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Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3183-3190
IMMUNOBIOLOGY
Interleukin-2 enhances the response of natural killer cells to
interleukin-12 through up-regulation of the interleukin-12 receptor
and STAT4
Kathy S. Wang,
David A. Frank, and
Jerome Ritz
From the Center for Hematologic Oncology, Department of Adult
Oncology, Dana-Farber Cancer Institute, and the Department of Medicine,
Brigham and Women's Hospital, Harvard Medical School, Boston, MA
02115.
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Abstract |
Interleukin (IL)-12 plays a critical role in modulating the
activities of natural killer (NK) cells and T lymphocytes. In animal
models, IL-12 has potent antitumor effects that are likely mediated by
its ability to enhance the cytotoxic activity of NK cells and cytotoxic
T lymphocytes, and to induce the production of interferon (IFN)- by
NK and T cells. In addition to IL-12, NK cells are responsive to IL-2,
and may mediate some of the antitumor effects of IL-2. In this study,
we examine the interaction between IL-2 and the signaling events
induced by IL-12 in NK cells. We find that IL-2 not only up-regulates
the expression of IL-12R 1 and IL-12R2, it also plays an important
role in up-regulating and maintaining the expression of STAT4, a
critical STAT protein involved in IL-12 signaling in NK cells. In
contrast to the effects of IL-2 alone, expression of IL-12 receptors
and STAT4 are unaffected or decreased by IL-12 or the combination of
IL-2 and IL-12. Through expression of high levels of IL-12 receptors
and STAT4, IL-2-primed NK cells show enhanced functional responses to
IL-12 as measured by IFN- production and the killing of target
cells. NK cells from cancer patients who received low-dose IL-2
treatment also exhibited increased expression of IL-12 receptor chains,
suggesting that IL-2 may enhance the response to IL-12 in vivo. These
findings provide a molecular framework to understand the interaction
between IL-2 and IL-12 in NK cells, and suggest strategies for
improving the effectiveness of these cytokines in the immunotherapy of cancer.
(Blood. 2000;95:3183-3190)
© 2000 by The American Society of Hematology.
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Introduction |
Interleukin (IL)-12 is a heterodimeric cytokine
consisting of p35 and p40 subunits. It was originally identified as
natural killer (NK) cell stimulatory factor (NKSF),1 and is
produced mainly by antigen-presenting cells. IL-12 plays an important
role in moduating both innate and adaptive immune responses. It
induces the production of interferon (IFN)- from human NK cells and
T cells, and also plays a key role in promoting the development of the Th1-type immune response.2 In addition, IL-12
enhances the cytotoxicity of NK cells and cytotoxic T lymphocytes
(CTL). NK cells activated by IL-12 exhibit enhanced cytotoxic
activity against NK cell sensitive and resistant target cells, both in vitro and in vivo.3-6
IL-12 exerts its functions by binding to specific cell surface
receptors and signaling through the Jak-STAT pathway. Two IL-12 receptor subunits, IL-12R1 and IL-12R2, have been identified. Each receptor subunit has a low IL-12 binding affinity, and neither is
capable of transmitting an IL-12 signal on its own.7,8 The
functional IL-12 receptor, which binds to IL-12 with high affinity, is
the heterodimeric receptor formed by IL-12R1 and IL-12R2.8 IL-12R1 is constitutively expressed on a
large portion of resting T cells and is relatively unaffected by
cytokines that induce Th1 or Th2 differentiation. On the other hand,
the expression of IL-12R2 is only detectable on activated T cells and is enhanced by cytokines that promote Th1 development and inhibited
by cytokines that promote Th2 development.8-12 Therefore, regulating the expression of IL-12R2 may be a key step in modulating IL-12 function. In addition to forming the high-affinity receptor with
IL-12R1, IL-12R2 also plays a unique role in IL-12 signaling. Unlike IL-12R1, the cytoplasmic domain of IL-12R2 becomes
tyrosine phosphorylated on IL-12 binding. In addition, IL-12R2
provides the binding sites for Tyk2 and Jak2, the kinases responsible
for activating STAT proteins.13 Several STAT proteins,
including STAT1, STAT3, STAT4, and STAT5, have been implicated in IL-12 signaling in T cells.8,14 Among these STATs, STAT4 is
essential in mediating IL-12 function in T and NK
cells.13,15-17 All major functions of IL-12 are
abrogated in STAT4-deficient mice, including production of
IFN- and NK cell cytotoxic activity.18,19 This suggests
that regulating the expression of STAT4 may also be an important
control point in modulating IL-12 function in NK and T cells.
