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Laboratory of Cellular Immunology, Division of Cellular and Gene Therapies, and Divisions of
Cytokine Biology and
Hematologic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD 20852
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-
, and granulocyte-macrophage CSF by NK cells. For the
first time, we demonstrate that freshly purified NK cells stimulated
with IL-2 proliferated and produced IL-10 in a dose-dependent manner.
IL-10 mRNA expression, as detected by semiquantitative reverse
transcription-PCR, reached peak levels at 24 h. IL-10 protein was
detectable on day 2 and further increased on days 3 and 6 as measured
by ELISA. However, IL-12 alone induced neither substantial
proliferation nor detectable IL-10 production by fresh NK cells, but it
synergized with IL-2 in inducing IL-10 mRNA expression and protein
synthesis. IL-10 production by activated NK cells was confirmed by
intracytoplasmic cytokine staining by three-color immunofluorescence of
CD16+ and/or CD56+ NK cells with
anti-IL-10 antibody. IL-10 production by NK cells was further
confirmed in the NK-like cell line, YT, which constitutively expressed
IL-10 mRNA and protein. IL-12 alone did not induce NK proliferation,
but it inhibited IL-2-induced proliferation. Neutralization of
endogenously produced IL-10 with anti-IL-10 antibodies did not
overcome the inhibition of IL-2-induced proliferation by IL-12.
Together, these results demonstrate that IL-2 and IL-12 synergize to
induce IL-10 production by human NK cells and that IL-12 inhibits IL-2
induced NK cell proliferation by an IL-10-independent mechanism. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF- (13),
GM-CSF,3 IL-3 (14), and
IL-5 (15). NK cells are also regulated by numerous other cytokines
(16). IL-12 up-regulates lytic activity and IFN- production by both
NK and T cells (17, 18, 19) and facilitates the development of Th1-type
responses by T cells (20, 21).
The immunoregulatory cytokine, IL-10, inhibits the Ag-dependent
proliferation of T cells (22, 23). Recent evidence has suggested that
mononuclear cells activated with mitogen rapidly produce IFN-,
TNF-, GM-CSF, IL-1, and IL-12 (24, 25). IL-10 is produced later
after stimulation by T cells or monocytes/macrophages in the population
(24) and down-regulates the production of other cytokines (25, 26).
However, production of IL-10 by other cell types is poorly defined.
Recently, it has been shown that IL-12 can prime T cells for high IL-10
production (27, 28), which then inhibits IL-12-induced T cell responses
(29). In this study we analyzed the effects of IL-2 and IL-12 on IL-10
production by human NK cells. We found that IL-2 stimulates freshly
purified human peripheral blood NK cells to produce low levels of
IL-10. In contrast to T cells, IL-12 did not stimulate detectable IL-10
production by NK cells, but synergized with IL-2 to enhance IL-10
production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human rIL-2 was provided by Chiron Corp. (Emeryville, CA), human rIL-12 was supplied by Hoffmann-La Roche, Inc. (Nutley, NJ), and rIL-10 was provided by Schering Corp. (Kenilworth, NJ). Monensin was kindly provided by Ms. Jill Johnson (National Cancer Institute, National Institutes of Health, Bethesda, MD). Anti-CD5 and anti-CD3 Abs were produced and purified as previously described (19, 30). OKM5 (anti-CD36) was purchased from Ortho (Raritan, NJ), and Leu 14 (anti-CD22) was obtained from Becton Dickinson (Mountain View, CA). FITC and phycoerythin (PE)-conjugated Abs to cell surface markers were obtained from Becton Dickinson. Cytochrome 5/PE (Cy5)-conjugated anti-CD56 was provided by Dr. Calman Prussin (National Institute of Allergy and Infectious Diseases, National Institutes of Health). Anti-IL-10-PE, isotype control rat IgG-PE and unconjugated anti-IL-10 Abs were purchased from PharMingen (San Diego, CA). Neutralizing anti-IL-10 mAbs were purchased from BioSource International (Camarillo, CA) and PharMingen (San Diego, CA).
