Poly-I:C Decreases Dendritic Cell Viability Independent of PKR Activation

doi:10.4236/jibtva.2012.11001

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Journal of Immune Based Therapies, Vaccines and Antimicrobials, 2012, 1, 1-6

http://dx.doi.org/10.4236/jibtva.2012.11001 Published Online April 2012 (http://www.SciRP.org/journal/jibtva)

Poly-I:C Decreases Dendritic Cell Viability Independent of

PKR Activation

Hjalte List Larsen, Anders Elm Pedersen

Department of International Health, Immunology and Microbiology, The Panum Institute,

University of Copenhagen, Copenhagen, Denmark

Email: hjaltel@sund.ku.dk, [email protected]

Received March 1, 2012; revised April 2, 2012; accepted April 12, 2012

ABSTRACT

Vaccination with tumor-antigen pulsed, monocyte-derived dendritic cells (DCs) has emerged as a promising strategy in

cancer immunotherapy. The standard DC maturation cocktail consists of a combination of tumor necrosis factor-α

(TNF-α)/interleukin (IL)-1β/IL-6 and prostaglandin E2 (PGE2) for generation of standard DCs (sDCs). In order to im-

prove IL-12p70 production and cytotoxic T-lymphocyte (CTL) induction, a novel cocktail composed of TNF-α/IL-1β/

interferon (IFN)-α/IFN-γ and polyinosinic:polycytidylic acid (Poly-I:C) has been introduced to generate so-called

α-Type-1 polarized DCs (αDC1s). We and others have previously performed a comprehensive comparison of sDCs and

αDC1s. Here we demonstrate that the viability of αDC1s is lowered compared to sDCs and that DC apoptosis is medi-

ated by Poly-I:C. We speculated that activation of protein kinase R (PKR) could mediate the observed apoptosis, but

despite significantly higher PKR expression in αDC1s compared to sDCs and induction of active threonine (Thr)446

autophosphorylation of PKR in αDC1s, Poly-I:C did not influence total PKR expression or autophosporylation, indi-

cating PKR-independent Poly-I:C-induced DC apoptosis.

Keywords: Cancer Immunotherapy; Dendritic Cells; Poly-I:C; PKR

1. Introduction

In therapeutic cancer vaccination with monocyte-derived

DCs [1], maturation is mandatory for proper T-cell

stimulation [2]. DCs maturated according to the standard

cytokine cocktail consisting of TNF-α/IL-1β/IL-6 and

PGE2 [3] show high expression of maturation markers

CD80, CD83, CD86, CD134L and CCR7, but low secre-

tion of IL-12p70 [4] which may have negative conse-

quences for activation of appropriate T cell responses

such as TH1 cells and cytotoxic T lymphocytes (CTLs).

In the search of cytokine cocktails that increase DC

IL-12p70 secretion, DCs maturated according to the

α-Type-1 polarizing cocktail containing TNF-α/IL-1β/

Poly-I:C/IFN-α and IFN-γ were developed, as these DCs

produce large amounts of IL-12p70 [4,5]. We and others

have characterized cytokine secretion and capacity for T

cell activation in details elsewhere [4,5] and it has been

found that αDC1s may have a reduced capacity for T cell

activation under certain conditions [4]. In addition to the

criterias for successful DC generation investigated else-

where, viability is an important factor. Here, we report a

decreased viability of αDC1s and investigate a potential

role of PKR mediated mechanisms.

The double-stranded RNA (dsRNA)-analog Poly-I:C

included in the α-Type-1 polarizing cocktail is a potential

inducer of apoptosis, as cytosolic Poly-I:C is capable of

activating the dsRNA-activated protein kinase (PKR) [6].

