Defence Research and Recherche et développement

Development Canada pour la défense Canada

Flow cytometric quantification of lung

natural killer cell activity associated with
TLR-3 signalling pathway activation

X. Dai, L.J. McLaws, G.J. Schnell, S. Viswanathan, C.C. Hu, H.S. Bhogal, and J.P. Wong
DRDC Suffield

Defence R&D Canada

Technical Memorandum
DRDC Suffield TM 2010-188
November 2010
Flow cytometric quantification of lung
natural killer cell activity associated with
TLR-3 signalling pathway activation

X. Dai, L.J. McLaws, G.J. Schnell, S. Viswanathan, C.C. Hu, H.S. Bhogal, and J.P. Wong
DRDC Suffield

Defence R&D Canada – Suffield

Technical Memorandum
DRDC Suffield TM 2010-188
November 2010
 Principal Author

J.P. Wong
Defence Scientist

Approved by

L. Nagata
Head, Biotechnology Section

Approved for release by

R. Clewley
Acting Chair, Document Review Panel

In conducting the research described in this report, the investigators adhered to the 'Guide to the
Care and Use of Experimental Animals, Vol. I, 2nd Ed.' published by the Canadian Council on
Animal Care.

© Her Majesty the Queen in Right of Canada, as represented by the Minister of National Defence, 2010
© Sa Majesté la Reine (en droit du Canada), telle que représentée par le ministre de la Défense nationale,
2010
Abstract ……..

The objective of this study was to evaluate natural killer (NK) cell activation as a potential
immunological biomarker for antiviral protection provided by pretreatment with liposome-
encapsulated (LE) Poly ICLC, a toll-like receptor-3 agonist. To achieve this, lungs of mice
intranasally treated with LE Poly ICLC or free Poly ICLC (1 mg/kg body weight) were
aseptically harvested at various time points post drug treatment and NK cells were then isolated
and used as effector cells in cytotoxicity assay. A flow cytometric assay using dual fluorescent
dyes was developed to examine the duration of lung-associated NK cytotoxic activity in mice.
Two methods of flow cytometric analysis were employed: one based on 7-amino-actinomycin D
(7-AAD) viability dye incorporation and the other combining 7-AAD incorporation with forward
scatter (FSC) parameters. The results from both methods demonstrated that LE PolyICLC and
free PolyICLC-induced NK cytotoxicity was significantly higher than the basal levels from the
PBS-treated lungs at 7 days post intranasal administration (p  0.05). However, LE PolyICLC-
augmented cytotoxicity remained elevated through 14 days, significantly higher than the basal
levels from the PBS-treated lungs. In comparison, PolyICLC-induced cytotoxicity gradually
declined at 14 days (p ! 0.05 vs PBS control). Our results demonstrated that LE Poly ICLC
induced a potent augmentation of lung-associated NK activity for 14 days post intranasal
treatment. The correlation between the duration of NK activation and the previously reported
window of antiviral protection suggests that NK activation may be a good predictive biomarker
for antiviral protection by LE Poly ICLC.

Résumé ….....

La présente étude avait pour objectif d’évaluer la possibilité d’utiliser l’activation des cellules NK
comme biomarqueur immunologique de la protection antivirale offerte par un prétraitement avec
le poly ICLC, un agoniste du récepteur de type toll 3, encapsulé dans des liposomes (EL). Pour ce
faire, nous avons administré un traitement intranasal de poly ICLC EL ou de poly ICLC libre
(1 mg/kg de poids corporel) à des souris. Nous avons ensuite prélevé les poumons de ces
dernières de façon aseptique à différents moments après le traitement et isolé les cellules NK, que
nous avons utilisées comme cellules effectrices pour des tests de cytotoxicité. Nous avons élaboré
un test de cytométrie de flux à deux fluorochromes afin d’examiner la durée de l’activité
cytotoxique des cellules NK associées aux poumons chez la souris. Nous avons utilisé deux
méthodes d’analyse de cytométrie de flux : l’une repose sur l’incorporation du colorant
d’évaluation de la viabilité 7-amino-actinomycine D (7-AAD) et l’autre combine l’incorporation
de la 7-AAD à des paramètres de diffusion vers l’avant. Les résultats obtenus à l’aide des deux
méthodes ont montré qu’au jour 7 après l’administration intranasale, la cytotoxicité des
cellules NK induite par les deux formes de poly ICLC (EL et libre) était significativement plus
élevée que celle observée dans les poumons des témoins ayant reçu un traitement au PBS
(p < 0,05). Toutefois, la cytotoxicité stimulée par le poly ICLC EL est demeurée élevée durant
14 jours; elle était significativement supérieure à celle générée par le traitement au PBS. Par
comparaison, la cytotoxicité générée par le poly ICLC a diminué progressivement à partir du
jour 14 (p > 0,05 par rapport au témoin PBS). Nos résultats ont montré que le poly ICLC EL
entraîne une forte augmentation de l’activité des cellules NK associées aux poumons durant

DRDC Suffield TM 2010-188 i

14 jours après le traitement intranasal. Le lien observé entre la durée de l’activation des
cellules NK et la possibilité de protection antivirale par le poly ICLC EL évoquée par le passé
laisse croire que l’activation des cellules NK pourrait être un bon biomarqueur prédictif pour la
protection antivirale conférée par le poly ICLC EL.

ii DRDC Suffield TM 2010-188

Executive summary

Flow cytometric quantification of lung natural killer cell activity

associated with TLR-3 signalling pathway activation:
Xiaojiang Dai; Lori J. McLaws; Glen J. Schnell; Satya Viswanathan; Charles C.
Hu, Hardeep S. Bhogal; Jonathan P. Wong; DRDC Suffield TM 2010-188;
Defence R&D Canada – Suffield; September 2010.