Preparation of cells
Highly purified human NK cells were isolated from either buffy coats or leukapheresis products obtained from healthy volunteers (Department of Transfusion Medicine, National Institutes of Health). PBMC were separated by density gradient centrifugation over Lymphocyte Separation Medium (Organon Teknika Corp., Durham, NC). PBMC (108 cells) were treated with sterilized carbonyl iron (100 mg; Sigma Chemical Co., St. Louis, MO) for 30 min at 37°C. The cell suspension was then subjected to a magnet (MCP-6, Dynal A.S., Oslo, Norway) to deplete monocytes, and unbound cells were layered over 47.5% Percoll to remove high density cells. The NK-enriched low density cells were incubated with anti-CD5, anti-CD3, anti-CD22, and anti-CD36 for 2 to 3 h at 4°C, followed by incubation with goat anti-mouse IgG-coated magnetic beads (Perseptive Diagnostics, Inc., Cambridge, MA) and exposure to a magnet to remove residual T cells, B cells, and monocytes. Unbound cells were treated with a second cycle of goat anti-mouse IgG-coated Dynal magnetic beads at a 1:1 ratio (Dynal) and exposed to a magnetic field. In replicate experiments, the purity of NK cells (CD16+ and/or CD56+) was approximately 90 to 95%, with <1% monocytes, 1 to 3% T cells (often positive for CD56 Ag), and 1 to 2% B cells as analyzed by FACScan (Becton Dickinson).
Cell culture
NK cells were cultured in RPMI 1640 plus 10% FCS (BioWhittaker, Walkersville, MD), 50 µg/ml gentamicin sulfate, 0.1 mM nonessential amino acids, and 2 mM L-glutamine. For activation, purified NK cells were incubated with various concentrations of IL-2 and/or IL-12 for the indicated time periods as described in Results and figure legends. For proliferation assays, cells were cultured at 106 cells/ml in 0.2 ml/well in 96-well microtiter plates with different cytokines and/or Abs to cytokines for 1 to 6 days. Wells were pulsed with 1 µCi of [3H]TdR (DuPont-New England Nuclear, Boston, MA) for 6 to 7 h and harvested onto glass-fiber filters (Wallac, Inc., Gaithersburg, MD). Proliferation was assessed by measuring [3H]TdR incorporation and was expressed as counts per minute. YTN10 and YTN17, two subclones of YT, an NK cell-like cell line obtained from Dr. Junji Yodoi, Kyoto University (Kyoto, Japan) (31), was maintained in RPMI 1640 medium containing 10% FCS.
Intracytoplasmic staining
Freshly purified NK cells were activated for 24 h in the presence of IL-2 and IL-12. Monensin was added at a final concentration of 2 µM, 3 h before termination of the culture. Cells were stained for intracellular cytokine and for cell surface Ags using a modified method of Prussin and Metcalf (32). Briefly, cells were washed in FACS buffer (1% FCS and 0.1% sodium azide in PBS) and stained for NK cell surface markers with CD16-FITC and CD56-Cy5 Abs (45 min, 4°C). Cells were washed twice, resuspended in 1% paraformaldehyde for 20 min at 4°C, washed again, and kept overnight in FACS buffer at 4°C. The next day, cells were washed twice in permeabilization buffer (1% FCS, 0.1% saponin, and 0.1% sodium azide in PBS, pH 7.2 (PB)). Unlabeled anti-IL-10 Ab or isotype control rat IgG at 100 µg/ml in a total volume of 50 µl of PB was incubated with the cells for >1 h in control tubes at 4°C. Anti-IL-10-PE (0.25 µg) was then added, and cells were incubated for an additional 45 min at 4°C. For blocking studies, a 1000-fold excess (1–2 µg) of human rIL-10 was mixed with anti-IL-10-PE Ab 1 h before addition to the cells. Cells were washed twice in PB buffer and once in FACS buffer, and then analyzed by flow cytometry.
