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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4210-4219
Interleukin-15 as an Activator of Natural Killer Cell-Mediated
Antiviral Response
By
Jean Gosselin,
Andru TomoÏu,
Robert C. Gallo, and
Louis Flamand
From the Laboratory of Viral Immunology, Laboratory of Virology,
Rheumatology and Immunology Research Center, Centre de Recherche du
CHUL and Anatomy and Physiology Department, Faculty of Medicine, Laval
University, Sainte-Foy, Quebec, Canada; and the Institute of Human
Virology, University of Maryland, Baltimore, MD.
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ABSTRACT |
Natural killer (NK) cells are large granular lymphocytes capable of
efficient killing of virus-infected and tumor cells in a major
histocompatibility complex-independent manner. The
cytotoxic killing potential of NK cells can be modulated by a variety
of factors, including cytokines such as interleukin-12 (IL-12), IL-15, and interferon (IFN). IL-15 also plays an important role in NK cell
development and survival. Killing of virally infected cells by NK cells
is likely to represent an important antiviral defense mechanism,
especially during the early phase of infection when antigen-specific
immunity has yet to be generated. In the present work, we studied the
potential of IL-15 to act as a modulator of NK cell-mediated antiviral
defense. Our results clearly indicate that IL-15 can curtail infections
by 3 human herpesviruses: Herpes simplex virus type 1, Epstein-Barr
virus, and human herpesvirus 6. The antiviral activity of IL-15 is
dose-, time-, and NK cell-dependent. IL-15-treated NK cells showed an
increased killing potential against a variety of cells, including
virus-infected target cells. Lastly, using highly purified cell
population, we report that IL-15 triggers the synthesis of IFN- from
both CD4+ and NK cells, which can act in both autocrine
and paracrine fashion to modulate NK cells cytotoxic potential. In
conclusion, IL-15 is a cytokine that can contribute to the
establishment of an antiviral state in 2 ways: first by increasing the
killing ability of NK cells and second by stimulating the synthesis and
secretion of IFN.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DURING THE EARLY PHASE of a viral
infection, the nonspecific immune defense mechanisms play a crucial
role in limiting viral spread. These mechanisms include interferon
(IFN) production, activation of the complement cascades, and activation
of phagocytes and natural killer (NK) cells. The role of NK cells is
best evidenced in patients having reduced or absent NK cell activity
that suffer from multiple and recurrent herpesvirus
infections1-7 and in infections of experimental animal
models.8-10 NK cells are large granular lymphocytes capable
of killing tumor and virus-infected cells, independently of major
histocompatibility complex (MHC) restriction.11,12 A number of cytokines have been shown to modulate NK cell activity. For example, the cytotoxic potential of NK
cells is augmented after exposure to IFN- and
IFN- ,13-15 whereas the secretion of IFN- by NK cells
is induced after treatment with interleukin-12
(IL-12).16,17 IL-15 is another cytokine that has a major
impact on NK cell biology. IL-15 shares many biological properties with
IL-2, such as a growth factor for activated T cells and the activation
of NK and cytotoxic T lymphocytes (CTL).18-23 IL-15 is
produced by a variety of cell types, including monocytes/macrophages (MO/Mø), bone marrow stromal cells, keratinocytes, dendritic cells, and synovial-derived cells from patients with rheumatoid
arthritis.24-31 T cells are not a source of
IL-15.22,28 Although the primary sequence of IL-15 does not
share homologies with IL-2, its modeled 3-dimensional structure closely
resembles that of IL-2, a member of the 4--helix bundle family of
cytokines.22 Structural and biological similarities can be
partly attributable to receptor subunit sharing between IL-2 and IL-15.
In fact, both cytokines bind and signal through the IL-2 receptor chain (CD122) and the common chain (CD132).21,22,32 A
third component of the IL-15 receptor, the chain, binds IL-15 with
very high affinity (1011 mol/L 1) and
makes up the third moiety of the heterotrimeric IL-15 receptor.
The effects of IL-15 on cells of the immune system have been relatively
well studied. However, the role of IL-15 in antimicrobial defense is
much less documented. Several reports have shown an increase in IL-15
mRNA or protein in response to lipopolysaccharide or after bacterial or
viral infections.22,26-28 These results suggest that this
cytokine may play an important role in the generation of an effective
immune response against invading pathogens. The general concept is that
IL-15 is rapidly produced by MO/Mø in response to aggression, and then
it acts primarily on NK cells, but also on activated T cells, which
renders them more responsive to IL-2 (via upregulation of IL-2R
chain).33 Later, once activated T cells produce IL-2, it
becomes the principal T-cell growth factor. IL-15 also has T-cell
chemoattractant properties, recruiting T lymphocytes to the site of
inflammation.34 To learn more about the biological
properties of IL-15, we undertook a study to determine the potential
antiviral activity of this cytokine. Our results indicate that IL-15
has an antiviral activity against human Herpesviruses, but its
effectiveness is dependent on the presence of NK cells.