The ability of IL-12 to augment the cytotoxicity of NK cells and CTL
suggested a therapeutic role for this cytokine in cancer treatment. In
preclinical studies using several murine tumor models, IL-12
demonstrated potent antitumor activity. Administration of IL-12 to
tumor-bearing mice resulted in the regression of established tumors,
systemic antitumor activity, and long-term survival.20-22 IL-12-induced augmentation of cytolytic activity and proliferation of
lymphocytes from cancer patients has also been demonstrated both in
vitro and in vivo.23 NK cells have been implicated as playing a major role in the antitumor effect of IL-12. Depletion of NK
cells abolishes IL-12-induced cytotoxicity and IFN- production in
mouse models, and abrogates the antimetastatic effect of IL-12 in these
mice.24
Although the antitumor effect of IL-12 in murine models is promising,
the use of IL-12 as a therapeutic anticancer agent in humans has been
disappointing because of its toxicity and decreased responsiveness over
time.25 Several approaches have been considered to improve
the effectiveness of IL-12 in cancer therapy, including the combination
of IL-12 with other immunomodulatory cytokines such as IL-2. IL-2 has
been shown to enhance the proliferation and cytotoxicity of NK cells in
vitro and in vivo.26-28 In vitro studies demonstrate that
the combination of IL-2 and IL-12 has a synergistic effect in promoting
IFN- production by T and NK cells, and the cytotoxic activity of NK
cells.29-31 Although the combination of IL-12 and IL-2 has
been associated with severe toxicity in mouse models,32,33
the treatment of mice bearing metastatic Renca tumors with IL-12 and
pulse IL-2 was shown to be less toxic and more effective than
IL-12 or IL-2 alone in inducing tumor regression.32 This
suggests that the anticancer activity of the combination of IL-12 and
IL-2 may rely on the strategic design of a regimen, which
ultimately should be based on an understanding of the mechanisms by
which IL-2 affects IL-12 function.
In light of the important role that NK cells play in IL-12-induced
antineoplastic effects and the potential importance of combination
therapy with IL-12 and IL-2, we investigated the effect of IL-2 on
IL-12 activated signaling pathways and functions in NK cells. Our data
show that pretreatment with IL-2, but not IL-12 or the combination of
IL-12 and IL-2, significantly enhances IL-12 signaling in NK cells.
Priming NK cells with IL-2 before IL-12 treatment leads to elevated
expression of both the IL-12 receptor chains and STAT4, and enhances
the response of NK cells to IL-12. These findings provide evidence that
IL-2, when used alternately with IL-12 in immunotherapy, may
significantly enhance the antitumor activity of IL-12.
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Materials and methods |
Cytokines, antibodies, and reagents
Recombinant human IL-2 (specific activity
6 × 106 U/mL) was generously provided by Amgen
(Thousand Oaks, CA). Human recombinant IL-12 (specific activity
1.7 × 107 U/mg) was kindly provided by Genetics
Institute (Cambridge, MA). Purified unconjugated, phycoerythrin (PE) or
PC5-conjugated murine monoclonal antibodies (mAbs), including B1 (CD20,
IgG2a), My4 (CD14, IgG2b), NKH1 (CD56, IgG1), N901 (CD56, IgG1), T1
(CD5, IgG2a), T3 (CD3, IgG1), and isotype control MsIgG1, were obtained from Coulter Immunology (Hialeah, FL). PE-conjugated goat antimouse IgG1 was purchased from Southern Biotechnology Associates Inc (Birmingham, AL). The generation of anti-IL-12 receptor 1 subunit, 44 was described previously.11 STAT1, STAT5, and STAT4
antibodies were purchased from Santa Cruz Biotechnology Inc (Santa
Cruz, CA). Expression vector pRC42, which contains an EF-1 promoter and hygromycin resistant marker, was kindly provided by Dr Gordon Freeman (Dana-Farber Cancer Institute).
Generation of antihuman IL-12 receptor 2 chain antibodies
Human IL-12 receptor 2 subunit (IL-12R2) cDNA was generated by
reverse transcriptase-polymerase chain reaction (RT-PCR) with
IL-12R2 specific primers based on the sequence information in
GenBank (Accession U64198). The cDNA was then cloned into expression
vector pRC42. The newly constructed IL-12R2-expressing plasmid
RC-Rb2 was transfected into the murine cell line 300-19 and CHO cell
line. Transfected cells were selected with hygromycin B. Single clones
from hygromycin B-resistant cells were then screened for their IL-12
binding activity by labeling the cells with IL-12, mouse anti-IL-12
antibody (IgG1, Genetics Institute), and PE-conjugated goat antimouse
IgG1 sequentially. The fluorescence of each clone was analyzed on a
Coulter EPICS XL cytometer (Coulter, Miami, FL). IL-12R2-expressing
300-19 clones were used to immunize BALB/c mice to generate
anti-IL-12R2 antibodies. Two monoclonal antibodies against
IL-12R2, 5A7 (IgG1) and 7A8 (IgG1), were generated and bind to the
RC-Rb2 transfected CHO cells but not to CHO cells transfected with the
vector alone. Both antibodies also bind to PHA-activated T cells but
not resting T cells. The 5A7 but not 7A8 blocked IL-12-induced STAT4
activation in activated T cells, and IL-12 induced proliferation in
activated T cells (data not shown).