Extraction of mRNA and RT-PCR
Total cellular RNA was isolated from activated cells by acid
guanidium isothiocyanate/phenol chloroform extraction as previously
described (33). Levels of cytokine mRNA were assessed by a
semiquantitative RT-PCR (34). RNA (2 µg) was reverse transcribed,
using reverse transcriptase and oligo(dT) primers (Superscript, Life
Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s
protocol. The cDNA was diluted with water to a final volume of 100
µl. Each sample was subjected to an initial amplification using
primers specific for glyceraldehyde-3-phosphate dehydrogenase (G3PDH;
Table
). Based on the amount of amplified
G3PDH PCR product, an equal amount of reverse transcribed product was
amplified for human IL-10 (Table 1). Primers designed for human IL-10
do not cross-react with viral IL-10. PCR was performed in a reaction
mixture containing 5 µl of cDNA, 200 µl of each dNTP, 0.4 µmol/l
of each primer, 1.5 mmol/l MgCl2, 2.5 U of Taq DNA
polymerase (Life Technologies), and 1 µCi of [32P]dCTP
(3000 Ci/mmol; DuPont-New England Nuclear) in reaction buffer supplied
by the manufacturer. cDNAs were amplified in a thermocycler
(Robocycler, Stratagene, CA) as follows. Samples were initially
denatured at 95°C for 3 min, then at 95°C for 1 min, at 61°C for
1 min, and at 72°C for 1 min (20 cycles for G3PDH; 30 cycles for
IL-10), with a final extension at 72°C for 7 min. Samples were
analyzed by electrophoresis through a 6% acrylamide (Long Ranger, AT
Biochem, Malvern, PA) Tris-borate-EDTA gel, followed by autoradiography
and quantitation by PhosphorImager analysis (Molecular Dynamics,
Sunnyvale, CA).
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Human NK cells were stimulated with cytokines as indicated in
the figure legends. The levels of IL-10 protein in NK cell culture
supernatants were measured using an ELISA kit obtained from BioSource
International (Camarillo, CA). A human IFN- ELISA kit was purchased
from Incstar, Inc. (Stillwater, MN). Assays were performed according to
the manufacturer’s instructions. The lower limit of IL-10 detection
was 5 pg/ml. All samples were assayed in duplicate. Results are
expressed as the mean levels of IL-10 in picograms per milliliter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The activation of NK cells by IL-2 is accompanied by an increase
in proliferation and cytolytic function. IL-12 has previously been
shown to induce the proliferation of preactivated PBMC (17, 18, 35) and
preactivated NK cells (35, 36, 37). However, IL-12 alone has been observed
to induce little or no proliferation of fresh NK cells (38, 39) and
certainly less proliferation than IL-2 (40). We examined the ability of
IL-12 together with IL-2 to induce the proliferation of fresh NK cells.
NK cells were activated in the presence of different concentrations of
IL-2 and IL-12 for 1, 2, 3, or 6 days (Fig. 1). IL-2 alone induced NK cell
proliferation in a time- and dose-dependent fashion, and the
combination of IL-12 (1 or 10 U/ml) with a suboptimal concentration of
IL-2 (0.6 ng/ml) consistently produced an additive or greater than
additive (depending on donor) effect. In contrast, when IL-12 was
tested in combination with high concentrations of IL-2 (6.0 ng/ml),
proliferation was markedly inhibited after 2, 3, or 6 days of culture.
This inhibition was IL-12 dose and time dependent. A 30 to 35%
decrease in thymidine incorporation was seen on day 2, a 50% decrease
on day 3, and approximately a 70% inhibition on day 6 (to ensure
sufficient nutrient levels, 6-day cultures had been replenished with
fresh medium and cytokines on day 3; Fig. 1).
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It has been shown that human T cells activated with mitogens such
as Con A proliferate and produce a high level of IL-10 (24), a cytokine
with known inhibitory effects on T cells (41, 42). Recently, IL-12 has
been shown to induce IL-10 production by T cells (27, 28, 43).
Activated NK cells are known to produce a number of cytokines,
including IFN-, TNF-, and GM-CSF, in vitro (2, 13), but IL-10
production by NK cells has not been reported. IL-10 has previously been
shown either to augment IL-2-induced NK activity or proliferation (44)
or to have no effect on IL-2-induced NK activity or proliferation (45, 46). Because of conflicting effects of IL-10 on NK proliferation, we
hypothesized that the IL-12-induced inhibitory effect on NK
proliferation may be due to endogenous IL-10 production. We first
determined whether IL-10 mRNA was expressed in freshly purified NK
cells in response to IL-2 and IL-12. Activation of NK cells with IL-2
(6 ng/ml) for 16 to 96 h resulted in the induction of IL-10 gene
expression. IL-10 mRNA levels were detectable as early as 16 h,
reached peak levels at 24 h (10-fold), and later declined (Fig. 2A). Very low levels of
IL-10 mRNA were consistently detected in control unstimulated cells.