Upon dsRNA/Poly-I:C binding, PKR is autophosphory-

lated on multiple sites including Thr446 and Thr451 [7],

enabling signal transduction which can induce global

translation inhibition and apoptosis [8]. Furthermore,

Poly-I:C can be detected by toll-like receptor 3 (TLR3)

in the endosomal compartment of DCs [9] and it has

been reported that PKR is recruited to a large signaling

complex following Poly-I:C binding to TLR3 [10]. We

thus speculated that Poly-I:C is the cause of lowered

αDC1 viability as compared to sDCs, and that such a Poly-

I:C-mediated induction of apoptosis was mediated by

PKR activation. In this case, siRNA mediated silencing

of PKR-dependent apoptotic pathways could be a future

strategy for improved efficacy of therapeutic αDC1s.

Here, we report that absence of Poly-I:C from the

α-Type-1 polarizing cocktail significantly improved αDC1

viability to levels comparable to sDCs without signifi-

cantly decreasing CD80, CD83 and CD86 expression.

αDC1s expressed significantly higher amounts of PKR

than iDCs and sDCs, with PKR expression being de-

pendent on the presence of IFNs in the maturation cock-

tail but not on Poly-I:C. We were also unable to detect an

H. L. LARSEN ET AL.

effect of Poly-I:C on PKR autophosphorylation in mature

DCs, suggesting that the observed lowered viability of

αDC1s was PKR-independent.

2. Methods

2.1. Generation of Human Monocyte-Derived

DCs

Human monocyte-derived DCs were generated as previ-

ously described with GM-CSF + IL-4 [11] from buffy

coats after informed consent from healthy donors at the

local department of clinical immunology. Maturation of

iDCs was performed on day 7. sDCs were maturated

with TNF-α (10 ng/mL), IL-1β (10 ng/mL), IL-6 (10

ng/mL) (all cytokines from Peprotech) and 1 μg/mL

PGE2 (Prostin E2, Pfizer). The αDC1 phenotype was

induced by TNF-α (10 ng/mL), IL-1β (10 ng/mL), IFN-α

(10 ng/mL), IFN-γ (10 ng/mL) (all cytokines from

Peprotech) and 20 μg/mL Poly-I:C (Sigma Aldrich). DCs

were maturated 48 hours prior to analysis.

2.2. Flow Cytometry

Flow cytometry was performed using antibodies against

the following surface proteins: CD11c, CD80, CD83 and

CD86 (BD-Pharmingen). Isotope control antibodies were

obtained from DAKO. Detection of apoptotic DCs were

performed using annexin V-FITC apoptosis detection Kit

I (BD Pharmingen) according to the manufacturer’s pro-

tocol. Data acquisition and analysis was performed on a

FACSCalibur flow cytometer (BD) using CellQuest

software (BD Biosciences). DCs were identified as the

large granular cell population based on forward scatter

(FSC)/side scatter (SSC) linear dot plots.

2.3. Western Blotting

DCs were lysed in RIPA buffer supplemented with pro-

teinase inhibitor cocktail (Sigma Aldrich), PMSF, NaF

and Na3VO4 during 20 minutes of centrifugation at

14,000 g, 4˚C. Lysates were treated with SDS and heated

for 5 minutes prior to loading onto 4% - 12% Bis-Tris

gels (Invitrogen). Electrophoresis was performed in MES

running buffer (Invitrogen) for 1 hour at 200 V. Blotting

was performed at 30 V for 1 hour onto nitrocellulose

membranes (Invitrogen) in transfer buffer (Invitrogen).

Membranes were blocked and probed with polyclonal

goat anti-pPKR (Thr446) antibody, polyclonal rabbit

anti-PKR antibody (both Santa Cruz Biotechnology, Inc.)

or goat anti-GAPDH antibody (AbCam) over night at 4˚C.

Secondary horse radish peroxidase (HRP)-conjugated

polyclonal antibodies (DAKO) were added and the

membrane was incubated 1 hour at room temperature.

The membrane was washed and visualized using an en-

hanced chemiluminescence detection system (ECL plus

Western Blotting Detection system, GE Healthcare) ac-

cording to the manufacturer’s instructions. Detection of

HRP-generated precipitates was performed on a Typhoon

Scanner 9410 with Typhoon Scanner software (both from

Amersham Biosciences). The same software was used to

analyze band intensity, with PKR and pPKR intensity

being normalized to GAPDH expression.

