cells stably transfectedwith empty vector (1 Vec) or complemented with a Noxa expressionplasmid (1 Noxa) were treated with synthetic dsRNA (poly(I:C)) orinfected with VSV (MOI 0.1). Cell death was monitored 16 h post-infection by (A) morphological changes or (B) caspase-3 cleavage byimmunoblot analysis of WCE using actin as internal control. (C) Theexpression of VSV-G mRNA was assayed by RT-PCR 24 h after VSVinfection in these cells. Gapdh was used as an internal control. Data arerepresentative of three independent experiments.



restricted OVA-specific T cell recptor(OT-1) at an effector:target

(E:T) ratio of 1:2. Supernatants were assayed for IL-2 production

by activated OT-I T lymphocytes by ELISA at 48 h. The processing

and presentation of OVA by Ad-F7 MF increased the production of

IL-2 by OT-I cells in a dose-specific response; differences with Ad-

GFP-transduced MF were strongest when 0.5 and 1.0 mg/mL

of OVA were used (p-values 0.0049 and 0.049, respectively)

(Fig. 6B). Similar results were obtained at an E:T ratio of 1:6 (data

not shown). The increased cross-presentation capacity of Ad-F7-

transduced MF was mediated by type I IFN, as blocking the IFN

response using a neutralizing antibody specific for the murine

IFNAR1 strongly inhibited IL-2 production by OT-1 lymphocytes

(p-value 0.0006) (Fig. 6B). Proteasome-mediated proteolysis was

required for the increased antigen processing induced by active

IRF-7 as pre-treatment of cells with a proteasome inhibitor,

lactacystin, before OVA exposure completely blocked the cross-

presentation capacity of Ad-F7 MF (data not shown).

Next, we wanted to determine if the selective effect of Ad-F7

on cross-presentation may be skewed by higher antigen capture.

To evaluate antigen uptake, the capture of red-labeled (using

PKH-26 fluorescent dye) apoptotic CTLL2 cells (CTL line) by the

GFP-expressing MF was monitored by flow cytometry (Support-

ing Information Fig. 4). No significant differences in antigen

capture were observed in MF transduced with Ad-GFP or Ad-F7

(65 and 58% of double-positive (GFP and PKH-26) MF, respec-

tively). Taken together, these data demonstrated that Ad-F7

transduction and subsequent type I IFN production increased the

expression of components of the MHC class I peptide loading

machinery and the cross-presentation capacity of MF.

Increased activation of surrounding MU byAd-F7-transduced MU

As successful immunotherapeutic approaches rely on enhancing

immunogenicity to promote a protective host response to

malignancies, we next evaluated whether Ad-F7 transduction

increased the expression of immunomodulatory genes. Array

results demonstrated that Ad-F7 induced the expression of various

cytokines and chemokines including CXCL10 (IP-10) and CCL5

(RANTES) – monocytic and T-cell chemoattractants, IL15 and

IL15RA – critical regulators of NK and CD81 T-cell proliferation,

activation and survival [35] and TNFSF13B (also known as B cell-

activating factor (BAFF) or BLyS) and pre-B-cell-enhancing factor

(PBEF) – B-cell survival and growth factors [36, 37] (Fig. 2). The

expression of the IL-6/IL-12 familly member, IL27p28, was also

induced by Ad-F7 transduction (Fig. 2). With the exception of

TNFSF13B, expression of these genes was highly dependent on

IFN-a/b triggering of the IFNAR (Supporting Information Fig. 3).

To determine whether Ad-F7-transduced MF could induce the

maturation of surrounding MF through secretion of soluble

factors, primary human MF were transduced with Ad-GFP or

Ad-F7 or pre-treated with recombinant IFN-a-2b for 24 h,

extensively washed and placed in an upper chamber overlaying

non-treated MF for an additional 24 h; up-regulation of

maturation markers CD40, CD80 and CD86 was assessed by real-

time PCR. MF transduced with Ad-F7 (in the upper chamber)

displayed increased expression of CD40, CD80 and CD86, at

levels higher than observed with 1000 IU/mL of IFN-a-2b.