Cells that were stimulated with different concentrations of IL-2 (0–60
ng/ml) for 24 h showed an increase in IL-10 mRNA (Fig. 2B). After normalizing IL-10 mRNA expression levels
to levels of mRNA encoding for G3PDH, a housekeeping gene, the ratio
demonstrated that IL-10 mRNA expression was induced after stimulation
with IL-2 in a dose-dependent manner (Fig. 2C).
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). IL-10 mRNA expression
levels (quantified by phosphoimage analysis) induced by IL-2 and IL-12
together were compared with the sum of the IL-10 mRNA (quantified)
levels produced by each cytokine alone. The ratio was 2.11 ± 0.36
for three experiments (p < 0.02, by one-sample
t test on logarithms of the ratio). Thus, a >2-fold
increase in IL-10 mRNA was observed when IL-2 and IL-12 were used
together over what would be expected if the effect were merely
additive.
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and data not shown).
Consistent with detection of IL-10 mRNA, YTN10 or YTN17 cells
constitutively produced IL-10 as measured by ELISA (1286 or 634 pg/ml).
Neither expression nor production of IL-10 by YT cells was altered by
IL-2 treatment. Synergistic effects of IL-2 with IL-12 on IL-10 secretion
To determine whether secretion of IL-10 was associated with IL-10
mRNA expression, highly purified fresh NK cells were stimulated with
different concentrations of IL-2 and IL-12. Two days later,
supernatants were collected and assayed for IL-10 by ELISA. NK cells
stimulated with IL-12 produced no detectable IL-10, but cells
stimulated with IL-2 alone (0.6 or 6.0 ng/ml) produced low levels of
IL-10 protein (10 and 30 pg/ml, respectively, after 2 days of culture;
Fig. 4A).
However, when NK cells were activated with IL-2 plus IL-12, IL-10
production was synergistically enhanced with as little as 1 U/ml of
IL-12 and 0.6 ng/ml of IL-2. The combined effect of IL-2 and IL-12
increased with the duration of culture; after 3 days the synergistic
effect on IL-10 secretion was even more pronounced, especially with
higher doses of IL-2 (6 ng/ml; Fig. 4B). Figure 4C shows a direct comparison between NK cells from a single
donor stimulated with IL-2 and/or IL-12 for 3 and 6 days. As cells were
refed on day 3, IL-10 levels on days 3 and 6 each represent cumulative
IL-10 accumulated over 3-day periods (0–3 and 3–6 days,
respectively). Under these conditions, the IL-10 production in response
to IL-2 plus IL-12 increased dramatically with time (150 pg/ml for days
0–3 and 400 pg/ml for days 3–6). IL-10 was not detectable after 1 day
of stimulation with IL-2 plus IL-12 (data not shown). To exclude the
possibility of IL-10 production by contaminating T cells or monocytes,
we also examined the effects of IL-2 alone or in combination with IL-12
on IL-10 production by purified NK and T cells obtained from same donor
and by monocytes purified by elutriation from a different donor. NK
cells stimulated with IL-2 together with IL-12 produced IL-10 protein,
but similarly stimulated T cells or monocytes did not produce
detectable levels of IL-10, as measured by ELISA (data not shown).
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IFN- was detectable as early as 1 day after culture. However, while
levels of IL-10 continued to increase after 3 days of stimulation with
either IL-2 or IL-2 plus IL-12, IFN- production declined (Fig. 4D). Intracytoplasmic staining for IL-10 by flow cytometry
To assure that IL-10 was produced by NK cells and not by
contaminating T cells, monocytes, or B cells within freshly purified NK
cell populations, IL-10 production by activated NK cells was further
investigated by fluorescent staining of intracellular IL-10 and flow
cytometry. NK cells were stimulated with IL-2 plus IL-12 for 24 h,
and the cells were stained with anti-CD16-FITC and
anti-CD56-Cy5, permeabilized, and then stained with
anti-IL-10-PE. Frequencies of IL-10-producing cells were then
enumerated among CD16+ and CD56+ NK cells.