Immunofluorescence analysis of the binding specificity of
anti-IL-12 receptor antibodies to IL-12 receptor chains
Murine cell line 300-19 transfected with IL-12R1 (300-191) or
IL-12R2 (300-192) were labeled with isotype control mouse IgG1,
anti-IL-12R1 antibody 44, or anti-IL-12R2 antibody 5A7. All
samples were then labeled with secondary antibody PE-conjugated goat
antimouse IgG1. The binding of the antireceptor antibodies to cell
surface receptors was analyzed by flow cytometry. As shown in Figure
1, 5A7 interacted only with 300-192
cells and 44 interacted only with 300-191 cells. This indicates
that the 5A7 monoclonal antibody we developed for these experiments
specifically interacts with IL-12R2 and does not cross-react with
IL-12R1.
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| Fig 1.
Binding of anti-IL-12 receptor antibodies to IL-12
receptors.
The 300-19 cell lines transfected with IL-12R1 (300-191) or
IL-12R2 (300-192) were labeled with mouse IgG1, anti-L-12R1
antibody, 44 or anti-IL-12R2 antibody, 5A7 and analyzed by flow
cytometry.
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Purification and culture of natural killer cells
Blood samples enriched with white blood cells were obtained from
healthy donors undergoing platelet pheresis in the Dana-Farber Cancer
Institute Blood Donor Center. Peripheral blood mononuclear cells
(PBMCs) were isolated from blood samples by Ficoll-Hypaque (Pharmacia
LKB) gradient centrifugation. Primary NK cells (more than 85% purity)
were obtained by depleting monocytes, B cells, and T cells from PBMCs
with B1 (CD20, IgG2a), My4 (CD14, IgG2b), T1 (CD5, IgG2a), and T3 (CD3,
IgG1) antibodies and immunomagnetic anti-IgG beads (PerSeptive
Biosystems, Framingham, MA). Purified NK cells were cultured at
2 × 106 cells per millilter in RPMI 1640 medium
containing 15% fetal calf serum, with or without cytokines as
indicated. All cytokines were used at 100 U/mL, if not specified.
Immunofluorescence analysis of IL-12 receptor expression
Primary or cultured T cells and NK cells were incubated with 20%
goat serum in RPMI to reduce the background binding by the secondary
antibody, goat antimouse IgG1-PE. Cells were then labeled with IgG1
isotype control, anti-IL-12R1 antibody 44, or anti-IL-12R2 antibody 5A7 and secondary antibody PE-conjugated goat antimouse IgG1.
Samples were incubated with PC5-conjugated CD56 for testing on NK cells
or PC5-conjugated CD3 for T cells. The expression of IL-12 receptors on
NK cells was analyzed by flow cytometry by gating on the CD56+
population, and the expression on T cells was analyzed by gating on the
CD3+ population. In the IL-12 competition assay, NK cells were first
incubated with IL-12 (100 U/mL) for 15 minutes, washed, and then
labeled with antibodies as describe above.
Whole-cell extracts and Western blotting
Whole-cell extracts were prepared as described
previously.17 Protein was separated on a 9% gel by
SDS-PAGE, then electrotransferred to nitrocellulose. The membrane was
blocked with 5% dry milk in TBST (100 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, and 0.05% Tween-20) and probed with primary antibody
diluted 1:10 000 in 5% bovine serum albumin (BSA). After incubation
with horseradish peroxidase-conjugated secondary antibody, specific
protein was detected by chemiluminescence (ECL, NEN Life Science Products).
Nuclear extracts and electrophoretic mobility shift assay
The STAT binding oligonucleotide used,
5'-GAGCCTGATTTCCCCGAAATGATGAGC-3' and its complement, is
derived from the IFN- responsive factor 1 gene
promoter.34 NK cells were primed as indicated for 3 days.