Background: Cell-mediated cytotoxicity has been traditionally measured by the radioactive

chromium (Cr51)-release assay. Although this method has been considered as the standard
method, it has a number of disadvantages. The major disadvantage is the concern about handling
and disposal of radioactive materials. Recently, non-radioactive flow cytometric assays have
been developed as more sensitive and safer alternatives to the Cr51-release assay. A FACSAria
flow cytometer was purchased by DRDC Suffield in 2004 and development of flow cytometric
assays is under way for several research projects at DRDC Suffield.

Results: A dual-fluorescent dye flow cytometric assay has been developed to examine the
duration of lung associated NK activity in mice treated with liposome encapsulated (LE) Poly
ICLC and free Poly ICLC using the FACSAria flow cytometer. Our results demonstrate that LE
Poly ICLC induces a potent augmentation of lung-associated NK activity for at least 14 days post
intranasal treatment.

Significance: This is the first study to successfully use FACSAria flow cytometer in NK-
mediated cytotoxicity at DRDC Suffield. This is also the first study to apply a dual-dye flow
cytometric assay in a precise and reliable analysis of the in vivo Poly ICLC augmentation of NK
activity. These techniques have the potential to improve the ability of DRDC Suffield to develop
and identify surrogate biomarker for antiviral agents that broadly protect against influenza viruses
and other biothreat agents.

Future plans: Flow cytometric assays for other formulations of LE Poly ICLC and Poly ICLC
are currently ongoing and will be made available for use in the study of LE Poly ICLC against
influenza virus infection.

DRDC Suffield TM 2010-188 iii

Sommaire .....

Flow cytometric quantification of lung natural killer cell activity

associated with TLR-3 signalling pathway activation:
Xiaojiang Dai; Lori J. McLaws; Glen J. Schnell; Satya Viswanathan; Charles C.
Hu; Hardeep S. Bhogal; Jonathan P. Wong; DRDC Suffield TM 2010-188R & D
pour la défense Canada – Suffield; Août 2010.

Introduction ou contexte : La cytotoxicité à médiation cellulaire est habituellement mesurée à

l’aide du test de libération de chrome radioactif (51Cr). Bien que cette méthode soit considérée
comme la méthode classique, elle présente plusieurs inconvénients. Le principal concerne les
préoccupations au sujet de la manipulation et de l’élimination de la matière radioactive.
Récemment, des tests de cytométrie de flux sans radioactivité ont été élaborés dans le but d’offrir
des options de rechange plus sensibles et plus sécuritaires que le test de libération du chrome 51Cr.
RDDC Suffield s’est procuré, en 2004, un cytomètre de flux FACSAria, et l’élaboration de tests
de cytométrie de flux se poursuit pour plusieurs projets de recherche à RDDC Suffield.

Résultats : Nous avons mis au point un test de cytométrie de flux à deux fluorochromes afin
d’examiner, à l’aide du cytomètre de flux FACSAria, la durée de l’activité des cellules NK
associées aux poumons chez les souris ayant reçu un traitement de poly ICLC EL et de poly ICLC
libre. Nos résultats ont montré que le poly ICLC EL entraîne une forte augmentation de l’activité
des cellules NK associées aux poumons durant au moins 14 jours après l’administration par voie
intranasale.

Importance : Cette étude est la première à utiliser avec succès le cytomètre de flux FACSAria
pour évaluer la cytotoxicité associée aux cellules NK à RDDC Suffield. Il s’agit également de la
première étude à utiliser un test de cytométrie de flux à deux fluorochromes pour effectuer une
analyse précise et fiable de l’augmentation in vivo de l’activité des cellules NK stimulées par le
poly ICLC. Ces techniques pourraient améliorer la capacité de RDDC Suffield à élaborer et à
identifier d’autres biomarqueurs d’agents antiviraux offrant une protection générale contre les
virus de l’influenza et d’autres agents de bioterrorisme.

Perspectives : Nous travaillons présentement à l’élaboration de tests de cytométrie de flux pour

d’autres préparations de poly ICLC EL et de poly ICLC libre, qui serviront à l’étude du
poly ICLC EL contre le virus de l’influenza.

iv DRDC Suffield TM 2010-188

Table of contents

Abstract …….. ................................................................................................................................. i

Résumé …..... ................................................................................................................................... i
Executive summary ........................................................................................................................ iii
Sommaire ....................................................................................................................................... iv
Table of contents ............................................................................................................................ v
List of figures ................................................................................................................................ vi
Acknowledgements ....................................................................................................................... vii
1 Introduction............................................................................................................................... 1
2 Materials and Methods.............................................................................................................. 3
2.1 Instrumentation .............................................................................................................. 3
2.2 Animals and treatment ................................................................................................... 3
2.3 Effector cells.................................................................................................................. 3
2.3.1 Preparation of single cell suspension from mouse lungs ................................ 3
2.3.2 Isolation of NK cells ....................................................................................... 4
2.4 Target cells .................................................................................................................... 4
2.5 Flow cytometry.............................................................................................................. 4
2.5.1 Compensation for CFSE and 7-AAD .............................................................. 4
2.5.2 Cytotoxicity assay ........................................................................................... 5
2.6 Statistical analyses ......................................................................................................... 5
3 Results....................................................................................................................................... 6
3.1 Optimization of CFSE and 7-AAD concentrations for cytotoxicity ............................. 6
3.2 Two methods of analysis for measuring cytotoxicity .................................................... 7
3.3 Flow cytometric quantitation of lung NK-mediated cytotoxicity.................................. 9
4 Discussion ............................................................................................................................... 11
5 Conclusions............................................................................................................................. 13
References ..... ............................................................................................................................... 14
List of abbreviations ...................................................................................................................... 17

DRDC Suffield TM 2010-188 v

List of figures

Figure 1: Optimization of CFSE concentrations for staining YAC-1 cells. .................................... 6

Figure 2: Discrimination of effector cells (NK) and target cells (YAC-1) on the CFSE/SSC
dot plots. ........................................................................................................................ 7
Figure 3: Dead cell quantification by the 7-AAD/FSC method of analysis. ................................... 8
Figure 4: Dead cell quantification by the 7-AAD method of analysis. ........................................... 9
Figure 5: Cytotoxic activity of effector NK cells on YAC-1 target cells analysed by the 7-
AAD/FSC and 7-AAD methods. ................................................................................. 10

vi DRDC Suffield TM 2010-188

Acknowledgements

This work was funded by CRTI-06-0301 TD (Development of nasal spray formulated anti-viral
drug against avian influenza virus).