The data (Fig. 5) clearly show that
most CD16+ and CD56+ NK cells contain
cytoplasmic IL-10. The specificity of the IL-10 staining was
demonstrated in two ways. First, addition of a molar excess of rIL-10
to the anti-IL-10-PE Ab before staining completely blocked the
intracellular IL-10 staining. Second, preincubation of cells with
unlabeled Ab blocked intracellular IL-10 staining. Contaminating T
cells, monocytes, and B cells, identified by CD3, CD14, and CD22 cell
surface markers, respectively, exhibited low or no staining with
anti-IL-10-PE (data not shown). Intracellular IL-10 staining was
further confirmed in YT cells using the same protocol. All YT cells
stained positive for both CD56 and IL-10 (Fig. 5).
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Previously, it has been shown that IL-10 can directly inhibit T
cell proliferation by a monocyte-independent pathway (47). Because its
production is synergistically enhanced by IL-2 and IL-12 treatment of
NK cells, we investigated whether endogenously produced IL-10 is
responsible for the inhibition of proliferation of NK cells by IL-12.
NK cells were activated with IL-2 and/or IL-12 for 3 to 6 days.
Neutralizing anti-IL-10 Abs (clone JES3-9D7) from two different
commercial sources or normal rat IgG were added at the beginning of
culture. The ability of the anti-human IL-10 mAbs at 2 µg/ml to
neutralize the IL-10 activity was previously confirmed by their ability
to neutralize the endogenously produced IL-10 in cultures of
LPS-stimulated human monocytes (48). Moreover, these anti-IL-10 Abs
prevented the measurement of IL-10 protein by ELISA in the supernatants
from NK cells treated with IL-2 and IL-12 (data not shown).
Nevertheless, the addition of neutralizing Abs to the culture did not
alter the inhibition of IL-2-induced proliferation by IL-12 (Fig. 6). These data suggest that endogenous
IL-10 is probably not responsible for IL-12-induced inhibition of
IL-2-enhanced proliferation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-, GM-CSF (13), and CSF-1
(14) when stimulated with IL-2 and CD16 ligand. Similarly,
IL-2-activated NK cells produced TNF- when stimulated with
anti-CD94 Ab (52). Recently, Warren et al. (15) have shown that
either fresh NK cells isolated from peripheral blood or NK cells
obtained from 17-day cultures, when cocultured with
gamma-irradiated MM-170 or JY stimulator cells in the presence of IL-2,
produced IL-5. The results from the present study extend the list of
cytokines that NK cells produce to include IL-10. Earlier studies have been unable to demonstrate IL-10 production by NK cells (44, 45). Spagnoli et al. (45) studied IL-10 expression in PBMC stimulated with IL-2. Carson et al. (44) characterized IL-10R expression and its potential functional role on human NK cells. They reported that neither resting nor activated NK cells produced human IL-10, although data were not shown. In contrast, we observed that IL-2-stimulated NK cells expressed IL-10 mRNA and protein in a dose-dependent manner. However, the sensitivity of ELISA used by Carson et al. (44) was 40 pg/ml, eightfold less sensitive than that used in the current study, and they did not examine mRNA expression. It is thus possible that the level of IL-10 secreted may not have been detectable with the low sensitivity ELISA.
Our time-course studies showed that expression of IL-10 mRNA expression occurs relatively late, peaking at 24 h, with a subsequent decline. This is consistent with findings in T cells (24, 27) and monocytes (53), in which IL-10 is produced late and inhibits the synthesis of other cytokines produced earlier (42, 53, 54). The production of IL-10 protein increased in a time-dependent manner, was detectable as early as 48 h following IL-2 stimulation, increased at 72 h, and further increased on days 3 to 6 after NK cells were replenished with fresh medium and cytokines. Similarly, Warren et al. (15) have shown that restimulation of NK cells with IL-2 resulted in much higher levels of IL-5 compared with those after the initial stimulation.