Cells were then washed and treated with IL-12 for 30 minutes. Nuclear
extracts were prepared and 5 µg of nuclear extract was used in the
electrophoretic mobility shift assay (EMSA) as described
previously.17
Interferon- enzyme-linked immunosorbent assay
The NK cells in equal numbers were primed for 3 days with or without
cytokines as indicated, and supernatants were collected on the third
day. Cells were then washed and plated at 30 000 cells per well in
duplicate into a 96-well U-bottom plate and cultured with or without
IL-12 for 3 days, after which the supernatants were collected. The
concentration of IFN- in all supernatants was determined by IFN-
enzyme-linked immunosorbent assay (ELISA) (Endogen, Boston, MA).
Cytotoxicity assay
The NK cells were primed for 3 days with or without cytokines as
indicated. Cells were then washed and cultured with or without IL-12
overnight. 51Cr-labeled Colo target cells were plated into
a 96-well plate with overnight cultured NK cells at a 5:1
effector-to-target ratio and incubated for 4 hours at 37°C.
Supernatants were then harvested and the release of 51Cr
was measured with a gamma counter. The percentage of specific cytotoxicity was calculated as previously described.3
Analysis of IL-12 receptor expression on natural killer cells from
patients receiving IL-2 treatment
Samples of PBMCs were cryopreserved from patients with metastatic
cancer enrolled in a clinical trial to examine the toxicity and
efficacy of continuous infusion IL-2, followed by bolus infusion of
IL-2.35 This clinical trial was approved by the Human
Subjects Protection Committee of the Dana-Farber Cancer Institute and
informed consent was obtained from each patient. Samples obtained after 4 weeks of continuous infusion IL-2 contained increased numbers of
CD56+ NK cells and were used in the current experiments to examine the
expression of IL-12 receptors. All samples were labeled with
anti-IL-12R1 or anti-IL-12R2 antibodies as described above and
with PC5-conjugated CD56. The expression of IL-12R1 and IL-12R2 on NK cells from these samples was determined with flow cytometry by
gating on the CD56+ population.
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Results |
Natural killer cells differ from T cells in IL-12 receptor chain
expression.
With the use of the monoclonal antibodies to IL-12R1 and IL-122
that we developed, the expression of these receptor chains on human NK
and T cells was examined. Resting T cells showed significant expression
of IL-12R1 as detected by the binding of anti-IL-12R1 antibody,
44, whereas the expression of IL-12R2 was not detectable (Figure
2A). This result is in agreement with
previous reports, which showed that, although a large portion of
resting PBMCs express IL-12R1, they do not bind IL-12, and the mRNA
for the IL-12R2 is not detectable in this population.8,9
The expression of IL-12R2 on T cells was markedly enhanced after
activation with the mitogen phytohemagglutinin (PHA) (Figure 2B),
consistent with the known increase in mRNA for this
subunit.8 The expression of IL-12R1 was also elevated by
PHA activation. In comparison with resting T cells, the expression of
IL-12R1 and IL-12R2 on resting NK cells showed notable
differences. The level of IL-12R1 expression on resting NK cells was
significantly higher than that on resting T cells, and was comparable
to the expression on PHA-activated T cells (Figure 2B and C). Unlike
resting T cells, resting NK cells express IL-12R2 (Figure 2C), which
correlates with our previous observations that IL-12 induces STAT4
activation, specific cytotoxic killing, and proliferation in resting NK
cells.3,17 This suggests that the expression of IL-12R2
on resting NK cells, although at low level, is sufficient for resting
NK cells to respond to IL-12. In both resting T cells and resting NK
cells, which have a high expression of IL-12R1, IL-12R2 appeared
to be the limiting component of the functional IL-12 receptor.
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| Fig 2.
Expression of IL-12R1 and IL-12R2 on T cells and NK
cells.
Cell surface IL-12R1 and IL-12R2 was measured by flow cytometry
on purified resting T cells (A), PHA-activated T cells (B), or purified
resting NK cells (C). The shaded area represents the isotype negative
control.
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IL-2 and IL-12 modulate the expression of IL-12 receptor chains on
NK cells.
Resting NK cells express both IL-12R1 and IL-12R2 and are
responsive to IL-12. Resting NK cells also express
intermediate-affinity IL-2 receptors and respond directly to IL-2
without prior activation.36,37 To investigate the effect of
IL-2 and IL-12 on the expression of IL-12R1 and IL-12R2 on NK
cells, we purified NK cells from the PBMCs of healthy donors and
cultured them in vitro with medium alone or supplemented with IL-2,
IL-12, or IL-2 plus IL-12. After 3 days of incubation, the expression
of IL-12R1 and IL-12R2 was tested by immunofluorescence analysis.