3. Results

3.1. DC Phenotype after Maturation

Day 7 IL-4 and GM-CSF differentiated monocyte-de-

rived DCs were identified as large granular cells ex-

pressing the DC marker CD11c (Figures 1(A) and (B)).

Maturation using the two established cocktails for gen-

eration of sDCs and αDC1s both significantly increased

the expression of CD80 (p < 0.05 and p < 0.01 respec-

tively) and CD83 (p < 0.05 and p < 0.05 respectively)

compared to iDCs, while a stable high expression level

of CD86 was maintained from iDCs into mature DCs.

CD83 expression in sDC was less pronounced in these

DCs from healthy donors as compared to our previous

results from cancer patient derived DCs [4]. Removal of

individual cytokine components from the α-Type-1 po-

larizing cocktail did not induce any significant changes

in the expression of the three maturation markers, how-

ever absence of Poly-I:C or IFN-α reduced the expres-

sion of both CD80 and CD83 compared to expression

levels in αDC1s. These observed reductions in CD80 and

CD83 expression were not statistical significant but con-

sistent in all donors. It was previously published that

IL-12p70 secretion was dependent on IFN-γ or Poly-I:C

with the largest contribution from IFN-γ [5]. For T cell

activation, αDC1s have been shown to be superior for

CTL activation [5], whereas assays mostly reflecting

CD4+ T cell responses has demonstrated a decreased

capacity for T cell activation as compared to sDCs [4].

3.2. Viability of ex Vivo Generated DCs

Annexin V-FITC and propidium iodide (PI) staining re-

vealed a tendency toward that sDCs were more viable

(mean of all donors: 79.4% living cells) as compared to

αDC1s (mean of all donors: 71.7% living cells), a dif-

ference that was not statistical significant (p = 0.069) but

consistent in all donors. Data from one donor is shown in

Figure 2(A) and mean of all donors in Figure 2(B). In

order to identify the cause of the viability changes in

αDC1s, all single components from the cocktail was sys-

tematically excluded (Figure 2(B)). Exclusion of Poly-I:C

from the α-type-1 polarizing cocktail significantly im-

proved the viability of DCs from a mean of 71.7%

(αDC1s) to 79.9% living cells (p = 0.02), thus increasing

DC viability to a level comparable to sDCs. The viability

H. L. LARSEN ET AL.

(A) (B)

(C)

Figure 1. DC Phenotype. (A) DCs were initially identified as the large granular population based on flow cytometry FSC/SSC

plots. Flow cytometric analysis was performed in order to determine the surface expression of the maturation markers CD80,

CD83 and CD86 on CD11c + DCs; (B) The percentage of CD11c+ DCs staining positive of individual maturation markers in

one representative of 4 donors. a: iDCs, b: sDCs: c: αDC1s, d: αDC1s – Poly-I:C, e: αDC1s – TNF-α, f: αDC1s – IL-1β, g:

αDC1s – IFN-α, h: αDC1s – IFN-γ; (C) Mean percentage of maturation marker positive CD11c+ DCs maturated according to

the indicated cytokine cocktails (n = 4). (*) indicates significant different maturation marker expression compared to iDCs (p

< 0.05), two-sided Students t-test.

of DCs maturated with all other α-Type-1 polarizing

cocktail derivatives containing Poly-I:C was also reduced

compared to sDCs and αDC1s without Poly-I:C (Figure

2(B)). Exclusion of other cytokines from the α-Type-1

polarizing cocktail did not significantly alter the per-

centage of viable DCs. However, inclusion of Poly-I:C in

a maturation cocktail does not seem to reduce DC capa-

city for overall in-vitro allogeneic T cell stimulation

(data not shown).