IL-10 production by activated NK cells was further confirmed by intracytoplasmic staining using flow cytometry with fluorescence-conjugated Abs to IL-10 and NK cell surface markers (CD16 and CD56). No fundamental differences in the proportion of IL-10-positive cells among CD16+ (89%) or CD56+ (86%) NK cells was observed. Specific blocking with either a molar excess of rIL-10 or the unconjugated anti-IL-10 Ab with conjugated anti-IL-10 Ab before staining supported the conclusion that IL-10 is present in NK cells. Finally, IL-10 production by NK cells was confirmed in the NK-like cell line, YT (N10 and N17 clones), which constitutively expresses IL-10 mRNA and protein under standard culture conditions.
The expression and production of IL-10 in IL-2-activated NK cells are
synergistically enhanced by IL-12. These results suggest that
regulation of IL-10 production in NK cells may be similar to that
reported in T cells (43). In addition to augmenting IL-10 production,
the combination of IL-2 and IL-12 augmented IFN- production in NK
cells, but IFN- production occurs much earlier than that of IL-10.
Recently, Windhagen et al. (55) have reported that stimulation of an
Ag-specific T cell clone with anti-CD3 Ab induced the secretion of
both IL-10 and IFN-, and the effect was enhanced by the addition of
IL-12. The present observations extend the previous reports of
synergistic interactions of IL-2 with IL-12 in inducing IFN- (19, 56, 57, 58) or IL-10 (43) production by PBL and T cells.
Of the known cytokines, IL-2 (59, 60), IL-7 (61), and IL-15 to some
extent (62) can induce significant proliferation of resting NK cells.
Our results support and extend the finding that IL-12 in combination
with low dose IL-2 can invariably enhance the proliferation of NK cells
(38) in an additive or synergistic manner, but with higher IL-2
concentrations, it substantially inhibited NK cell proliferation. This
is consistent with previous findings in T cells (63), an effect that
has been reported to be mediated in part by TNF- (35, 64). However,
the reversal of the effect by blocking with anti-TNF- Ab was
never complete. Therefore, Perussia et al. (35) speculated that IL-2
together with IL-12 may induce the production of other soluble factors
that may be involved in the inhibition of proliferation. Recently,
Jeannin et al. (43) reported that IL-12 directly, independently of APC,
can induce IL-10 production by human T cells. IL-10 has been shown to
suppress both cytokine production (41, 65, 66) and Ag-specific
proliferation (51) of Th1-type clones in an accessory cell-dependent
(47) or -independent (67) manner. Previously, conflicting reports have
appeared showing that IL-10 augments IL-2-induced NK proliferation (44)
or has no effect on it (45, 46). Therefore, we investigated whether the
inhibitory effect of IL-12 on IL-2-induced NK proliferation was in part
due to endogenously produced IL-10. However, neutralizing Abs against
IL-10 failed to block IL-12-induced growth inhibition, suggesting that
IL-10 production, augmented in response to IL-12, does not contribute
to the inhibition by IL-12 of IL-2-induced proliferation of NK
cells.
Previous studies have demonstrated that certain viral infections
activate NK cells to produce IFN- (68, 69), which has been shown to
be a consequence of virus-induced IL-12 production (70), resulting in
increased antiviral activity of NK cells. However, inappropriately
up-regulated levels of IL-12 may have detrimental effects on the host
(71, 72, 73). It can synergize with endogenously produced IL-2 and can
down-modulate CD8+ T cells responses (72, 73). However,
overexpression of IL-12 is controlled through a negative feedback
mechanism involving IL-10 production (54). Although the amounts of
IL-10 produced by NK cells are small compared with those produced by T
cells or monocytes, it is possible that in certain viral infections in
vivo, IL-10 produced by NK cells in response to IL-2 and IL-12
may have a feedback inhibitory effect on cytokines produced within the
microenvironment of NK cells. In summary, our studies suggest that
human NK cells can produce IL-10 and IFN- upon IL-2 stimulation, and
the production can be enhanced synergistically by costimulation with
IL-12.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eda Bloom, Laboratory of Cellular Immunology (HFM-518), Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, Rockville, MD 20852-1448.