The expression of IL-12R1 was slightly increased in NK cells treated
with IL-2 in comparison with medium alone (Figure
3A and B and Figure
4A). By contrast, IL-12 or the
combination of IL-2 and IL-12 reduced the expression of IL-12R1
(Figure 4A). To test whether IL-12, which was present in these culture
conditions, would compete with the anti-IL-12R antibodies in binding
to the IL-12 receptor, we incubated IL-2-cultured NK cells with IL-12
before antibody labeling. The addition of IL-12 did not significantly
affect the binding of either anti-IL-12R1 antibody or
anti-IL-12R2 antibody to the cell surface receptor (Figure 3B and
C). This indicated that the reduced expression of IL-12R1 in IL-12-
or IL-2 plus IL-12-cultured NK cells was not due to competition
between IL-12 in the culture medium and the anti-IL-12R1 antibody.
Although the expression of IL-12R1 was reduced by IL-12 or the
combination of IL-2 and IL-12, there was still significant expression
of IL-12R1 on NK cells (Figure 4A). It is not clear whether the
reduction in the expression of IL-12R1 would significantly impact
IL-12 signaling in NK cells. The expression of IL-12R2 on NK cells
was slightly increased by priming with IL-12 or the combination of IL-2
and IL-12, but it was significantly enhanced when NK cells were primed
with IL-2 (Figure 4B). The expression of IL-12R2 on NK cells
cultured with IL-2 increased 10-fold in comparison with that on NK
cells cultured without cytokines, and 3-fold over NK cells primed with
IL-12 (Figure 4B).
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| Fig 3.
Expression of IL-12R1 and IL-12R2 on
cytokine-primed NK cells.
Purified NK cells were incubated for 3 days in the absence or presence
of IL-2. Expression of IL-12 receptor chains was then determined by
flow cytometry. Representative histograms demonstrate the expression of
IL-12R1 and IL-12R2 on NK cells cultured without IL-2 (A) or with
IL-2 (B). In (C), NK cells cultured with IL-2 were incubated with IL-12
before antibody labeling. The shaded area represents the isotype
negative control.
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| Fig 4.
Summary of the expression of IL-12R1 and IL-12R2.
A summary of the expression of IL-12R1 (A) and IL-12R2 (B) on
cultured NK cells with or without cytokines from 3 healthy donors. MFI:
mean fluorescent intensity.
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IL-2 and IL-12 modulate the expression of STAT4 in natural killer
cells.
Because STAT4 is the only known STAT protein that mediates IL-12
signaling in NK cells, we investigated whether IL-2 treatment would
alter the expression of STAT4. After culture with or without cytokines,
cell lysates were prepared from highly purified NK cells, and the
expression of STAT4 in the cells was monitored by Western blot. The
expression of STAT4 was enhanced by IL-2, compared with freshly
isolated NK cells, and was maintained at a similar level over 4 days of
culture (Figure 5A). In NK cells cultured
with medium alone, IL-12, or IL-2 plus IL-12, the level of STAT4 was
dramatically reduced in 1 day and further decreased over the time,
indicating that the expression of STAT4 is dependent on IL-2 but not
IL-12. The slower migrating band that appears in IL-12- or IL-2 plus
IL-12-treated NK cells is the phosphorylated STAT4 induced by IL-2 or
IL-12 in NK cells.17 In contrast to the dependency of STAT4
on IL-2, the level of STAT5 is stable in NK cells in all culture
conditions examined over the 4 days (Figure 5C). The expression of
STAT1 appeared to be dependent on either IL-2 or IL-12, and STAT1
decreased in cells cultured without either cytokine (Figure 5B). These
data suggest that there are different mechanisms regulating the
expression of these STAT proteins in NK cells.
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| Fig 5.
Expression of STAT proteins in cytokine- primed NK cells.
Purified NK cells were primed with cytokines as indicated. Cells were
collected at the indicated times, whole-cell extracts were prepared,
and Western blots were performed with anti-STAT4 (A), and reprobed with
anti-STAT1 (B) and anti-STAT5 (C). Representative data from 1 of 3 experiments is shown.
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When used at a concentration of 100 U/mL, IL-2 consistently promotes
the expression of STAT4 in our culture systems. To determine whether
lower concentrations of IL-2 also promote the expression of STAT4, we
incubated NK cells with different concentrations of IL-2 for 2 days,
and examined the expression of STAT4 by Western blot. As shown in
Figure 6, enhanced expression of STAT4 was
noted at an IL-2 concentration of 1 U/mL and reached a maximum at 100 U/mL (Figure 6 upper panel). IL-2 induced the phosphorylation of STAT5,
resulting in the appearance of a slower migrating band. The level of
phospho-STAT5 increased with higher concentrations of IL-2 but the
overall expression of STAT5 showed no increase with higher
concentrations of IL-2 (Figure 6 lower panel). These results indicate
that very low levels of IL-2 can promote the expression of STAT4, and
this promotion is dose-dependent.