DRDC Suffield TM 2010-188 vii

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viii DRDC Suffield TM 2010-188

1 Introduction

In recent years, significant advances have been made in the discovery of Toll-like receptors
(TLRs) signalling pathway, which has a crucial role in early host defence against invading
pathogens. TLRs are pattern recognition receptors whose ligands represent conserved features of
bacteria, viruses and other microorganisms. Thirteen mammalian TLRs have been identified and
each TLR acts as a primary sensor of conserved microbial components and drives the induction of
specific immune responses. For specific recognition of viral components, it has been established
that TLR-3 recognizes dsRNA, TLR-7 ssRNA, TLR-8 ssRNA, and TLR-9 unmethylated CpG
motifs.

Poly ICLC, a TLR-3 agonist, is a synthetic double-stranded RNA consisting of a complementary

strand of polyriboinosinic-polyribocytidylic acid (Poly I:C) stabilized with poly-L lysine and
carboxymethyl cellulose (LC). A previous study at DRDC Suffield has shown that the mRNA
expression of IFN-ȕ in the mouse lung treated with Poly ICLC was 200-fold higher than the PBS
control group at 72 h post intranasal treatment (Wong et al., 2009). Furthermore, another
previous research at DRDC Suffield has indicated that intranasally administered liposome-
encapsulated (LE) Poly ICLC provided a 3-week prophylactic window against a lethal challenge
with influenza A/PR/8/34 (H1N1) in mice, compared to a 2-week window for free Poly ICLC
(Wong et al., 1999). Even though LE Poly ICLC and free Poly ICLC are known to elicit anti-
influenza activity by activation of TLR-3 signalling pathway, little is known about the protective
component of the antiviral immunity which would account for the prolonged window of antiviral
state observed in the previous studies.

Natural killer (NK) cells represent the major component of innate immunity against viruses and
tumors due to their potent cytotoxic activity and rapid production of cytokines. Although Poly
ICLC or PolyI:C-induced augmentation of NK activity has been widely studied, most of these
studies were performed using cultured lymphocytes or NK cells in vitro (Sivori et al., 2004;
Yang, Q., 2006). Among the few in vivo studies, NK activity was monitored in easily sampled
organs and compartments, including spleen, blood and peritoneal cavity (Vaknin et al., 2008;
Akazawa et al., 2007; Chirigos et al., 1985; Nassiry et al., 1987). Other organs, such as lungs, are
difficult to sample and isolation of lung NK cells involves the usage of enzymatic digestion. It
has been reported that the NK activity is only seen after collagenase treatment of the mouse lung,
suggesting that NK cells are entrapped in pulmonary interstitial collagen (Stein-Streilein et al.,
1983). Though the lung is the target organ of many highly infectious agents by the respiratory
route, such as influenza virus, respiratory synthial virus and severe acute respiratory syndrome
(SARS), few studies with lung-associated NK cytotoxic activity have been reported in the last
two decades. In addition, there have not been any published studies on the effects of liposome
delivery of Poly ICLC on NK cytotoxic activity nor correlative studies on NK activation and
antiviral protection.

Cell-mediated cytotoxicity has been traditionally measured by the radioactive chromium (Cr51)-
release assay (Brunner et al., 1968; Strober, 2001). In this assay, target cells labeled with Cr51 are
incubated with effector cells for 4-6 hours, and the culture supernatants are quantified for Cr51
released from lysed targets in a gamma counter. Although this method has been widely used and
considered as the standard method, it has a number of disadvantages. The major disadvantage is

DRDC Suffield TM 2010-188 1

the concern about handling and disposal of radioactive materials. In addition, target cell death is
not measured at the single-cell level but based on lytic-unit calculations.

Recently, non-radioactive flow cytometric assays have been developed as more sensitive and
safer alternatives to the Cr51-release assay (Kim et al., 2007; Lecoeur et al., 2001; Liu et al.,
2002). An essential element of flow cytometric assays is the ability to discriminate target cells
from effector cells and dead cells from live cells. In most flow cytometric assays, effector cells
and target cells are distinguished through the use of different fluorescent dyes such as
carboxyfluorescein diacetate succinimidyl ester (CFSE, also called CFDA-SE), 3,3’-
diotadecyloxacarbocyanine perchlorate, Cell Tracker Orange (Kim et al., 2007, Lecoeur et al.
2001, Liu et al., 2002). Two strategies have been commonly used in distinguishing dead cells
from live cells in flow cytometric assay. One is based on an alteration of membrane permeability
(Lecoeur et al., 1997; Schmid et al., 1992; 1994a); loss of membrane permeability can be
evaluated by incorporating fluorescent viability dyes in dead cells, such as 7-amino-actinomycin
D (7AAD) and propidium iodide (PI). The other strategy for dead cell discrimination is based on
morphological modification of dead cells; cell shrinkage (low forward scatter, FSC) and increased
cellular granularity (high side scatter, SSC) have been shown to be the markers of dead cells
(Carbonari et al., 1995; Petit et al., 1995; Schmid et al., 1994a & b). Furthermore, the
combination of FSC or SSC parameters with 7-AAD incorporation allows more precise analysis
and reliable quantification of dead cells (Lecoeur et al., 1997 & 2001; Marin et al., 2003; Schmid
et al., 1992; 1994a & b).