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MATERIALS AND METHODS |
Cell lines and culture conditions.
The HSB-2, K562, and B95-8 cell lines were obtained from the American
Type Culture Collection (Rockville, MD). HSB-2 is an immature human
T-cell line susceptible to HHV-6 infection, K562 is an erythroleukemia
cell line sensitive to NK cell lysis, and the B95-8 cells are
chronically Epstein-Barr virus (EBV)-infected B cells of monkey origin.
Lymphoblastoid cell lines (LCL) were obtained after immortalization of
peripheral blood B lymphocytes by the B95-8 strain of EBV. All cell
lines were cultured in RPMI 1640 medium supplemented with 10%
heat-inactivated fetal bovine serum (FBS), 50 U/mL of penicillin, 50 µg/mL of streptomycin, 30 µg/mL of gentamicin, and 10 mmol/L
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)
buffer, all purchased from GIBCO BRL (Gaithersburg, MD).
Peripheral blood mononuclear cell (PBMC) isolation and
cell population purification.
PBMC were obtained from healthy donors after centrifugation of venous
blood over Ficoll-Hypaque gradients obtained from Pharmacia Biotech Inc
(Piscataway, NJ). Cells were washed 3 times in phosphate-buffered saline (PBS). In some experiments, PBMC were depleted of NK cells (CD16+/CD56+) using anti-CD16 and anti-CD56
monoclonal antibodies (MoAbs) and goat antimouse IgG-coated magnetic
beads according to the manufacturer's technical guidelines (Immunotech
Inc, Westbrook, ME). Effectiveness of NK cell depletion from PBMC was
determined by flow cytometry. No significant changes in remaining cell
populations (T cells, B cells, and monocytes) were observed. Human
CD4+ T cells were enriched by negative selection using a
cocktail of antibodies (anti-CD8, -CD14, -CD16, -CD19, -CD56, and
-glycophorin A; StemCell Technologies, Vancouver, British Columbia,
Canada). NK cells were also enriched by negative selection using a
cocktail of antibodies (anti-CD3, -CD4, -CD14, -CD19, -CD66b, and
-glycophorin A; StemCell Technologies). Human CD8+ T cells
were enriched by negative selection using an antibody cocktail
(anti-CD4, -CD14, -CD16, -CD19, -CD56, and -glycophorin A). CD4, CD8,
and NK cell enrichment procedures consistently yielded population of
cells expressing (98%) the CD4, the CD8, or the CD56 marker, respectively.
Virus production.
HHV-6 (GS strain) was propagated in HSB-2 cells as described
previously.35 The HHV-6 titer, expressed as the 50% tissue culture infective dose, was determined by scoring the number of HSB-2
cells exhibiting cytopathic effect (CPE). The virus stock had a titer
of 106 50% tissue culture infective doses/mL. The
mock-infected control was prepared from uninfected HSB-2 culture
supernatant and processed as for the infected supernatant.
EBV was produced from the chronically infected B95-8 cell
line.36 Briefly, B95-8 cells were seeded at 2 × 105 cells/mL and phorbol 12-myristate
13-acetate (TPA)-activated (20 ng/mL) to induce the EBV replicative
cycle. When cell viability was less than 10%, supernatant was
harvested and passed through a 0.45-µm membrane filter, and virions
were pelleted by centrifugation at 25,000g for 45 minutes.
Viral pellets were resuspended in RPMI-1640. Infectivity and titer of
preparations were determined by anti-Epstein-Barr nuclear antigen
(EBNA) immunofluorescence after infection of PBMC.
Herpes simplex virus type 1 (HSV-1) was produced from
infected Vero cells. Briefly, Vero cells were infected with HSV-1
(multiplicity of infection [moi], 0.01) and monitored
regularly for CPE. When greater than 85% of the culture was destroyed,
supernatant was filtered and stored frozen at  80°C. Titer of
preparations was determined by plaque assay formation on Vero cells.
Infection of cells and culture conditions.
In the case of infection by HHV-6, PBMC were activated with
phytohemagglutinin (PHA; 1 µg/mL) obtained from Sigma (St Louis, MO)
for 2 days before infection. Activated cells were then pelleted by
centrifugation, infected with HHV-6 (moi, 0.1) for 2 hours at 37°C,
and subsequently washed twice with PBS to remove unadsorbed virions.
Cell density was adjusted to 106 cells/mL in complete
medium supplemented with 5 U/mL of IL-2 (Boehringer Mannheim,
Indianapolis, IN). Selected cultured were further complemented with 10 to 50 ng/mL of IL-15 or IL-6 (R&D Systems, Minneapolis, MN). In some
experiments, HSB-2 cells were infected with HHV-6 (moi, 0.1) for 2 hours at 37°C, washed twice with PBS, and resuspended at 4 × 105 cells/mL.
In the cases of EBV and HSV-1, freshly isolated PBMC were infected with
EBV (104 TFU/106 cells) or HSV-1 (moi, 0.05)
for 2 hours at 37°C, washed with PBS, and resuspended in complete
medium with or without 25 to 50 ng/mL of IL-15. In some experiments,
cultures were supplemented immediately after infection with 10 µg/mL
of anti-IFN- and anti-IFN- neutralizing MoAbs (Serotec Inc,
Raleigh, NC). In previous experiments, 10 µg/mL of these antibody
preparations was proven to effectively neutralize the antiviral
activity of 1,000 pg/mL of recombinant IFN- or IFN- (data not shown).
Flow cytometry and immunofluorescence.
HHV-6 infection of cells was monitored by flow cytometry. Briefly,
cells were harvested, washed in PBS, pelleted by centrifugation, and
surface-stained for HHV-6 antigen expression using the anti-gp106 MoAb
(Advanced Biotechnologies Inc, Columbia, MD). After 1 hour at 4°C,
cells were washed with PBS and labeled with fluorescein isothiocyanate
(FITC)-labeled goat antimouse antibodies for 45 minutes at 4°C.
After washing with PBS, cells were fixed with 1% paraformaldehyde in
PBS and analyzed using the FACScalibur flow cytometer (Becton
Dickinson, Mountain View, CA). Results were calculated
after the accumulation of 10,000 events using the CellQuest software
(Becton Dickinson).
For EBV and HSV-1, infectivity was determined by standard
immunofluorescence. Briefly, cells were harvested, washed with PBS, and
deposited on glass microscope slides. Cells were fixed in cold acetone
for 5 minutes, air-dried, and stained with MoAb H62 against HSV-1
(BioSource International, Camarillo, CA) or with a positive reference
serum for EBV. Statistics were calculated using the Student's
t-test, and the results were considered significant when
P < .05.
DNA slot blot analysis.
Cells were harvested and washed in PBS, and genomic DNA was isolated as
described.28 Genomic DNA at 5, 1, and 0.2 µg was transferred to nylon membranes with the use of a slot blot apparatus (Schleicher and Schuell, Keene, NH). After UV
cross-linking of the DNA, the membranes were incubated for 1 hour at
68°C in prehybridization buffer (6× SSC, 0.1% sodium dodecyl
sulfate [SDS], 1× Denhardt's solution, and 100 µg/mL salmon
sperm DNA) and then hybridized overnight at 68°C in
prehybridization buffer containing the pZVH14 32P-labeled
HHV-6 probe.37 Membranes were washed twice with 1× SSC, 0.1% SDS and once at 68°C with 0.1× SSC, 0.1% SDS
before being exposed to x-ray films.
IFN- and IFN- determination.
To determine the effects of IL-15 on IFN synthesis, PBMC were either
treated with IL-15 (50 ng/mL), EBV, HSV-1, or in combination of IL-15
with virus for varying periods of time. Cell-free supernatants were
collected at time points and stored frozen at 80°C until assayed for IFN- (sensitivity, 25 pg/mL; Biosource International) and IFN- (sensitivity, 4 pg/mL; Pharmingen, Mississauga, Ontario, Canada) by enzyme-linked immunosorbent assay (ELISA), according to the
manufacturer's instructions (Pharmingen). Kinetics of IFN- synthesis from purified CD4+, CD8+, and
CD56+ cell populations after IL-15 (50 ng/mL) stimulation
were determined by ELISA (Pharmingen). Statistics were calculated using
the Student's t-test, and the results were considered
significant when P < .05.
NK cell cytotoxicity assay.
Purified CD56+ NK cells (98%) were cultured
(106 cells/mL) in absence or presence of IL-15 (50 ng/mL)
for 3 days. Two million target cells (K562, LCL, HSB-2, and HSB-2
infected with HHV-6) were labeled by incubation with 200 µCi of
sodium chromate (DuPont, Mississauga, Ontario, Canada) for
1 hour at 37°C. After 3 washes with PBS, target cells (5 × 103 cells) were mixed with effector NK cells (in
triplicate) at ratios ranging from 0.1:1 to 10:1 in V-bottomed wells
and were incubated for 16 hours at 37°C in a CO2
incubator. Plates were centrifuged and the radioactivity of supernatant
(0.1 mL) was determined using a -counter (LKB, Uppsala,
Sweden). Data are expressed as the percentage of
cytotoxicity after calculation using the following formula: (cpm
experimental cpm spontaneous)/(cpm maximum - cpm spontaneous) × 100.