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| Fig 6.
Dose response of IL-2-induced STAT4 expression.
NK cells were cultured with IL-2 at the indicated concentration for 2 days. Whole-cell extracts were prepared, and Western blots were probed
with anti-STAT4 (A) and reprobed with anti-STAT5 (B).
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IL-12-induced STAT4-DNA-binding activity is enhanced in natural
killer cells primed with IL-2.
IL-12 exerts its functional effects by inducing tyrosine
phosphorylation of STAT4, which leads to its translocation to the nucleus, binding specific promoter regions, and activation of its
target genes. Because IL-2-primed NK cells express high levels of
IL-12 receptors and STAT4, we investigated whether this would affect
the IL-12-induced STAT4-DNA-binding activity in NK cells. Purified NK
cells were primed for 3 days with or without cytokines, and
IL-12-induced DNA-binding activity was examined by EMSA with an oligo-ucleotide containing a STAT4 binding site.17
IL-12 induced a prominent STAT4-DNA complex only in IL-2-primed NK
cells (Figure 7, lane 2, S4). The
involvement of STAT4 in this complex was confirmed by the disappearance
of complex S4 and the appearance of a slower migrating complex, SS4,
after the anti-STAT4 antibody was introduced into the binding reaction
(Figure 7, lane 6). The IL-12-induced STAT4-DNA complex S4 was nearly
undetectable in NK cells primed under all other conditions. The
presence of a prominent IL-12-induced STAT4-DNA complex in
IL-2-primed NK cells suggests that the priming of NK cells with IL-2
may have a prominent impact on the functional response of these cells
to IL-12.
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| Fig 7.
IL-12-induced STAT4-DNA-binding activity in NK cells
primed with cytokines.
NK cells were primed with or without cytokines as indicated for 3 days
and then all cells were treated with IL-12 for 30 minutes. Nuclear
extracts were prepared and EMSA was performed in the absence or
presence of antibody to STAT4, as indicated.
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Enhanced IL-12-induced natural killer cell function after priming
with IL-2.
Previous evidence suggests that the antitumor effect of IL-12 is
mediated by the ability of IL-12 to induce IFN- production in NK
cells and to enhance the cytotoxicity of NK cells toward target cells.
To investigate the effect of IL-2 priming on the functions of
IL-12-treated NK cells, purified NK cells were primed with or without
cytokines, and IL-12-induced IFN- production and cytotoxicity were
tested. Both IL-2 and IL-12 induced IFN- production in NK cells
during the priming period and, as previously reported, the combination
of IL-2 and IL-12 induced synergistic IFN- production in NK cells
(Figure 8A).29,30 Although a
significant amount of IFN- was produced in all primed cultures with
cytokines, the majority of the IFN- induced by these cytokines was
produced within the first 24 hours (data not shown). When these primed NK cells were then treated with IL-12, maximal production of IFN- was detected only in the NK cells primed with IL-2 (Figure 8B). The NK
cells primed with IL-12 showed a significant reduction in IFN-
production in response to additional IL-12 (Figure 8B). This indicates
that continued IL-12 treatment of NK cells does not lead to continued
production of IFN-. The specific cytotoxicity of the primed NK cells
in response to IL-12 was also tested with Colo target cells.
IL-2-primed NK cells showed stronger cytotoxic killing against these
target cells, compared with NK cells primed under other conditions,
particularly in the presence of IL-12 (Figure
9). IL-12 priming of NK cells led only to a
weak enhancement of cytotoxic killing in response to IL-12.
IL-2- and IL-12-primed NK cells did not exhibit any enhancement
of specific cytotoxic killing in response to IL-12 compared with
cells primed with medium alone. Similar results were obtained at
varying effector-to-target ratios (data not shown). Thus,
in addition to potentiating IL-12-mediated signaling events, IL-2
priming enhances the functional stimulation of NK cells by IL-12.
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| Fig 8.
IFN- production in cytokine-primed NK cells.
NK cells were primed with or without cytokines as indicated for 3 days.
The INF- produced in the medium at the end of 3 days was quantified
by ELISA (A). These primed cells were then washed and treated with or
without IL-12 for 3 days. The IFN- produced from the cells was
quantified by ELISA (B). Net IFN- production, representing the
difference between that produced in the presence and absence of IL-12,
is shown.
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| Fig 9.