3.3. Poly-I:C Mediated Effects on PKR

Expression and Activation

Reports of PKR activation upon direct binding of cytoso-

lic Poly-I:C, and recruitment of PKR to a signaling com-

plex binding to the cytosolic tail of TLR3 following

Poly-I:C binding in the endosomal compartment, led to

the speculation that the apparent Poly-I:C mediated re-

duction in DC viability might be caused by activation of

PKR. Here we demonstrate that PKR expression in sDCs

was not elevated as compared to iDCs (Figures 3(A) and

(B)). In contrast, PKR expression was elevated in αDC1s

as compared to iDCs and sDCs (p < 0.05). However, no

significant difference in PKR expression was found be-

tween DCs maturated using the αDC1 cocktail with (14.7

fold increase) or without Poly-I:C (15.2 fold increase),

suggesting that Poly-I:C has no effect on total DC PKR

expression. In contrast, PKR expression of αDC1s was

dependent on IFN-α- (p < 0.05) (Figure 3(A) and (B)).

Other subtractions did not lead to significant changes as

compared to PKR expression in αDC1s.

As the role of Poly-I:C in PKR dynamics was expected

to be found at the level of activation rather than on tran-

scriptional regulation we next tested the levels of PKR

Thr446 autophosphorylation in DCs and how absence of

Poly-I:C affected PKR activation. Here, αDC1 matura-

tion resulted in autophosphorylation in 2 out of 4 donors,

but we were unable to detect any differences in the levels

H. L. LARSEN ET AL.

(A)

(B)

Figure 2. Exclusion of Polyα-Type-1 polariz-

f Thr446 phosphorylated PKR in αDC1s and αDC1s

4. Discussion

uality of ex vivo generated DCs for

-I:C from the

ing cocktail increases αDC1 viability. DCs maturated with

the indicated cytokine cocktails were stained with annexin

V FITC and PI, and subjected to flow cytometric analysis.

(A) Dot plots of annexin V FITC and PI stained DCs from

one representative of 8 donors. The percentage of viable

cells is indicated in the lower left corner; (B) Mean per-

centage of viable cells from all donors (n = 8). (*) indicates

significant different levels of viability (p < 0.05), one-sided

Students t-test.

maturated without Poly-I:C for 24 or 48 hours (Figure 3(C)

and data not shown) or during the first 6 hours after Poly-

I:C addition to DCs maturated with IFN-α (data not shown).

Improving the q

(A)

(B)

(C)

Figure 3. PKR expression is unaffected by

erapeutic cancer vaccination is of paramount impor-

and activation

extracellular Poly-I:C. PKR expression was investigated by

western blotting in DCs maturated with the indicated cyto-

kine cocktails for 48 hours. (A) One representative blot is

shown (n = 3); (B) PKR expression levels were normalized

to GAPDH levels and the mean fold change in PKR expres-

sion compared to iDCs is plotted (n = 3). (*) indicates sig-

nificant different PKR expression (p < 0.05), two-sided

Students t-test; (C) Active Thr446 phosphorylated PKR

detection by western blotting was performed 24 and 48

hours after addition of the indicated maturation cocktails to

iDCs. 2 out of 4 donors demonstrated Thr446 phosphory-

lated PKR. A western blot from one of these two donors is

shown.

tance and may increase the efficiency of this vaccine

strategy. αDC1s have been shown to be superior in

IL-12p70 secretion and CTL activation [5]. Viability of

therapeutic DCs is another factor of potential importance

as DCs may exert their effect in vivo after more than 24

hours upon injection. In order to improve αDC1 yields,

we tried to identify the cause of Poly-I:C mediated DC

death, in the attempt of identifying a target for future

siRNA-mediated silencing strategies as a mean of gener-

ating functional αDC1s with increased viability. In par-

ticular we investigated the potential role of PKR induc-

tion and autophosphorylation.

H. L. LARSEN ET AL. 5

Poly-I:C-mediated IL-12p70 secretion and induction

n of PKR in αDC1s

pression levels in αDC1s, we ob-

sion, we identified a Poly-I:C dependent

5. Acknowledgements

the Faculty of Health Sci-

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