The goals of this study are (i) to develop a flow cytometric assay to examine the lung-associated
NK cytotoxic activity and, (ii) to evaluate whether lung NK-mediated cytotoxicity is a reliable
biomarker which correlates with antiviral protection in mice intranasally treated with LE poly
ICLC or Poly ICLC. To achieve these, a dual-dye flow cytometric assay was developed to
measure the duration of lung-associated NK cytotoxic activity. Our results demonstrated that LE
Poly ICLC induces a potent augmentation of lung-associated NK cytotoxic activity for at least 14
days post intranasal treatment. The correlation between the duration of NK activity and the
previously reported window of antiviral protection suggests that NK activation may be a reliable
biomarker for antiviral protection elicited by LE Poly ICLC.

2 DRDC Suffield TM 2010-188

2 Materials and Methods

2.1 Instrumentation

FACSAria flow cytometer was procured from Becton Dickinson (San Jose, California).

2.2 Animals and treatment

Female BALB/c mice (6-7-week-old) were obtained from the mouse breeding colonies at DRDC
Suffield with the original breeding pairs from Charles River Canada (St. Constant, Quebec,
Canada). All animal studies were approved by the Animal Care Committee of DRDC Suffield
and the guideline of the Canadian Council on Animal Care was followed for caring and handling
mice.

Poly ICLC used in this study was supplied by Oncovir Inc. (Washington, DC). Preparation of LE
Poly ICLC has been previously described (Wong et al., 1999). Groups of mice were given two
doses of LE Poly ICLC or free Poly ICLC (1 mg/kg body weight) or phosphate-buffered saline
(PBS) 48 h apart by the intranasal route. The volume of the inoculum used was 50 Pl.

2.3 Effector cells

NK cells isolated from mouse lungs were used as the source of effector cells. Preparation of NK
cells was performed as described as follows.

2.3.1 Preparation of single cell suspension from mouse lungs

Mice were euthanized by cervical dislocation at 7 days and 14 days post second treatment. At
each time point, 2 mice were samples from each group. Lungs were aseptically removed and
washed twice with cold 10 mM HEPES (pH 7.4). Lungs were then minced finely by end-frosted
slides and incubated in 5 ml of HEPES containing 100 Pl of 100 mg/ml collagenase D (Roche
Applied Science, Laval, Quebec) and 20 Pl of 20,000 Unit/ml DNase I (Roche Applied Science)
for four lungs (from two mice) at 37oC for 35 min with constant rotation by using the MACSmix
Tube Rotator (Miltenyi Biotec Inc., Auburn, Calif). Cell suspensions were applied to a 70 Pm
mesh cell strainer; cells were washed in 45 ml of HEPES and then in 20 ml cold serum-free and
phenol red-free RPMI 1640 medium (Invitrogen, Burlington, Ontario) by centrifugation at 300 u
g for 9 min. Cells were counted by a Cell Viability Analyzer (Beckman Coulter). When properly
performed, this procedure yielded an average of 5 u 107 lung cells per two lungs (from one
mouse).

DRDC Suffield TM 2010-188 3

2.3.2 Isolation of NK cells

NK cells were isolated from the single cell suspensions using a positive selection microbeads
DX49b (DX5) (Miltenyi Biotec Inc.) according to the manufacturer’s instructions. In brief, 1 u
108 lung cells were resuspended in 900 Pl of PBS-0.5% bovine serum albumin (BSA)-2 mM
EDTA, pH 7.2 (PEB buffer) and then incubated with 100 Pl of CD49b (DX5) microbeads for 15
min at 4-8oC. After washing once, cells were resuspended in 500 Pl of PEB and applied onto a
MACS MS column placed in the magnetic field of a MACS separator (Miltenyi Biotec Inc.). The
column was washed three times with 500 Pl of PEB and enriched NK cells were eluted with 1 ml
of PEB. Cells were counted by a Cell Viability Analyzer, and then centrifuged at 300 u g for 10
min and resuspended in phenol red-free RPMI 1640 medium supplemented with 10% fetal bovine
serum (FBS) (Hyclone, Ottawa, Ontario) to a final concentration of 2 u 106 cells /ml.

2.4 Target cells

YAC-1 lymphoma of mouse strain A/Sn origin, used as target in this study, was purchased
commercially (American Type Culture Collection, Hornby, Ontario). The cells were cultured in
suspension in RPMI 1640 medium supplemented with 10% FBS and 100 Pg/ml kanamycin
(Invitrogen) at 37oC in 5% CO2.

CFSE (Invitrogen) was dissolved in dimethyl sulfoxide at a concentration of 20 mM as stock

solution, aliquoted into single-usage vials and stored at 20oC for no more than two months. For
the 2 days prior to the assay, YAC-1 cells were subcultured every 24 h to ensure that they were in
log phase. For labeling of YAC-1 cells with 2 PM CFSE, 5 ml of 4 PM CFSE solution was
prepared in PBS-0.1% BSA from the stock solution and added to the same volume of cell
suspension at a concentration of a5 u 105 cells /ml in a 50 ml Falcon tube. The cells were gently
mixed and incubated for 10 min at 37oC in 5% CO2. Immediately after incubation, 10 ml of
RPMI 1640 medium (phenol-red free) was added to the labeling tube and the cells were
centrifuged at 1000 rpm for 6 min at room temperature. The cells were resuspended and washed
three times with 10 ml of RPMI 1640 medium (phenol-red free) by centrifugation at 1000 rpm for
6 min at room temperature. Cell number was adjusted to 2 u 105 cells/ml in RPMI 1640 medium
(phenol red-free).

2.5 Flow cytometry

2.5.1 Compensation for CFSE and 7-AAD

CFSE and 7-AAD fluorescence emissions were detected in the FITC and PI channels,
respectively. To reduce spectral crossover between CFSE and 7-AAD emissions, compensation
for FITC and PI channels were calculated according to the manufacturer’s protocols. In brief,
CFSE-labelled or unlabeled YAC cells were co-incubated with NK cells for 4 h at 37oC in 5%
CO2. Cell mixtures were then incubated with 7-AAD (Sigma, Oakville, Ontario) or without 7-
AAD for 20 min on ice protected from light. Unlabeled cells, cells labelled with CFSE alone, and
cells labelled with 7-AAD alone, were used to set spectral compensation in the FACSAria flow

4 DRDC Suffield TM 2010-188

cytometer. A minimum of 30,000 events were collected for each sample and analysis was
performed using BD FACSDiva version 5.0.3 software (BD Biosciences, San Jose, California).

When similar samples were analyzed, spectral compensation between CFSE and 7-AAD were
consistent between experiments; 5.7-6.0% CFSE emission was subtracted from 7-AAD (PI)
channel, and 1.5-2.5% 7-AAD emission was subtracted from CFSE (FITC) channel.

2.5.2 Cytotoxicity assay

CFSE-labelled YAC-1 cells (1 u 104) were seeded with NK cells (2 u 105) at an effector : target
(E :T) ratio of 20:1 in U-bottom 96-well plates (BD FalconTM, Missisauge, Ontario) in a total
volume of 200 Pl complete RPMI 1640 medium (phenol-red free) per well. In parallel, YAC-1
cells or NK cells were seeded alone in the same volume of medium to measure spontaneous cell
death. Each cell sample was plated in triplicate. After incubation for 4 h at 37oC in 5% CO2,
each sample was transferred to a 4 ml BD Falcon tube containing 400 Pl of serum-free and
phenol red-free RPMI 1640 medium. 7-AAD was added to each sample at a concentration of 6
Pg/ml and cells were incubated with the dye for at least 20 min on ice protected from light. Cell
samples were loaded onto FACSAria flow cytometer. Events were acquired for a fixed number
of 2,000 CFSE positive cells. Data analysis was performed with the BD FACSDiva version 5.0.3
software. The percentage of killing was calculated with 7-AAD/FSC and 7-AAD methods of
analysis using the following equation:

[(the cell number acquired in P2 + P3 or Q1 from the sample)  (the cell number acquired in P2 +
P3 or Q1 from YAC-1 alone) / 2000  (the cell number acquired in P2 + P3 or Q1 from YAC-1
alone)] u 100

2.6 Statistical analyses

GraphPad PRISM 5 was used for statistical analysis. Differences in mean cell number between
groups were compared by a two-way ANOVA. A p value of  0.05 is considered as significant.

DRDC Suffield TM 2010-188 5

3 Results

3.1 Optimization of CFSE and 7-AAD concentrations for

cytotoxicity

Initial experiments were performed to optimize the concentration of CFSE used for distinguishing
YAC-1 cells (target) from NK cells (effector). YAC-1 cells were labeled with different
concentrations of CFSE (0.25, 0.5, 1.0, 2.0, 3.0, and 5.0 PM) before co-culture with effctor cells.
The fluorescence intensity of CFSE was determined by FACS analysis. Cells with a fluorescence
intensity of  103 were considered to be CFSE negative (Fig. 1A); otherwise intensities above this
value were consider CFSE positive as shown in the histograms of Fig. 1. The results indicated
that 0.25 PM of CFSE was not enough to discriminate YAC-1 cells from unstained cells (Fig.
1B). At 2 PM of CFSE, YAC-1 cells were easily differentiated from the unstained cells (Fig.

Figure 1: Optimization of CFSE concentrations for staining YAC-1 cells.

YAC-1 cells were stained with CFSE at concentrations ranging from 0.25 to 5 PM and further
cultured for 4 h. The fluorescence intensity of CFSE was determined by FACS analysis. Cells
with a fluorescence intensity of  103 were considered to be CFSE negative; otherwise CFSE
positive cells in the histograms. A. Unstained YAC-1 cells. B. YAC-1 cells stained with 0.25
PM CFSE. C. YAC-1 cells stained with 2 PM CFSE.

6 DRDC Suffield TM 2010-188

 Figure 2: Discrimination of effector cells (NK) and target cells (YAC-1) on the CFSE/SSC dot
plots.

YAC-1 cells, labeled with 2 PM CFSE, were incubated for 4 h in the presence of lung NK cells.
7-AAD at a concentration of 6 Pg/ml was added and cell samples were loaded onto FACSAria
flow cytometer. Events were acquired for a fixed number of 2,000 CFSE positive cells. YAC-1
cells were selected in the gate P1. NK cells were excluded from the gate P1 according to their
CFSE negativity on the CFSE vs SSC dot plots.

1C). At high concentrations (5 PM or higher) of CFSE, YAC-1 cells were intensely stained, but
cell viability was decreased due to a toxic effect by CFSE (data not shown). Therefore, the
concentration of 2 PM of CFSE was chosen to label YAC-1 cells in subsequent experiments.

Similar experiments were performed to optimize concentrations of 7-AAD to distinguish live

YAC-1 cells from unviable (dead and dying) YAC-1 cells. 7-AAD concentrations at 2.5, 4, 6, 8,
10, 12, 15, and 20 Pg/ml were examined for optimization. As a result of optimization, a
concentration of 6 Pg/ml was determined for use in subsequent experiments (data not shown).

3.2 Two methods of analysis for measuring cytotoxicity

Two methods of flow cytometric analysis were compared and evaluated for measuring NK-
mediated cytotoxicity by flow cytometry. One was based on combination of 7-AAD
incorporation with FSC parameters (7-AAD/FSC method of analysis) and the other on 7-AAD
incorporation (7-AAD method of analysis).

YAC-1 cells were discriminated from NK cells by setting a gate P1 according to their CFSE
positivity on the CFSE vs SSC dot plots (Fig. 2). With the 7-AAD/FSC method of analysis as
shown on the 7-AAD vs FSC dot plots in Fig. 3, YAC-1 cells were further divided into three
populations: (1) dead YAC-1 cells, exhibiting a cell shrinkage (gate P2, green dots, FSClow, 7-
AAD); (2) dead YAC-1 cells, exhibiting a loss of membrane integrity (gate P3, red dots, 7-
AAD+); (3) live YAC-1 cells with a normal membrane integrity and a normal cell size (gate P4,

DRDC Suffield TM 2010-188 7

blue dots, FSChigh, 7-AAD). ~7% dead YAC-1 cells were detected after 4 h incubation in the
absence of NK cells, corresponding to spontaneous cell death (Fig. 3A). In contrast, incubation
of YAC-1 cells with NK induced a strong cell shrinkage and significant 7-AAD staining, leading
to ~35% specific cell death (Fig. 3B).

The data were further analyzed with a second method, in which only 7-AAD incorporation was
applied to discriminate dead and live cells (7-AAD method of analysis). On the 7-AAD vs CFSE
dot plots in Fig. 4, YAC-1 and NK cells were discriminated according to their CFSE staining; live
and dead cells (NK and YAC-1 cells) were distinguished by their 7-AAD incorporation. With
this method of analysis, NK and YAC-1 cells were divided into four populations: (1) dead YAC-1

Figure 3: Dead cell quantification by the 7-AAD/FSC method of analysis.

YAC-1 cells, labelled with 2 PM CFSE, was incubated for 4 h in the absence of lung NK cells
(A) or presence of lung NK cells (B). 7-AAD at a concentration of 6 Pg/ml was added after
incubation and cell samples were loaded onto FACSAria flow cytometer. Events were acquired
for a fixed number of 2,000 CFSE positive cells. Live YAC-1 cells were distinguished from dead
cells by setting gates P2, P3and P4 based on the 7-AAD incorporation plus FSC parameters.

cells, CFSE positive cells with a loss of membrane integrity (gate Q1, red dots, 7-AAD+, CFSE+);
(2) dead NK cells, CFSE negative cells with a loss of membrane integrity (gate Q2, purple dots,
7-AAD+, CFSE). (3) live NK cells, CFSE negative cells with a normal membrane integrity (gate
Q3, grey dots, 7-AAD, CFSE); (4) live YAC-1 cells, CFSE positive cells with a normal
membrane integrity (gate Q4, blue dots, 7-AAD, CFSE+). With the 7-AAD method of analysis,
a3% dead YAC-1 cells (spontaneous death) were detected after 4 h incubation in the absence of
NK cells (Fig. 4A). In comparison, a significant cell death (~22%) of YAC-1 cells was induced
after incubation with lung NK cells (Fig. 4B).

8 DRDC Suffield TM 2010-188

 Figure 4: Dead cell quantification by the 7-AAD method of analysis.

YAC-1 cells, labelled with 2 PM CFSE, were incubated for 4 h in the absence of lung NK cells
(A) or presence of lung NK cells (B). 7-AAD at a concentration of 6 Pg/ml was added after
incubation and cell samples were loaded onto FACSAria flow cytometer. Events were acquired
for a fixed number of 2,000 CFSE positive cells. YAC-1 and NK cells were discriminated
according to their CFSE staining, and live and dead YAC-1/NK cells were distinguished by
their 7-AAD incorporation.

3.3 Flow cytometric quantitation of lung NK-mediated

cytotoxicity

Lung NK cytotoxic activities in BALB/c mice, treated with LE Poly ICLC, free Poly ICLC, or
PBS, were examined with two methods of analysis. With the 7-AAD method of analysis, LE
Poly ICLC and Poly ICLC stimulated a virtually identical lung NK activity at 7 days post
treatment (p  0.05 vs PBS control; p ! 0.05 LE Poly ICLC vs Poly ICLC) (Fig. 5). With the 7-
AAD/FSC method of analysis, however, LE Poly ICLC induced a potent NK activity at 7 days
and 14 days post treatment (38% and 25%, respectively), significantly higher than that by Poly
ICLC (31% and 20%, respectively) (p  0.05) (Fig. 5). Furthermore, the results from both the
methods indicated that LE Poly ICLC-augmented cytotoxicity remained elevated through 14
days, significantly higher than the basal levels from the PBS-treated mice (p  0.05). In
comparison, the data from either the 7-AAD or 7-AAD/FSC method of analysis showed that Poly
ICLC-induced cytotoxicity gradually declined by 14 days, not significantly higher than those
from the PBS-treated mice (p ! 0.05) (Fig. 5).

Variable levels of NK activity in the lungs treated with PBS, ranging from 17% to 23%, were
observed (Fig. 5). This was not unexpected as the lung that was exposed to the environment may
harbour some active NK cells even without any treatment. Similar observations have been
previously reported by several groups (Nogusa et al., 2008; Stein-Streilein et al., 1983; Wiltrout
et al., 1985a). Stein-Streilein et al. (1983) reported a NK activity at a20% from the untreated
mouse lungs using a Cr51-release assay. Our results indicated that different flow cytometric

DRDC Suffield TM 2010-188 9

methods exhibited different levels of NK activity from the PBS-treated lungs. As shown in Fig.
5, a basal level of only ~17% was observed with the 7-AAD method, but the basal level increased
to ~23% with the 7-AAD/FSC method.

Collectively, these data from both 7-AAD/FSC and 7-AAD methods of analysis demonstrated
that LE Poly ICLC induced a potent augmentation of lung-associated NK activity up to at least 14
days post intranasal treatment. These results also suggest liposome-encapsulation of Poly ICLC
has profound effect on the duration of NK activation by prolonging the NK activation up to at
least 14 days.

Figure 5: Cytotoxic activity of effector NK cells on YAC-1 target cells analysed by the 7-
AAD/FSC and 7-AAD methods.