IL-12 induced cytotoxicity in cytokine-primed NK cells.
NK cells were primed with or without cytokines as indicated for 3 days.
Cells were then washed and incubated with or without IL-12 for 18 hours. Specific cytotoxicity of the NK cells against Colo cells at a
5:1 ratio was tested. A summary of experiments with NK cells from 3 different donors is presented.
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Patients treated with low-dose IL-2 show increased expression of
IL-12 receptor chains on natural killer cells.
Our data have shown that IL-2 can enhance the expression of IL-12R1,
IL-12R2, and STAT4 in NK cells in vitro, and lead to enhanced
IL-12-induced function of NK cells. This suggests that the immune
response of NK cells to IL-12 could be modulated by exposure to IL-2.
To explore whether IL-2 has a similar effect on NK cells in vivo, we
examined the expression of IL-12 receptors on NK cells from patients
treated with IL-2. These patients had metastatic cancer and received
low-dose continuous infusion of IL-2 for at least 4 weeks.35 The NK cell population in these patients was
expanded during the treatment and constitutes about 45% to 85% of the
PBMCs (data not shown; Soiffer et al35). The expression of
IL-12R1 and IL-12R2 was assessed on NK cells obtained from 6 patients after continuous IL-2 treatment and compared with enriched NK
cells from 4 healthy donors. NK cells from these 6 patients exhibited
elevated expression of both IL-12R1 and IL-12R2, compared with
the NK cells from the healthy donors (Figure
10A, B, and C). The expression of
IL-12R1 in the 6 patients treated with IL-2 was twice that in the
healthy donors. The expression of IL-12R2 on the NK cells from the
patients treated with IL-2 was comparable to that on IL-2-primed NK
cells in vitro, and was 3-fold higher than the expression level on NK
cells from healthy donors (Figures 4B and 10C).
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| Fig 10.
Expression of IL-12R1 and IL-12R2 on NK cells from
healthy donors and patients treated with IL-2.
(A) Flow cytometric histograms of the expression of IL-12 receptor
chains on NK cells from 3 patients and 1 healthy control. (B) and (C)
Summary of the expression of IL-12R1 and IL-12R2 on NK cells from
6 patients and 4 healthy donors. MFI: mean fluorescent intensity.
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Discussion |
Previous studies have demonstrated that the response of T cells to
IL-12 can be modulated by several other cytokines, such as IL-4, IL-18,
IFN-, and IFN-. These cytokines modulate IL-12 signaling
primarily by altering the expression of
IL-12R2.10,11,38,39 In this report, we investigated the
regulation of IL-12 signaling in human NK cells and demonstrated that
IL-2, a cytokine that directly modulates NK cell function both in vitro
and in vivo, has a critical effect on the ability of these cells to
respond to IL-12. IL-2 not only enhances the expression of IL-12
receptor 1 and 2, but also enhances the expression of STAT4, a
critical STAT protein that is known to be associated with IL-12
signaling in NK cells. The up-regulation of both IL-12 receptors and
STAT4 by IL-2 leads to an augmented response of NK cells to IL-12 in both cell-mediated cytotoxicity and IFN- production.
Our results show that the expression of STAT4 in NK cells is regulated
independently from that of STAT1 and STAT5, particularly with respect
to dependency on exogenous cytokines. The expression of STAT5 in NK
cells appears to be independent of cytokines. By contrast, the
expression of STAT1 is dependent on either IL-2 or IL-12, because only
NK cells cultured without IL-2 or IL-12 express low levels of STAT1.
The expression of STAT4 is unique in that it is dependent only on IL-2,
and the addition of IL-12 resulted in decreased expression of STAT4.
These 3 STATs participate in many cytokine signaling pathways. STAT4 is
involved in the signaling of IL-12, IFN-, and IL-2 in NK cells.
STAT1 is associated with IL-2, IFN-, IFN-, and IL-15 signaling,
and STAT5 can be activated by IL-2, IL-3, IL-5, and
granulocyte-macrophage colony-stimulating factor.15,17,40-44 Although all 3 STATs are involved in
multiple cytokine pathways in NK cells, the unique IL-2-dependent
expression of STAT4 may allow a more complex mechanism of regulation.
For example, the signals mediated by STAT4 in NK cells may be more dependent on the microenvironment in the immune system and could be
more precisely controlled by other factors, compared with the signals
relayed by other STATs. In the current study, we demonstrate that IL-2
can promote the expression of STAT4. Other cytokines such as IL-15 may
have a similar effect. IL-15 utilizes the IL-2 and receptor
chains and has many modulating effects on NK cells that are similar to
those of IL-2, such as inducing NK cell proliferation, enhancing target
specific cytotoxicity, and synergizing with IL-12 in inducing IFN-
production.45-47 Because IL-15 is present in human bone
marrow where NK cells are derived,48 IL-15 may play a role
in promoting the expression of STAT4 and in priming NK cells to respond
to IL-12 in vivo.