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RESULTS |
Several cytokines, such as IFN and CC-chemokines, have been shown to
possess, in addition to their immunomodulatory activities, antiviral
activities. In an effort to learn more about the immunobiological properties of IL-15, we studied its ability to affect the viral growth
of HSV-1 (a member of the herpesvirinae), HHV-6 (a member of the
herpesvirinae), and EBV (as a representative of the herpesvirinae).
Effects of IL-15 on infection of PBMC by HHV-6.
PBMC from normal individuals were activated with PHA for 48 hours
before infection with HHV-6, since productive infection of resting
cells by this virus is not very efficient. After infection, cells were
either cultured in absence or presence of recombinant IL-15 (10 or 50 ng/mL). After 5 to 7 days postinfection, cells were monitored for HHV-6
antigen expression by flow cytometry. As shown in
Fig 1A, in the absence of IL-15, more that
95% of the cells were positive for HHV-6 antigen 7 days postinfection (PI), with a mean fluorescence intensity (MFI) of 135. In the presence
of 10 ng/mL of IL-15, the percentage was reduced minimally to 85%,
whereas the MFI decreased more than half to 65 (Fig 1B). Moreover, in
the presence of 50 ng/mL of IL-15, HHV-6 antigen expression was
drastically reduced to a minimum, with less than 10% of the cells
found to be expressing HHV-6 antigens (Fig 1C). To determine whether
this effect was specific to IL-15, we performed the same type of
experiment using IL-6, another monocyte-derived cytokine. In
contrast to IL-15, cultures treated with 50 ng/mL of IL-6 supported
viral replication to the same extent as those without IL-6 (data not
shown).
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| Fig 1.
Effect of IL-15 on infection of PBMC by HHV-6. PBMC were
PHA-activated, infected with HHV-6 for 2 hours, and cultured in the
absence (A) or presence of 10 ng/mL (B) or 50 ng/mL (C) recombinant
IL-15 (rIL-15). On day 7 postinfection, cells were harvested and tested
for cell surface expression of HHV-6 antigen by flow cytometry. Results
are presented as overlay histograms, with the black histograms
representing cells stained with anti-HHV-6 MoAb, whereas the white
histograms represent cells stained with an irrelevant MoAb. (D) depicts
the results obtained when HHV-6-infected cells, treated or not with
IL-15, were analyzed for HHV-6 DNA content. Genomic DNA was hybridized
with the 32P-labeled HHV-6 pZVH14 probe.
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The inhibitory effects of IL-15 on HHV-6 expression were further
confirmed by analyzing the HHV-6 DNA content in the various cultures.
At day 5 PI, cells were harvested and genomic DNA was isolated. The DNA
was probed for HHV-6-related sequences using the pZVH14 probe. As
shown in Fig 1D, a reduction in HHV-6 DNA was observed in both
IL-15-treated cultures, with the most striking effect observed with
the 50 ng/mL dose, for which near complete elimination of HHV-6 genomic
DNA is observed, in accordance with the antigen expression results
presented above (Fig 1C).
Effects of IL-15 on infection of NK-depleted PBMC and enriched
CD4+ T-cell cultures by HHV-6.
To determine whether IL-15 is acting directly or indirectly on the
HHV-6 replicative cycle, we tested IL-15 activity in NK-depleted PBMC
cultures as well as in enriched CD4+ T-cell cultures. We
opted to deplete NK cells from PBMC cultures (<0.5% residual
CD16+/CD56+ cells), because one of the effects
of IL-15 is to increase the killing potential of NK cells, an effect
that may account for the observed decrease in the number of
HHV-6-infected cells. NK-depleted PBMC were therefore infected with
HHV-6 and cultured in the absence or presence of IL-15 (10 or 50 ng/mL). Both HHV-6 antigen expression (Fig
2A through C) and HHV-6 genomic DNA (Fig 2D) were monitored as
described above. As with unfractionated PBMC, successful HHV-6 infection of NK-depleted PBMC was observed, with more than 95% of the
cells infected by day 7 PI (Fig 2A). The addition of 10 ng/mL of IL-15
to the cultures had no effect on HHV-6 antigen expression (Fig 2B),
with more than 95% of the cells expressing HHV-6 antigen and with an
MFI comparable to that of cultures with no IL-15. In the presence of 50 ng/mL of IL-15, more that 81% of the cells were found to express HHV-6
antigens (Fig 2C), which is in striking contrast with unfractionated
PBMC, for which less that 10% of the cells were infected (Fig 1C).
This result was also confirmed by viral DNA analysis. Genomic DNA was
extracted from untreated and IL-15-treated (50 ng/mL) cultures and
probed for HHV-6. As shown (Fig 2D), a strong hybridization signal is observed in untreated HHV-6-infected cultures, with a slightly reduced
signal in IL-15-treated cultures. Again, this is contrast to
unfractionated PBMC, for which virtually no hybridization signals could
be detected in IL-15-treated (50 ng/mL) cultures (Fig 1D). The effects
of IL-15 on HHV-6 infection of purified (>95%) CD4+ T
cells was also tested. No difference between IL-15-treated (50 ng/mL)
and nontreated cultures were noticed, with more than 95% of the cells
infected with HHV-6 (data not shown).
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| Fig 2.
Effect of IL-15 on infection of NK-depleted PBMC by
HHV-6. PHA-activated NK-depleted PBMC were infected with HHV-6 and
cultured in the absence (A) or presence of 10 ng/mL (B) or 50 ng/mL (C)
rIL-15. HHV-6 antigen expression and HHV-6 DNA content (D) were
determined as described in Materials and Methods and as in the legend
to Fig 1.
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To determine whether NK cells needed a direct contact with their
targets for inhibition of viral growth or whether they secreted an
inhibitory soluble factor in response to IL-15, 2 types of experiments
were conducted. First, PBMC depleted of NK cells were infected with
HHV-6 and cocultured with unfractionated, uninfected PBMC. Both
populations were physically separated by a 0.45-µm filter. The
cultures were supplemented with 50 ng/mL of IL-15 and monitored for
HHV-6 antigen expression by flow cytometry. Results indicate that
HHV-6-infected cells were not affected or lysed by the
release of soluble factors from PBMC, suggesting that a direct contact
between the NK cells and the infected target cell is necessary for
inhibition of viral growth (data not shown). Second, the possibility
exists that a contact between the NK cells and their targets may be
necessary for the induction and release of factors with antiviral
properties. To verify this hypothesis, purified NK cells (98%
CD56+) were cultured with IL-15 (50 ng/mL) for 2 days and
subsequently mixed with K562 or HHV-6-infected HSB-2 cells. After 8 hours, the supernatants were harvested and tested for their potential cytolytic and antiviral activity. The supernatants were not found to
contain soluble factors susceptible of causing lysis of K562 cells and
neither were they found to inhibit HHV-6 growth (data not shown). These
results suggest that IL-15 enhances the direct NK cell killing of
infected cells and that the release of soluble antiviral mediator by
such cells is marginal.
We next determined the effect of time of addition of IL-15 to the
cultures on HHV-6 expression. PBMC were infected with HHV-6 for varying
times before the addition of IL-15 (50 ng/mL) and were monitored for
antigen expression by flow cytometry on day 7 PI. In the absence of
IL-15, more than 90% of the cells were infected with HHV-6 (Fig 1 and
data not shown). The addition of IL-15 on day 0 (same day as infection)
resulted in a complete elimination of the HHV-6-infected cells from
the cultures, whereas the addition of IL-15 on the second day PI
resulted in partial suppression of HHV-6 growth, with 26% of the cells
being infected. The addition of IL-15 on day 4 PI was unable to prevent
HHV-6 infection, with more than 87% of the cells being infected (data not shown).
Effects of IL-15 on HSV-1 infection of PBMC.
Having determined that IL-15 can prevent the spread of HHV-6 in PBMC,
we next determined whether this effect is limited to HHV-6 or can be
extended to other viruses. A similar experimental procedure to that of
HHV-6 was used, with the exception that resting, freshly isolated PBMC
were used to monitor the effect of IL-15 on HSV-1 infection. PBMC from
3 healthy donors were infected with HSV-1 and cultured in the absence
or presence of IL-15 (25 or 50 ng/mL) for 5 days before evaluating the
percentage of HSV-1-infected cells by conventional immunofluorescence
assay using the anti-HSV-1 H62 MoAb. As shown in
Fig 3, 10% to 12% of the cells were
positive for HSV-1 antigens in the absence of IL-15. In the presence of 25 ng/mL IL-15, a low decrease in the percentage of infected cells is observed, with 4% to 8% of the cells being infected with
HSV-1. Finally, a 60% to 75% reduction in cells expressing
HSV-1 antigens is recorded in culture treated with 50 ng/mL rIL-15.
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| Fig 3.
Effects of IL-15 on infection of PBMC by HSV-1. PBMC from
3 healthy blood donors were infected with HSV-1 and cultured in the
absence or presence of 25 to 50 ng/mL. On day 4 PI, cells were analyzed
by standard immunofluorescence with anti-HSV-1 MoAb. The percentage of
infected cells was calculated after counting a minimum of 500 cells.
*P < .05.
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Effects of IL-15 on EBV infection of PBMC.
Unlike HHV-6 and HSV-1, EBV does not lytically infect PBMC, but rather
immortalizes B lymphocytes. We therefore tested whether IL-15 may play
a role in the control of EBV infection of PBMC. Resting PBMC from 3 healthy donors were infected with the B95-8 transforming strain of EBV
and cultured in the absence or presence of IL-15 (25 or 50 ng/mL) for 7 and 10 days, at which times the percentage of EBV antigen-positive
cells was determined by immunofluorescence. By day 7, in cultures
without IL-15, EBV infected 10% of the cells (Fig 4). The percentage of EBV-infected
cells decreased by more than half to 3% to 5% in the presence of 50 ng/mL IL-15. On day 10 PI, 12% to 14% of the cells were infected with
EBV. However, in presence of IL-15, a reduction in the percentage of
EBV-infected cells, ranging from 75% to 85%, was observed. As a
control experiment, treatment of cultures with 50 ng/mL IL-6 had no
effect on the percentage of EBV-infected cells (data not shown). The
effects of IL-15 on the chronically EBV-infected B95-8 cell line were also studied. Cells were TPA-activated to induce EBV replication and
cultured in the absence or presence of IL-15 (50 ng/mL). EBV gp350 antigen expression was moni- tored by flow cytometry 4 days after TPA activation and were found not to be influenced by IL-15, suggesting that IL-15 does not directly inhibit the EBV replicative cycle (data not shown).
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| Fig 4.
Effects of IL-15 on infection of PBMC by EBV. PBMC from 3 healthy blood donors were infected with EBV and cultured in the absence
or presence of 25 to 50 ng/mL rIL-15. On days 7 and 10, the percentages
of EBV-infected cells were determined using the ACIF test, with the use
of a human reference serum. Results were calculated after counting a
minimum of 500 cells. *P < .05.
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Effects of IL-15 on IFN- and IFN-
secretion by PBMC in response to viral infections.
Knowing that many cytokines can modulate the cytolytic activity of NK
cells, including IFN, we studied the effects of IL-15 on IFN- and
IFN- secretions by PBMC. Our results indicate that IL-15 per se is
not able to induce the secretion of IFN- from PBMC
(Fig 5). Viral infection of PBMC by EBV and
HSV-1 leads to significant levels of secreted IFN- on day 3 PI that
last up to day 7 PI. A combination of EBV and IL-15 leads to the
secretion of more than twice IFN- into the culture medium.
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| Fig 5.
IFN- production in response to IL-15 and viral
infections. PBMC were either treated with rIL-15 (50 ng/mL), EBV,
HSV-1, or a combination of virus plus IL-15. On the indicated days,
cell-free supernatants were harvested and tested for the presence of
IFN- using commercial ELISA kits. Results are representative of 3 donors. *P < .05.
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In contrast to IFN-, IL-15 was able to induce the secretion of
IFN- from PBMC (Fig 6). IFN- could be
detected (110 pg/mL) in the culture supernatant of IL-15-treated PBMC
on day 3, with increasing IFN- levels produced on days 5 (725 pg/mL) and 7 (934 pg/mL). Both EBV and HSV-1 were found
to induce low levels of IFN-, with maximal secretion observed at day
7 PI. However, combining EBV or HSV-1 with IL-15 lead to a synergistic
induction of IFN-, reaching levels as high as 4,000 pg/mL (Fig 6).
By combining virus and IL-15, IFN- is detected to significant levels
as early as the first day of culture, with a 3-fold synergistic effect
observed later during infection.
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| Fig 6.
IFN- production in response to IL-15 and viral
infections. PBMC were either treated with rIL-15 (50 ng/mL), EBV,
HSV-1, or a combination of virus plus IL-15. On the indicated days,
cell-free supernatants were harvested and tested for the presence of
IFN- using commercial ELISA kits. Results are representative of 3 donors. *P < .05; **P < .02.
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Effects of IL-15 on IFN- secretion by purified cell
populations.
Knowing that IL-15 can induce the synthesis and release of IFN- from
PBMC, we sought to identify the cell type capable of secreting IFN-
in response to IL-15 stimulation. Highly enriched (98%)
CD4+, CD8+, and CD56+ cells were
cultured in absence or presence of IL-15 (50 ng/mL) for 8 days.
Supernatants were collected every 2 days, starting on the second day of
culture, and were assayed for IFN- production. As shown in
Fig 7, both CD4+ and
CD56+ cells were capable of producing IFN- in response
to IL-15. During the first 4 days of culture, CD56+ cells
produced 3 to 5 times more IL-15 than CD4+ cells. However,
at later time points, CD4+ cells were found to produce
significantly more IFN- (~15 times more) than CD56+
cells whose IFN- production appeared to have reached a plateau. In
contrast to CD4+ and CD56+, CD8+
cells were not found to secrete any detectable IFN- in response to
IL-15 stimulation.
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| Fig 7.
IFN- production by purified cell populations in
response to IL-15 stimulation. CD4+ T cells,
CD8+ T cells, and CD56+ NK cells were
purified from PBMC by negative selection as described in Materials and
Methods. The top panels represent the flow cytometry analyses of
purified cell population (all 98% pure; black histograms) using
anti-CD4 (left panel), anti-CD8 (middle), and anti-CD56 (right)
antibodies. The isotype control antibody is represented by the white
histograms. The bottom panels represent kinetics of IFN- production
by corresponding cell population after IL-15 stimulation. Cells were
seeded at 106/mL in RPMI medium supplemented or not with
IL-15. Cell-free supernatants were collected every 2 days starting on
day 2 and were tested by ELISA for IFN- production. ( ) Mock;
( ) 50 ng/mL IL-15. Results are expressed as picograms per milliliter
of IFN- (mean ± SD of triplicate cultures).
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Modulation of NK cell killing potential by IL-15.
We have so far shown that IL-15 is able, in presence of NK cells, to
limit the viral infection of 3 distinct human herpesviruses. In
addition, IL-15 induces the release of IFN- from both NK and CD4+ T cells. To clearly establish that it is the increased
killing ability of NK cells that is mainly responsible for the
antiviral effects of IL-15, we performed an additional experiment.
Purified NK cells (98% CD56+) were cultured for 3 days in
the absence or presence of IL-15 (10 or 50 ng/mL). NK cells were then
mixed with 4 different target cells and tested for their ability to
efficiently cause cell lysis (Fig 8). Our
results indicate that IL-15-treated cells are much more potent killers
than are NK cells that have not been stimulated with IL-15. This
increased killing is observed not only with the K562 reference target
cells (Fig 8A), but also with an EBV-immortalized lymphoblastoid cell
line (Fig 8B) and the HSB-2 T-cell line (Fig 8C). Not surprisingly,
HHV-6-infected HSB-2 cells (Fig 8D) are more susceptible to NK cell
lysis when compared with uninfected HSB-2 cells. This can be observed
for NK cells that were treated or not with IL-15. However, the
observation that IL-15 caused a significant increased in lysis of
infected cells that is much higher than that of NK cells that were not
stimulated with IL-15 is important. Taken together, these
results suggest that an increase in killing potential of IL-15-treated
NK cells is responsible for the IL-15 antiviral activity.
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| Fig 8.
Increased killing of targets cells by IL-15-stimulated
NK cells. Purified CD56+ NK cells (98%) were cultured in
the absence () or presence of IL-15 ([ ] 10 or [] 50 ng/mL)
for 3 days. NK cells were washed with PBS and mixed, in triplicate,
with 51Cr-labeled target cells at effector:target ratios
varying between 0.1:1 and 10:1. Cells were incubated in V-bottomed
wells for 16 hours at 37°C, after which the supernatants (0.1 mL)
were analyzed for radioactivity using a -counter. Results are
expressed as the mean of triplicate cultures ± SD. (A) K562 target
cells; (B) EBV-immortalized lymphoblastoid cell line; (C) HSB-2 target
cells; (D) HHV-6-infected HSB-2 target cells.
|
|
| |
DISCUSSION |
The control and elimination of viral agents is achieved by both
specific and nonspecific defense mechanisms. The specific arm of the
immune response includes the generation of antigen-specific antibodies,
T-helper cell expansion and recruitment, and activation of
CD8+ T lymphocytes capable of lysing infected cells in an
MHC class 1-restricted manner. The nonspecific arm includes phagocytes, complement proteins, cytokines, and NK cells. The nonspecific immune
mechanisms play a pivotal role in the outcome of an infection, because
they represent the first line of defense against invading pathogens.
Early cytokine secretion may act directly as antiviral agents, such as
the IFNs, tumor necrosis factor- (TNF-), and IL-12.38-43 In addition, cytokines such as IFNs and IL-2
can act indirectly by promoting the proliferation and enhancing the
cytolytic functions of effector cells such as NK or
CTLs.13,14,44-46 In 1994, IL-15, a cytokine having
biological properties similar to that of IL-2, was
identified.20,22 One such attribute of IL-15 is its ability
to generate lymphokine-activated killers (LAK) from NK
cells.19-22 IL-15 was also shown to be secreted in response to various intracellular infectious agents and important for proper NK
cell activity.27,28,47-49 Furthermore, IL-15 plays an
important role in the in vitro survival of NK cells by preventing or
delaying apoptosis.50 Knowing this, we became interested in
determining whether IL-15 may play a role in the control of a viral
infections. The results presented here show that IL-15 can curtail
infections by herpesviruses belonging to the (HSV-1), (HHV-6),
and (EBV) herpesvirinae subfamilies. IL-15 does not act as an
antiviral agent per se, but rather is dependent on the presence of NK
cells for its antiviral effectiveness. In fact, the removal of
CD16+/CD56+ NK cells from cultures eliminated
the antiviral activity of IL-15. This is further supported by the
inability of IL-15 to inhibit infection of purified CD4+ T
cells infected by HHV-6. Inhibition of viral infection is IL-15 dose-dependent, with maximal activity in the range of 25 to 50 ng/mL.
Furthermore, the time at which IL-15 is added to the cultures is
important for maximal activity. If added more than 24 hours after the
initiation of the cultures, a dramatic loss of action is noticed. This
may represent the inability of NK cells to efficiently eliminate
infected cells once infection was allowed to be fully established. In
addition, the report that NK cells undergo rapid (within 24 hours)
programmed cell death in vitro in the absence of IL-2 or IL-15 may
suggest an additional hypothesis for this observation.50
IL-15 treatment of PBMC did not induce the secretion of IFN- in
contradistinction to IFN-, which is secreted in response to IL-15.
Our results are in agreement with those of others who have reported an
induction of IFN- secretion after IL-15
stimulation.23,30,50,51 Interestingly, when IL-15 was
combined with EBV or HSV-1, the IFN- productions were
synergistically enhanced by 3- to 4-fold. The observed synergy between
virus and IL-15 in IFN- production could be the result of autocrine
and paracrine stimulation from endogenously produced cytokines such as
IL-12. IFN- can, in turn, stimulate or prime monocytes/macrophages
to secrete IL-1252-54 and TNF-,55-58 which
can further activate NK cells. In fact, both EBV and HSV-1 were
reported to induce the secretion of IL-12.59,60 IL-12 is a
cytokine previously reported to act synergistically with IL-15 in
IFN- induction.61 It is likely that many of these cytokines can contribute to boost the NK cells' antiherpesvirus activity. However, surprisingly, neutralization of secreted IFN- had
little impact on the ability of IL-15 to restrict EBV and HHV-6
infection. This is most likely due to the fact that infections took
place before IL-15 stimulation and IFN secretion. IFN- is most
effective in establishing an antiviral state in cells that have yet to
be infected. In addition, EBV establishes a latent infection and does
not produce new viral progeny that would be more susceptible to IFN's
action upon infection of new target cells.
Of interest is the fact that IL-15-activated NK cells can control
infection by herpesviruses having very different biological properties.
HSV-1 has a very wide tropism and kills its target cell rapidly within
1 to 3 days. HHV-6 infects mostly cells of hematopoietic lineage, with
an intermediate replication time of 3 to 5 days. Lastly, unlike HSV-1
and HHV-6, EBV has a narrow cell tropism, infecting mainly B
lymphocytes and epithelial cells of the oropharynx. Furthermore,
EBV-infected B cells are immortalized and can survive indefinitely in
vitro. Cells infected with these herpesviruses were shown previously to
be efficiently recognized and killed by NK cells.62-77
Increasing the cytotoxic potential of NK cells may therefore represent
a useful strategy to help combat infections by this group of viruses.
Another mean by which IL-15 may promote antiviral activities is through
the induction and secretion of molecules having direct antiviral
properties. In fact, Oliva et al78 and Fehniger et
al79 reported that IL-15-stimulated NK cells, through the
secretion of C-C chemokines such as macrophage inflammatory proteins
(MIP) and RANTES, were capable of inhibiting HIV-1 infection.
Overall, the results presented indicate that IL-15 can reduce
herpesvirus infection through the activation of NK cells resulting in a
more efficient killing of the infected cells. Boosting of the innate
immune response by IL-15 during primary herpesvirus infections may
therefore prove valuable clinically to help reduce viral spread and
associated complications. The use of animal models experimentally
infected with herpesviruses will enable the testing of the in vivo
therapeutic efficacy of IL-15 and determine whether the potential for
selected clinical application for this cytokine exists.
| |
ACKNOWLEDGMENT |
The authors acknowledge the excellent technical expertise of Suzie Arsenault.
| |
FOOTNOTES |
Submitted May 13, 1999; accepted August 10, 1999.
Supported in part by a grant from the Medical Research Council of
Canada (MRCC) to L.F. L.F. and J.G. are both scholars from the MRCC.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Louis Flamand, PhD, Laboratory of Virology,
Rheumatology and Immunology Research Center Local T1-49, Centre de
Recherche du CHUL, 2705 Laurier Blvd, Sainte-Foy, Quebec, Canada G1V
4G2; e-mail: louis.flamand{at}crchul.ulaval.ca.
| |
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