3 Abbreviations used in this paper: GM-CSF, granulocyte-macrophage colony-stimulating factor; PE, phycoerythin; PB, 1% fetal calf serum, 0.1% saponin, and 0.1% sodium azide in phosphate-buffered saline, pH 7.2; G3PDH, glyceraldehyde-3-phosphate dehydrogenase.
Received for publication May 1, 1997. Accepted for publication November 19, 1997.
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References |
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 J. A. Gollob, K. G. Veenstra, R. A. Parker, J. W. Mier, D. F. McDermott, D. Clancy, L. Tutin, H. Koon, and M. B. Atkins Phase I Trial of Concurrent Twice-Weekly Recombinant Human Interleukin-12 Plus Low-Dose IL-2 in Patients With Melanoma or Renal Cell Carcinoma J. Clin. Oncol., July 1, 2003; 21(13): 2564 - 2573. [Abstract] [Full Text] [PDF]
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D. L. Hodge, W. B. Schill, J. M. Wang, I. Blanca, D. A. Reynolds, J. R. Ortaldo, and H. A. Young IL-2 and IL-12 Alter NK Cell Responsiveness to IFN-{gamma}-Inducible Protein 10 by Down-Regulating CXCR3 Expression J. Immunol., June 15, 2002; 168(12): 6090 - 6098. [Abstract] [Full Text] [PDF]
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 D. Haller, P. Serrant, D. Granato, E. J. Schiffrin, and S. Blum Activation of Human NK Cells by Staphylococci and Lactobacilli Requires Cell Contact-Dependent Costimulation by Autologous Monocytes Clin. Vaccine Immunol., May 1, 2002; 9(3): 649 - 657. [Abstract] [Full Text] [PDF]
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 P. Vigano, B. Gaffuri, E. Somigliana, M. Infantino, M. Vignali, and A.M. Di Blasio Interleukin-10 is produced by human uterine natural killer cells but does not affect their production of interferon-{gamma} Mol. Hum. Reprod., October 1, 2001; 7(10): 971 - 977. [Abstract] [Full Text] [PDF]
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H. Chakir, A. A. Camilucci, L. G. Filion, and J. R. Webb Differentiation of Murine NK Cells into Distinct Subsets Based on Variable Expression of the IL-12R{beta}2 Subunit J. Immunol., November 1, 2000; 165(9): 4985 - 4993. [Abstract] [Full Text] [PDF]
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T. Hoshino, R. H. Wiltrout, and H. A. Young IL-18 Is a Potent Coinducer of IL-13 in NK and T Cells: A New Potential Role for IL-18 in Modulating the Immune Response J. Immunol., May 1, 1999; 162(9): 5070 - 5077. [Abstract] [Full Text] [PDF]
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T. A. Fehniger, M. H. Shah, M. J. Turner, J. B. VanDeusen, S. P. Whitman, M. A. Cooper, K. Suzuki, M. Wechser, F. Goodsaid, and M. A. Caligiuri Differential Cytokine and Chemokine Gene Expression by Human NK Cells Following Activation with IL-18 or IL-15 in Combination with IL-12: Implications for the Innate Immune Response J. Immunol., April 15, 1999; 162(8): 4511 - 4520. [Abstract] [Full Text] [PDF]
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K. Bernard, A. Cambiaggi, S. Guia, F. Bertucci, S. Granjeaud, R. Tagett, C. N'Guyen, B. R. Jordan, and E. Vivier Engagement of Natural Cytotoxicity Programs Regulates AP-1 Expression in the NKL Human NK Cell Line J. Immunol., April 1, 1999; 162(7): 4062 - 4068. [Abstract] [Full Text] [PDF]
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T. Hoshino, R. T. Winkler-Pickett, A. T. Mason, J. R. Ortaldo, and H. A. Young IL-13 Production by NK Cells: IL-13-Producing NK and T Cells Are Present In Vivo in the Absence of IFN-{gamma} J. Immunol., January 1, 1999; 162(1): 51 - 59. [Abstract] [Full Text] [PDF]
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