BALB/c female mice were treated with LE Poly ICLC, Poly ICLC or PBS by the intranasal route.
At 7 days and 14 days post treatment, natural killer cells were isolated from the lungs of treated
mice and incubated with 2 PM CFSE labelled-YAC-1 cells. After 4 h incubation, 7-AAD at a
concentration of 6 Pg/ml was added to each sample and cell samples were loaded onto FACSAria
flow cytometer. Events were acquired for a fixed number of 2,000 CFSE positive cells. Specific
cytotoxicity was calculated with the 7-AAD/FSC and 7-AAD methods of analysis using the
equation described in Materials and Method (Section 2.5.2). The data shown were means r SEM
of results for three replicates. Results of a two-way ANOVA were shown (ns, not significant;
, p  0.05; , p  0.01; , p  0.001;

10 DRDC Suffield TM 2010-188

4 Discussion

Despite advances in antiviral chemotherapy and vaccine development, the world is still
vulnerable to threats from seasonal and pandemic influenza viruses. Due to the rapid increase in
clinical isolates of influenza viruses resistant to conventional anti-influenza drugs including
amantadines and oseltamivir, there is a compelling reason to consider fast track development of
novel antiviral agents which broadly protect against various influenza viruses, regardless of their
zoonotic origin or drug-resistant profile. In animal studies, LE Poly ICLC has been shown to
protect against all subtypes tested including H5N1, H1N1 and H3N2 (Wong et al., 1999, 2007).
In order to further develop this drug clinically, a better understanding of the drug’s mechanism of
action and identification of surrogate biomarkers for antiviral protection are fundamentally
important for regulatory review and for design of clinical studies. Even though LE Poly ICLC
and Poly ICLC are known to elicit anti-influenza activity by activation of TLR-3 signaling
pathway, little is known about the protective component of the antiviral immunity which would
account for the prolonged state of antiviral state observed in previous studies.

The results in this study which demonstrated the prolonged activation of lung NK cells were in
close correlation with the 3 week window of anti-influenza virus protection provided by
liposome-encapsulation of Poly ICLC. This correlation suggests that NK activation may likely be
a reliable biomarker for antiviral protection elicited by LE Poly ICLC and Poly ICLC. This study
will be important in providing guidance in the design of clinical trials in establishing the dosing
regiment and treatment schedule.

In this study, we have described a dual-dye flow cytometric assay to measure the duration of lung
NK activity in BALB/c mice treated with LE Poly ICLC and Poly ICLC. Target YAC-1 cells
were labeled with CFSE before the co-culture with effector cells. Lung NK cells from mice
treated with LE Poly ICLC or free Poly ICLC were used as effector cells. Following co-culture,
7-AAD viability dye was added for live and dead cell discrimination. Several reports described
the usage of Cr51-release assay to measure spleen and liver NK activity in mice treated with Poly
ICLC in the mid-80s (Chirigos et al., 1985; Nassiry et al., 1987; Wiltrout, RH et al., 1985a & b).
Recently, flow cytometry was applied to measure NK activity. Most of these studies were
performed using cultured lymphocytes or NK cells that were treated with Poly ICLC in vitro.
Nevertheless, to our knowledge, this is the first study to apply CFSE/7-AAD dual-dye flow
cytometric assay in a precise and reliable analysis of the in vivo Poly ICLC augmentation of NK
activity. Moreover, our results demonstrate, for the first time, that LE Poly ICLC induces a
potent augmentation of lung-associated NK activity for at least 14 days after a local (intranasal)
inoculation in mice.
Two methods of flow cytometric analysis were compared and evaluated for measuring NK-
mediated cytotoxicity: one combined 7-AAD viability dye incorporation with FSC parameters (7-
AAD/FSC method of analysis) and the other based on 7-AAD staining (7-AAD method of
analysis). Several groups have previously reported the usage of the 7-AAD/FSC criteria as a
reliable and precise strategy to discriminate live and dead cells (Lecoeur et al., 1997 & 2001;
Marin et al., 2003; Schmid et al., 1992; 1994a & b). Examination of the 7-AAD vs FSC and 7-
AAD vs CFSE dot plots shown in Fig. 3 & 4 indicated that most cells in the gate P4 (blue dots,
identified as live cells in the 7-AAD/FSC method of analysis) were present in the gate Q4 (cells
in the Q4 identified as live cells in the 7-AAD method of analysis), and most cells in the gates P3

DRDC Suffield TM 2010-188 11

(red dots, identified as dead cells in the 7-AAD/FSC method of analysis) were included in the
gate Q1 (cells in the Q1 identified as dead cells in the 7-AAD method of analysis). This suggests
a consistency on dead cell discrimination between the two methods of analysis. Our results
indicated that 7-AAD/FSC method of analysis exhibited a higher cytotoxicity than the 7-AAD
method of analysis. The reason for this is because dead cells were identified only based on 7-
AAD incorporation and those cells which exhibited low forward scatter were excluded from the
dead cell counting in the 7-AAD method. All together, these data suggest that 7-AAD/FSC
method of analysis might be the favourite but both these methods of analysis could be appropriate
for flow cytometric quantification of NK-mediated cytotoxicity and could be used
interchangeably for verification of cytotoxicity.

The lung is the target organ of many highly infectious agents by the respiratory route, including
influenza virus and SARS. Surprisingly, few studies have been reported to evaluate pulmonary
NK activity in the last two decades, although several groups described lung-associated NK
activity in the 80s. Wiltrout et al. (1985a & b) reported that NK activity was boosted to much
higher levels in lung and liver than those observed in spleen and blood after an intravenous
treatment with Poly ICLC or a biological response modifier (BRM) named MVE-2. Stein-
Streilein et al. (1983) demonstrated that a local inoculation of influenza virus led an augmentation
of NK activity in the mouse lung and not the spleen at 48 h post intratracheal treatment. Similar
results were described by Prichard et al. (1987), who reported that a localized augmentation of
NK activity was induced in the lung, but not in the spleen, in guinea pigs after 24 h post
intratracheal treatment with PolyI:C. Seo et al (2002) reported that a cross-protective immunity
involved in influenza viruses H9N2 and H5N in chickens was closely related to the percentage of
CD8(+) T cells expression IFN-J in the lungs, rather than in the spleen, suggesting that
pulmonary cellular immunity may be very important in protecting naïve natural hosts against
lethal influenza viruses. Interestingly, recent results in our group have demonstrated that the
mRNA expression of interferon ȕ in the lung was at least 200-fold higher than those observed in
spleen at 72 h post intranasal treatment with either LE Poly ICLC or free Poly ICLC in mice
(Wong et al., 2009). These data indicate that NK activity in target organs like lungs can be
augmented to much higher levels than in blood and lymphoid organs like spleen by a local
inoculation of Poly ICLC or other BRM. Therefore, highly efficient pulmonary cellular
immunity should be considered as a possible mechanism for the therapeutic effects of BRM
treatment.

12 DRDC Suffield TM 2010-188

5 Conclusions

NK cells represent the major component of innate immunity against viruses and tumors due to
their potent cytotoxic activity and rapid production of cytokines. Poly ICLC, a synthetic double-
stranded RNA, is a broad-spectrum antiviral agent which activates TLR-3 signaling pathway. To
evaluate natural cell activation as a potential immunological biomarker for antiviral protection
provided by pre-treatment with LE Poly ICLC and free Poly ICLC, lungs of mice intranasally
treated with LE Poly ICLC or free Poly ICLC (1 mg/kg body weight) were aseptically harvested
at various time points post drug treatment and NK cells were isolated and used as effector cells in
cytotoxicity assay. A dual-fluorescent dye flow cytometric assay was developed to examine the
duration of lung-associated NK cytotoxic activity in mice. Two methods of flow cytometric
analysis were employed: one based on 7-AAD viability dye incorporation and the other
combining 7-AAD incorporation with FSC parameters. The results from both methods indicated
that LE Poly ICLC-augmented cytotoxicity remained elevated through 14 days, significantly
higher than the basal levels from the PBS-treated lungs (p  0.01). In comparison, Poly ICLC-
induced cytotoxicity gradually declined by 14 days (p ! 0.05). Our results demonstrated that LE
Poly ICLC induced a potent augmentation of lung-associated NK activity up to at least 14 days
post intranasal treatment. The correlation between the duration of NK activation and the
previously reported window of antiviral protection suggests that NK activation may be a good
predictive biomarker for antiviral protection by LE Poly ICLC.

DRDC Suffield TM 2010-188 13

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16 DRDC Suffield TM 2010-188

List of abbreviations

DRDC Defence Research & Development Canada

Poly ICLC Polyriboinosinic-polyribocytidylic acid (Poly I:C) stabilized with poly-L
lysine and carboxymethyl cellulose (LC).
LE Poly ICLC Liposome-encapsulated (LE) Poly ICLC
NK cells Natural killer cells
7-AAD 7-amino-actinomycin D
CFSE Carboxyfluorescein diacetate succinimidyl ester
FSC Forward scatter
SSC Side scatter
IN Intranasal
BSA Bovine serum albumin
FBS Fetal bovine serum
BRM Biological response modifier

DRDC Suffield TM 2010-188 17

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18 DRDC Suffield TM 2010-188

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Flow cytometric quantification of lung natural killer cell activity associated with TLR-3 signalling
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Da i, X., McLa ws , L.J ., S chne ll, G.J ., Vis wa na tha n, S ., Hu, C.C., Bhoga l, H.S ., a nd Wong, J .P .
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The objective of this study was to evaluate natural killer cell activation as a potential
immunological biomarker for antiviral protection provided by pretreatment with liposome-
encapsulated (LE) Poly ICLC, a toll-like receptor-3 agonist. To achieve this, lungs of mice
intranasally treated with LE Poly ICLC or free Poly ICLC (1 mg/kg body weight) were
aseptically harvested at various time points post drug treatment and natural killer (NK) cells
were then isolated and used as effector cells in cytotoxicity assay. A dual-fluorescent dye flow
cytometric assay was developed to examine the duration of lung-associated NK cytotoxic
activity in mice. Two methods of flow cytometric analysis were employed: one based on 7-
amino-actinomycin D (7-AAD) viability dye incorporation and the other combining 7-AAD
incorporation with forward scatter (FSC) parameters. With the 7-AAD method of analysis,
virtually identical lung NK-mediated cytotoxicity was observed by LE Poly ICLC and free Poly
ICLC at 7 days post intranasal administration (p  0.05 vs PBS control; p ! 0.05 LE Poly ICLC
vs Poly ICLC). With the 7-AAD/FSC method of analysis, however, LE Poly ICLC-augmented
cytotoxicity was significantly higher than that by free Poly ICLC (p  0.05). Furthermore, the
results from both methods indicated that LE Poly ICLC-augmented cytotoxicity remained
elevated through 14 days, significantly higher than the basal levels from the PBS-treated lungs
(p  0.01). In comparison, Poly ICLC-induced cytotoxicity gradually declined by 14 days (p !
0.05). Our results demonstrated that LE Poly ICLC induced a potent augmentation of lung-
associated NK activity up to at least 14 days post intranasal treatment. The correlation between
the duration of NK activation and the previously reported window of antiviral protection
suggests that NK activation may be a good predictive biomarker for antiviral protection by LE
Poly ICLC.

14. KEYWORDS, DESCRIPTORS or IDENTIFIERS (Technically meaningful terms or short phrases that characterize a document and could be
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Cytotoxicity; Flow cytome try; LE P oly ICLC; BALB/c mous e ; Na tura l kille r ce ll; YAC-1 ce ll; 7-
AAD; CFS E
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