Furthermore, MF in the lower chamber acquired a more mature

phenotype when sharing supernatants with Ad-F7-transduced

MF compared with non-treated or IFN-a-2b-treated MF(Fig. 6C). Importantly, MF in the lower chamber did not express

GFP as asssessed by flow cytometry, demonstrating that increased

expression of co-stimulatory molecules was not a result of

adenovirus transduction in these cells (data not shown). As a

whole, these data demonstrated that Ad-F7-transduced MF were

Figure 5. Role of IRF-3 in dsRNA-mediated apoptosis and binding ofIRF-3 to the Noxa promoter. (A and B) WT and Irf3�/� MEF weretransfected with 10 mg of poly(I:C) using lipofectamine 2000 (Lipo).(A) Cell death was monitored by morphological changes at 20 h and(B) expression levels of murine Noxa and Ifnb mRNA was determined byreal-time PCR 12 h post-transfection. RQ was calculated by normalizingto Gapdh and Lipo samples. (C) IRF-3 binding to the NOXA and IFNBpromoter. HT1080 human fibrosarcoma cells were left untransfected ortransfected with dsRNA using Lipofectin for 2 or 4 h. ChIP studies wereperformed with normal rabbit serum (IgG) or IRF-3 polyclonal Ab. Dataare representative of three independent experiments.



capable of inducing the maturation of surrounding MF via the

secretion of immunostimulatory factors.

Increased anti-tumor properties of Ad-F7-transducedmurine MU in vivo

As Ad-F7 was proven efficient in increasing the in vitro cytostatic

activity of primary human MF against breast cancer cell lines

SK-BR-3 and MCF-7 (breast cancer cells) [32], the anti-tumor

activity of Ad-F7-transduced MF was assessed using the

aggressive and poorly immunogenic B16.F0 melanoma model.

First, the effect of IRF-7 on the in vitro inhibitory effect of MF was

tested in co-cultures of Ad-GFP or Ad-F7-transduced syngeneic

peritoneal murine MF with B16.F0 cells at different E:T ratios.

Ad-F7-transduced MF strongly inhibited the proliferation of B16

cells (>70% at E:T ratios of 1:1 and 3:1) after 4 days of co-

culture, whereas GFP-expressing MF displayed a very limited

Figure 6. Increased antigen cross-presentation by active IRF-7-expressing MF. (A) Ad-GFP, Ad-F3, Ad-F7-transduced or IFN-a-2b-treated primaryhuman MF were plated with an isotype control (IgG) or a neutralizing antibody for the human IFNAR (anti-IFNAR2). Expression of human LMP2,LMP7, LMP10, PA28B and TAP1 was analyzed 24 h later by real-time PCR. RQ was calculated by normalizing to GAPDH and Ad-GFP MF. (B) NT, Ad-GFP, Ad-F7-transduced or recombinant murine IFN-a-treated peritoneal MF were plated and cells in the right panel were treated with controlantibody (Ab) or a neutralizing antibody for the murine IFNAR (anti-IFNAR1). Twenty-four hours post-transduction, MF were presented with0.125–2.0 mg/mL (1.0 mg/mL for the right panel) of OVA for 8 h and washed thoroughly. OT-1-derived CD81 T cells were incubated at a 1:2 E:T ratiowith Ad-IRF-transduced MF. Graph represents the average murine IL-2 in pg/mL (7SD) in supernatants collected at 48 h as assayed by ELISA(�po0.05; ��po0.005 with n 5 3). Data are representative of three independent experiments. (C) Primary human MF were transduced with Ad-GFP,Ad-F7 or treated recombinant human IFN-a-2b. 24 h post-treatment, cells were harvested and extensively washed to remove IFN-a-2b from themedia. MF were then placed on 0.02 mm Anopores membranes over non-treated (NT) MF. Twenty four-hours post-co-culture, MF from top andbottom chambers were harvested and the expression of CD40, CD80 and CD86 mRNA was analyzed by real-time PCR as in (A). Data arerepresentative of three independent experiments.



ability (o18%) to inhibit their proliferation (Fig. 7A). Significant

growth inhibition (45%; SD79.8) by Ad-F7-transduced MF also

occurred at a ratio as low as 1:3 (Fig. 7A).

In light of these observations, immunocompetent C57BL/6

mice were inoculated subcutaneously with B16 tumor cells

together with Ad-GFP or Ad-F7-transduced syngeneic peritoneal

MF and tumor growth was monitored over time. Co-injection of

B16.F0 with Ad-F7-transduced MF but not Ad-GFP-transduced

MF resulted in a significant delay in tumor growth (p-value

B161MF Ad-GFP versus B161MF Ad-F7 5 0.04 on day 17 post-

injection) (Fig. 7B). Similar results were also observed in a

separate experiment (p-value 0.06 (data not shown)). In sum, the

constitutively active form of IRF-7 was able to increase the anti-

tumor effector functions of MF and limit tumor growth in vivo.

Discussion

Transcription factors IRF-3 and IRF-7 share many structural and

regulatory features [13]. Despite these similarities, functional

distinctions can be identified between these two anti-viral

regulators. In this report, we demonstrated that delivery of the

constitutively active IRF-3 to primary human MF using an

adenoviral vector rapidly induced cell death through the strong

up-regulation of the pro-apoptotic protein Noxa. In contrast,

expression of the active form of IRF-7 increased the expression of

a broad range of ISG, and augmented the cross-presentation

capacity and tumoricidal activity of MF, leading to an anti-tumor

response against B16 melanoma in vivo.

Genome-wide microarray analysis revealed that Pmaip1/

Noxa, a pro-apoptotic BH3-only Bcl-2 family member, was

preferentially up-regulated by Ad-F3 compared with Ad-F7.

Pmaip1/Noxa was previously identified as an IRF-3 5D target

gene in Jurkat T cells [29]. Noxa is proposed to mediate apoptosis

by interacting with and inhibiting pro-survival Bcl-2 family

members such as Mcl-1, which leads to the displacement and

activation of Bax/Bak [38, 39]. Activated Bax/Bak permeabilizes

the mitochondrial membrane resulting in cytochrome C release,

caspase-9 and -3 cleavage and cell death. Inhibition of Noxa

expression by siRNA delivery in human MF or gene ablation in

murine cells reduced apoptosis to levels seen in control samples

upon Ad-F3 tranduction, VSV infection or exposure to dsRNA.

Although NOXA had been implicated previously as a dsRNA/

virus-induced gene [40], the current studies implicated a direct

role for IRF-3 in this process by demonstrating binding of IRF-3 to

the NOXA promoter in the region spanning �1413 to �642.

These results are consistent with previous studies demonstrating

that the transcriptional activation of NOXA by VSV was required

for efficient virus-induced apoptosis and necessitated the binding

of IRF-1, IRF-3 and CREB [33]. Hence, the increased expression

of Noxa following the activation of IRF-3 in MF may tip the

balance among Bcl-2 family members toward a pro-apoptotic

response. Interestingly, we observed that re-introduction of Noxa

in Noxa�/� BMK cells restored apoptotic responses after virus

infection or dsRNA treatment, suggesting either that additional

pro-apoptotic factors are involved in the ability of virus/dsRNA to

induce apoptosis, or that secondary modification to Noxa is

important for full activation of the apoptotic pathway following

virus or dsRNA stimulation.

In contrast to Ad-F3, transduction of MF with Ad-F7 was not

pro-apoptotic but led to increased MF effector functions,

including an augmentation of tumoricidal activity, cross-presen-

tation capacity and in vivo protection against the aggressive B16

melanoma cell growth. Functional consequences of Ad-F7 trans-

duction in MF correlated with the broad and potent induction

capacity of this transcription factor as demonstrated by the

microarray data. Ad-F7 transduction in MF increased the

Figure 7. Ad-F7-transduced MF exert anti-tumor properties in vitroand in vivo. Murine peritoneal MF were harvested and transduced withAd-GFP or Ad-F7 as described in the Materials and methods. (A) MF wereseeded in 96-well plates together with B16.F0 tumor cells (1.25� 103) ateffector/target ratios of 1:3, 1:1 or 3:1 for 4 days. MTS tetrazoliumcompound was added to cells for 1 h and bioreduction to coloredformazan was assayed by recording the absorbance to determine thepercentage of inhibition of B16.F0 tumor-cell proliferation. Datarepresent the percentage inhibition of tumor-cell proliferation accord-ing to non-treated cells (7SD; n 5 3). (B) 5�105 Ad-GFP and Ad-F7-transduced MF were subcutaneously co-injected with 5�105 B16.F0tumor cells in C57BL/6 mice (n 5 4�5). Tumor growth, represented asmean tumor size 7SEM, was monitored over time following injectionusing a digital caliper. �po0.05; ��po0.005.



expression of tumoricidal molecules (TRAIL and IFN-a/b) [32]

and important immunomodulatory cytokines and chemokines

(IP-10, RANTES, IL-15, IL-27p28, BAFF and PBEF). In addition,

transduction with Ad-F7 in MF induced the expression of genes

involved at different steps of MHC class I-mediated antigen

processing, including genes regulating proteasome protein

degradation (LMP2, LMP10 and LMP7), proteasome activation

(PA28-a/b), and peptide translocation to the ER (TAP-1 and -2).

Increased expression of LMP2, LMP7 and LMP10 is particularly

important because these subunits displace the constitutive

proteasomal subunits b1, b2 and b5 and are incorportated

into an alternative form of the proteasome known as the immu-

noproteasome, which alters the catalytic site and possesses an

increased ability to produce peptides with a proper motif

for efficient binding to MHC class I molecule [41–45].

Increased expression of genes involved in antigen processing was

complemented by the up-regulation of T lymphocyte

co-stimulatory molecules CD40, CD80 and CD86, suggesting

that Ad-F7-transduced MF may acquire higher cross-priming

abilities.

Qualitative and quantitative differences in the type I IFN loop

induced following IRF-3 or IRF-7 activation may play a role in their

distinct biological functions. However, our data suggest that the

main mechanism by which IRF-3 or IRF-7 regulate cell viability

may not be, as previously reported, via the IFN-b-mediated up-

regulation of p53 [46] and/or p53-induced Noxa expression [34].

Ad-F7-transduced MF produced higher levels of IFN-b compared

with Ad-F3 MF. In addition, p53 mRNA up-regulation was not

observed in microarray and real-time PCR analyses (data not

shown). These data support work demonstrating that apoptosis

induced by dsRNA or Newcastle Disease Virus was independent of

type I IFN and that the activity of p53 was not required for IRF-3

5D-mediated apoptosis [47]. Furthermore, the expression of Noxa

does not appear to be regulated by the type I IFN loop, because

neutralizing the IFNAR did not alter Noxa expression levels

following Ad-F3 transduction and increased expression of Noxa

following recombinant IFN-a treatment was not observed. This

result is consistent with earlier studies using IFN-a-unresponsive

mutant human cell lines or Jurkat T cells treated with neutralizing

antibodies for IFN-a/b that still exhibited Noxa up-regulation in

response to dsRNA or IRF-3 5D expression, respectively [29, 40].

In contrast, many of the acquired immune and anti-tumor func-

tions of Ad-F7 MF are likely dependent on the type I IFN loop, as

demonstrated by the neutralization of IFNAR, consistent with

previous studies demonstrating that type I IFN favors the differ-

entiation of monocytes to DC [9–12], the generation of the

immunoproteasome following virus infection [48], the cross-

priming of CD81 T cells [49, 50], as well as increased in tumor-

icidal effector functions of DC [51–53].

The consequences of IRF-3- and Noxa-induced cell death,

appear independent of the type I IFN loop, and the relationship to

the control of viral replication and the development of an

immune response remain to be elucidated. One hypothesis is that

Noxa and the mitochondrial apoptotic pathway may be required

for the full activation of IRF-3, as this activation was reduced in

Bax�/�MEF following virus infection [54]. A second hypothesis is

that induction of Noxa by IRF-3 triggers apoptosis in infected

cells in order to clear these rapidly. It was recently reported that

IRF-3-deficient cells do not enter apoptosis when infected with

Sendai virus and continuously produce virus [55]. We have

observed a similar response to VSV in Noxa�/� cells, which fail to

undergo rapid apoptosis following infection and continue to shed

virus for many days thereafter (D.L., unpublished observations).

Together, these findings suggest that IRF-3 and Noxa may control

cell viability in order to avoid in vivo viral persistence.

Our data highlight the importance of the type I IFN loop

induced by IRF-7 in the acquisition of tumoricidal and cross-

priming functions by MF, suggesting their potential in the

development of anti-tumor immunotherapies. Compared with

systemic delivery of IFN-a or pre-treatment of MF with IFN-a, the

intra-tumoral injection of MF transduced with Ad-F7 may result

in an improved immunity and tumor growth inhibition via the

release of type I IFN in the tumor environment, the full induction

of MF effector functions, activation of surrounding APC and

effector immune cells capable of directly mediating tumor cell

killing, including cytotoxic T lymphocytes.

Materials and methods

MU preparation

PBMC were isolated by Ficoll gradient from fresh apheresis with

informed and institutionally approved consent forms from

healthy donors. Monocytes were isolated from PBMC by magnetic

cell sorting using anti-CD14-conjugated microbeads (Stem Cell

Technologies, Vancouver, Canada) and Automacs (Miltenyi

Biotec Auburn, CA, USA) and cultured for 7 days in Iscove media

(Wisent Technologies, Rocklin, CA, USA) supplemented with 2%

human serum type A/B (Wisent Technologies), 700 U/mL of GM-

CSF (a generous gift from Cangene Corporation, Mississauga,

Canada) and antibiotics in gas permeable thermoplastic non-

adherent culture bags (EVAMs, IDM, Paris, France). On day 7,

MF were harvested and re-suspended in complete McCoy’s 5A

medium (supplemented with 10% FBS and antibiotics) (Wisent

Technologies). MF differentiation and purity were analyzed by

flow cytometry as in [32]. Peritoneal MF were exuded from

C57BL/6 (retired breeders) mice (Charles River Laboratories,

Wilmington, MA, USA) by sterile lavage, plated in complete

DMEM (Wisent Technologies), allowed to attach for 2 h and

extensively washed to remove non-adherent cells.

Adenoviral vectors and transduction

Construction, production and purification of the three adenoviral

vectors Ad-GFP, Ad-GFPq/IRF-3 5D (Ad-F3) and Ad-GFPq/IRF-7

D247–467 (Ad-F7), as well as transduction of primary MF and

cell lines used in this study were performed as previously

described in [32].



Mice and cell lines

Retired breeders and 4–6-wk-old female C57BL/6 mice (Charles

River Laboratories) and OT-I mice: C57BL/6-Tg (TcraTcrb)

1100Mjb/J (Jackson Laboratory, Bar Habor, ME, USA) were

handled according to institutionally approved Animal Use

Protocol. Tumor size was measured using an electronic caliper

and calculated using the formula (length�width2/2). The

C57BL/6-derived B16.F0 melanoma cell lines, 293 human

embryonic kidney cells, HT1080 fibrosarcoma cells and WT and

Irf3�/� MEF [26] were cultured in complete DMEM (Wisent

Technologies). WT and Noxa knock-out BMK cells were a gift

from E. White (Rutgers University, Piscataway, NJ, USA), were

established as described previously [56] and cultured in complete

DMEM. Early passage Noxa knock-out BMK cells were stably

transfected with empty pBabepuro plasmid (Noxa�/�1 Vec cells),

or Noxa pBabepuro (Noxa�/�1 Noxa) as described previously

[40]. Results from representative clones are presented.

Reagents

Nuclear staining was done using To-PROs-3 iodide (Molecular

Probes, Invitrogen, Carlsbad, CA, USA) according to the

manufacturer’s instructions, and confocal analysis was

done as described in [57]. Apoptotic MF were analyzed by

MitoProbeTM DilC1(5) staining to detect active mitochondrial

membranes (Molecular Probes, Invitrogen). Fourteen percent

acrylamide gels were run when blotting for Noxa (114C307,

Calbiochem, Merck KgaA, Darmstadt, Germany) caspase-3,

cleaved-caspase-3 Asp175, caspase-9 (]9662, ]9661 and ]9502,

respectively, Cell Signaling Technology, Danvers, MA, USA) and

a-tubulin (B-7) (sc-5286, Santa Cruz, Santa Cruz, CA, USA).

Caspase activity was determined as previously described [31]

using Ac-DEVD-AFC (Biomol International, L.P. Plymouth

Meeting, PA, USA). MF were treated with 1000 IU/mL

of recombinant human IFN-a-2b (IntronsA, Schering, Pointe-

Claire, Canada) or 2000 IU/mL of recombinant murine IFN-a(Millipore, Temecula, CA, USA). Human IFNAR was neutralized

using mouse monoclonal antibody against human IFN-a/breceptor chain 2 (anti-IFNAR2) (PBL Biomedical Laboratories,

Piscataway, NJ, USA) or treated with control isotype (IgG2a)

(eBioscience, San Diego, CA, USA) at 20 mg/mL. For cross-

presentation study, purified mouse monoclonal antibodies

specific for the murine IFNAR1 (MARI-5A3) and control antibody

against human IFNGR-1 (GIR-208) (generous gifts from R.D.

Schreiber’s laboratory (St. Louis, MO, USA) [58]) were used at

10 mg/mL. Poly(I:C) (Sigma-Aldrich, St-Louis, MO, USA) and

lipofectamine 2000 (Invitrogen) were also used in this study.

BMK cells were infected with 0.1 MOI of VSV-HR strain (Indiana

serotype). Anopores 0.02 mm membranes (NuncA/S, Roskilde,

Denmark) were used for MF maturation study. Proliferation

assay was performed as described in [32] using CellTiter 96s

Aqueous One Solution Cell Proliferation Assay (MTS assay:

[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA) 4

days after B16 and murine MF co-culture.

RNA preparation and bead array gene expressionanalysis

DNase-treated total RNA was prepared as described in [32].

Sample amplification for microarray analysis was performed using

200 ng of total RNA using Illuminas Total Prep RNA Amplification

Kit (Ambion, Austin, TX, USA). Samples were labeled by

incorporating biotin-16-UTP (Enzo Life Sciences, Farmingdale,

NY, USA) at a 1:1 ratio with unlabeled UTP. Eight hundred and

fifty nanograms of labeled, amplified material was hybridized to

the Illuminas HumanRef-8 BeadChip according to the manufac-

turer’s instructions (Illumina, San Diego, CA, USA). The signal was

then developed with streptavidin-Cy3 (Amersham, GE Healthcare

Bio-sciences, Little Chalfont, UK) and the BeadChip was scanned

with the Illuminas BeadArray reader. After scanning, microarray

data quality was checked by evaluating chip-dependent (biotin,

hybridization, stringency) and sample-dependent (gene intensity,

labeling, background) controls and by comparing sample distribu-

tions and statistics.

Pre-processing, analysis and clustering of microarraydata

Pre-processing was done in R (http://www.r-project.org/) using

the Linear Models for Microarray Analysis (LIMMA) package.

Microarray data were quantile normalized and values smaller

than the background cutoff were replaced with this value. The

24 354-row matrix was reduced by filtering non-expressed genes,

i.e. genes with background value for all samples. A data matrix of

8519 genes (rows) by 28 samples, 4 conditions�7 donors

(columns) was constructed. LIMMA was used to identify

significant differentially expressed genes [59]. Four contrasts of

interest, i.e. NT versus Ad-GFP, Ad-F3 versus Ad-GFP, Ad-F7 versus

Ad-GFP and Ad-F7 versus Ad-F3 were defined. The data were

log2-transformed before fitting a linear model for each gene. To

control the proportion of false-positive calls in all positive calls at

the desired significance level, the p-value was adjusted by

applying a false discovery rate of 0.01. Hierarchical gene average

linkage clustering was performed using Genesis (developed by

Alexander Sturn, [60]). Microarray data have been deposited in

the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.-

gov/geo/) under accession no. GSE12120.

RT-PCR and real-time PCR

Total RNA was reverse transcribed as described in [32]. Real-time

PCR primers were designed using the primer3 website

(primer3_www.cgi v. 0.2) and listed in Supporting Information



Table 1. Human NOXA, GAPDH and murine Gapdh primers

used are described in [54, 61, 62], respectively. cDNA was

amplified using SyBR Green I PCR master mix (Applied

Biosystems, Foster City, CA, USA) and the data were collected

using the AB 7500 Real-Time PCR System (Applied Biosystems)

and analyzed by Comparative CT Method using the SDS v1.3.1

Relative Quantification (RQ) Software. For semiquantitative

RT-PCR, amplification products were resolved on an agarose

gel and digital image of the ethidium bromide stained bands

inverted for presentation.

ChIP

Cellular proteins were cross-linked to chromatin with 1%

formaldehyde for 30 min at 371C. After sonication, protein–DNA

complexes were immunoprecipitated from nuclear extracts by

using human IRF-3 antiserum (M. David, University of California,

San Francisco, CA, USA) or normal rabbit serum, followed by

capture on protein A sepharose beads (GE Healthcare). After IP

and elution from the resin, DNA–protein cross-linking was

reversed with 250 mM NaCl at 651C for 16 h and the DNA was

purified using the Qiaquick PCR Purification kit (Qiagen). PCR

amplification of NOXA and IFNB gene sequences was performed

using primers detailed in Supporting Information Table 1.

Cross-presentation assay

Twenty-four hours post-transduction and antibody treatments,

MF were presented with OVA (Sigma, Oakville, Canada) for 8 h

and washed thoroughly. CD81 T cells were isolated from the

spleen of OT-I mice and purified using the EasySeps mouse CD8

positive selection kit (Stem Cell Technologies). OT-1 mice

contain a transgenic T-cell receptor designed to recognize OVA

residues 257–264 in the context of H-2 Kb MHC class I molecules.

Supernatants were collected after MF and T-cell co-culture in

96-well plate and assayed by ELISA for IL-2 production

(eBioscience).

Luciferase assays, plasmids and siRNA

For reporter gene assay, 293 cells (1�105 in 24-well plate) were

transfected using lipofectamine 2000 (Invitrogen) with 500 ng of

IRF-3 WT, IRF-3 5D, IRF-7 WT and IRF-7 D247–467 pEGFP-C1

constructs as previously described in [16–18]. Two hundred

nanogram of pRLTK reporter (Renilla luciferase for internal

control) and 200 ng of pGL-3 plasmids harboring the Firefly

luciferase reporter gene expressed under the control of the NOXA

promoter. The full-length (1413 bp) fragment of the NOXA

promoter was amplified by PCR from human Hela cell genomic

DNA, and the PCR product cloned directly into pGEM-T easy

vector (Promega). After sequence confirmation, promoter

sequences were liberated from pGEM-T easy with EcoRI, blunted

with Klenow fragment and ligated into a blunted SacI site in

pGL3-Basic by blunt-end ligation. Proper promoter orientation

was confirmed by restriction analysis and sequencing. The Noxa

promoter/luciferase deletion constructs were generated by PCR

using the �1413 pGL3-Basic-Noxa-promoter as template and the

forward primers (Supporting Information Table 1). A common

reverse primer (Supporting Information Table 1) within the

luciferase gene was used to generate the truncated PCR products,

which were then digested with BglII and NcoI and directionally

ligated into pGL3-basic. Primary human MF were electroporated

as described previously in [62] using Noxa-specific RNAi

sequences described in [33], plated for 48 h and transduced for

24 h with Ad-vectors.

Acknowledgements: Grant support: Canadian Institutes of

Health Research (CIHR), the National Cancer Institute of

Canada, with the support of the Canadian Foundation for AIDS

Research. CIHR Senior Investigator Award (J. Hiscott) and Senior

Chercheur Boursier Award (R. Lin). NIH grant R01 AI068133

(D. Leaman). The authors thank the following people for their

help on specific assays: Alessandra Nardin with primay human

macrophage preparations, Moıra Franc-ois with cross-

presentation assays, Simon Leveille, Moutih Rafei and Tiejun

Zhao for the B16 murine model, Genevieve Boucher and Peter

Wilkinson for microarray data analysis, Tiannan Chen provided

help with Western blots and Guy Klaiman for caspase enzymatic

activity assays.

Conflict of interest: The authors declare no financial or

commercial conflict of interest.

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Abbreviations: BMK cells: baby mouse kidney cells � DEVD-AFC:

N-acetyl-Asp-Glu-Val-Asp-AFC (7-amino-4-trifluoromethyl coumarin) �IFNAR: type I IFN receptor � ISG: IFN-stimulated gene � MEF: mouse

embryonic fibroblasts � NT: non-transduced � RQ: relative

quantification � VSV: vesicular stomatitis virus � WCE: whole-cell

extracts

Full correspondence: Dr. John Hiscott, Lady Davis Institute, Jewish

General Hospital, McGill University 3999, Chemin de la Cote Ste-

Catherine Montreal, Quebec H3T 1E2, Canada

Fax: 11-514-340-7576

e-mail: [email protected]

Additional correspondence: Dr. Douglas Leaman, Department of

Biological Sciences, University of Toledo 2801, W. Bancroft Toledo, OH

43606, USA

e-mail: [email protected]

Supporting Information for this article is available at

www.wiley-vch.de/contents/jc_2040/2009/38832_s.pdf

Received: 15/8/2008

Revised: 13/11/2008

Accepted: 28/11/2008



Transcriptional re-programming of primary macrophages reveals distinct apoptotic and anti-tumoral...

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