It has previously been shown that IL-12 can synergize with IL-2 in
inducing IFN- and TNF- production from NK cells and up-regulating the expression of several cell surface molecules on NK
cells,29,33 but IL-12 has also been shown to inhibit the
proliferative effect of IL-2 on NK cells.3,49 Our results
indicate that the expression of IL-12R1 and STAT4 is significantly
reduced in NK cells treated with IL-2 plus IL-12, compared with cells
treated with IL-2 alone. This suggests that the addition of IL-12
should have an inhibitory effect on IL-2-induced functions rather than
a synergistic effect. This apparent contradiction was further examined
in our functional experiments in which we also demonstrated enhanced
production of IFN- from NK cells stimulated by both IL-2 and IL-12.
However, the synergy between IL-12 and IL-2 in inducing IFN-
production from NK cells largely occurred in the first 24 hours of
treatment, and diminished thereafter (data not shown). We have
previously demonstrated that, in addition to STAT1 and STAT5, STAT4 is
also activated by IL-2 in NK cells.17 The synergy between
IL-2 and IL-12 in inducing IFN- production in NK cells is likely due
to the initial enhanced activation of STAT4 by both cytokines. Although the expression of STAT1 and STAT5 remains the same, the expression of
STAT4 in NK cells treated with both IL-2 and IL-12 is significantly decreased after 24 hours, compared with that in NK cells treated with
IL-2 alone. The correlation between the reduced expression of STAT4 and
the inhibitory effects of IL-12- on IL-2-induced functions in NK
cells suggests that STAT4 may play an important role in IL-2-induced
functions such as proliferation and IFN- production in this unique
population. NK cells are known to play a critical role in the
antineoplastic effects of IL-12. In some animal models, the major
factors that contribute to the antitumor effect of IL-12 are NK cell
cytotoxicity and IFN- production.20,24 As a result of
the observation that IL-2 and IL-12 are synergistic in inducing IFN-
production and cytotoxic activity of NK cells in vitro, the combination
of IL-2 and IL-12 has become a more attractive alternative to the use
of IL-12 alone in models of cancer
immunotherapy.29,31,50,51 However, our data suggest that
the simultaneous use of IL-2 and IL-12 is unlikely to be effective in
cancer therapy because of the loss of STAT4 expression in NK cells
treated by both IL-12 and IL-2 and the subsequent loss of the
responsiveness to these cytokines. Therefore, improving the antitumor
effect of IL-12 in combination with IL-2 will likely require strategies
that would maintain the responsiveness of NK cells to IL-12. IL-2 has
been shown to enhance the proliferation of NK cells both in vitro and
in vivo, and patients who receive low-dose IL-2 have a greatly expanded
NK population in their peripheral blood.28,35 In this
study, we have demonstrated that in vitro priming of NK cells with IL-2
up-regulates the expression of IL-12 receptors and STAT4 and enhances
the response of NK cells to IL-12 in both cell-mediated cytotoxicity
and IFN- production. More importantly, NK cells from patients who
receive low-dose IL-2 treatment also exhibited enhanced expression of
IL-12 receptors, indicating the potential of enhanced response to IL-12
in vivo. The results of clinical trials using IL-2 or IL-12 alone have generally been disappointing, with few sustained responses in patients
with metastatic cancer. These results provide further insights into the
functional interactions between IL-2 and IL-12 that can be further
evaluated in animal models and used in the design of future clinical
immunotherapy trials. For example, continuous low-dose IL-2 treatment
can expand the NK cell population and also enhance the NK cell response
to IL-12. A treatment schedule using continuous low-dose IL-2, followed
by an intermittent dose of IL-12, may therefore be a more effective
strategy for enhancing the antitumor activity of these cytokines, and
this can be tested in future clinical trials.
| |
Acknowledgments |
We thank Dr Gordon Freeman for providing materials and advice in
generating receptor expressing cell lines, and Dr Edward Greenfield and
Khuong Nguyen for their assistance in generating monoclonal antibodies.
| |
Footnotes |
Submitted September 20, 1999; accepted January 17, 2000.
Supported by NIH grant CA41619.
Reprints: Jerome Ritz, Dana-Farber Cancer Institute, 44 Binney
St, Boston, MA 02115; email: jerome_ritz{at}dfci.harvard.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction