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Cancer Research Unit, Canberra Hospital, Canberra, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIIA), the low affinity receptor for IgG, is expressed
on the majority of human peripheral blood NK cells. Ligation of CD16
with mAb or immune complexes activates NK cell cytotoxicity and
cytokine secretion, and stimulates death of activated NK cells by
apoptosis. This study uses NK cells labeled with the stable
intracytoplasmic fluorescent dye 5- and 6-carboxyfluorescein diacetate
succinimidyl ester to provide quantitative data on the effect of CD16
ligation on NK cell division and NK cell survival. When NK cells are
cultured with rIL-2 and CD16 is ligated, NK cell division is
stimulated, but there also is a substantial loss of NK progenitor
cells. When NK cell proliferation is stimulated by coculture with
-irradiated MM-170 malignant melanoma cells and rIL-2, CD16 ligation
enhances entry of NK cells into division. In some cases, CD16 ligation
is essential for NK cell proliferation stimulated by MM-170 cells. In
these cultures, there is no loss of NK progenitor cells. This study
demonstrates that CD16 is an activation receptor for NK cell
proliferation, and suggests that cellular costimulation alters the
balance between NK cell death and NK cell proliferation stimulated by
CD16 ligation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(CD3)- and (Fc
RI)-chains
(2, 3, 4, 5). Ligation of CD16 stimulates cytotoxicity and cytokine secretion
(6) as a consequence of the activation of the
p56lck protein tyrosine kinase (7, 8) as
well as ZAP-70 and syk (9, 10). Recent studies show that
CD16 ligation of cytokine-activated NK cells leads to apoptosis
(11, 12, 13, 14), and it is proposed that this is a mechanism for
down-regulating the NK cell response. Although CD16 is an activation
receptor for NK cells, earlier attempts to demonstrate a role for CD16
ligation in stimulating NK cell proliferation were not successful
(15, 16, 17, 18). This study examines the effect of CD16 ligation on NK cell
proliferation using a quantitative method to measure cell division that
uses the stable intracytoplasmic dye 5- and 6-carboxyfluorescein
diacetate succinimidyl ester
(CFSE)3 (19). In addition to
demonstrating NK cell division, this technique allows the number of
progenitor cells that survive CD16 ligation to be estimated. We show
that CD16 ligation stimulates NK cell division, but also NK cell death.
In the presence of -irradiated MM-170 malignant melanoma stimulator
cells, NK progenitor cells survive CD16 ligation, and NK cell
proliferation is enhanced. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The culture medium was MEM (41500-034; Life Technologies, Grand Island, NY) supplemented with antibiotics (100 µg/ml penicillin, 100 µg/ml streptomycin, 60 µg/ml gentamicin), 24 mM NaHCO3, 0.1 mM 2-ME, 15% heat-inactivated FCS, and 200 U/ml (87 ng/ml) rIL-2. In some experiments, rIL-2 was replaced with rIL-15 (33 ng/ml). Recombinant human IL-2 and IL-15 were generously provided by Dr. G. Zurawski and Dr. R. Kastelein, respectively (DNAX Research Institute, Palo Alto, CA).
Isolation and culture of NK cells
NK cells were isolated from peripheral blood and stimulated to
proliferate, as described previously (16), using -irradiated (40 Gy,
from either a Co60 or a Cs137 source) MM-170
malignant melanoma cells (20) and rIL-2. Briefly, 2.5 x
103 NK cells were cultured with 3 x 103
-irradiated MM-170 cells in multiple 0.2-ml vol in round 96-well
plates (Linbro 76-042-05; ICN Biomedicals, Sydney, Australia). Cultures
were incubated at a temperature of 38°C and a gas phase of 7%
O2, 10% CO2, 83% N2. NK cells
were maintained in culture beyond day 8 by daily diluting twofold with
complete medium during exponential growth. NK cells become quiescent at
day 16 to 18, and are then maintained at a concentration of 0.5 x
106/ml for an additional 7 days by replacing one-half of
the culture medium with complete medium every 2 days. In some cases, NK
cell cultures were intiated and maintained with an IL-2-conditioned
medium prepared from PHA-stimulated tonsil lymphocytes (21), rather
than rIL-2. Cultured cells were phenotyped to establish that they were
entirely NK cells (membrane CD3-, CD56+/-,
CD16+, CD94+).
All experiments in this study used these culture-generated quiescent NK
cells and, except where indicated, medium supplemented with rIL-2. NK
cells were cultured in duplicate 0.2-ml vol in flat 96-well plates
(Linbro 76-032-05; ICN Biomedicals) at 5 x 104/well,
or at 104/well with 3 x 104
-irradiated MM-170 cells. In some studies, the MM-170 cells were
metabolically inactivated by treatment for 10 min with 0.25%
paraformaldehyde before culture (21). For costimulation with
plastic-bound mAb, culture wells were precoated with purified mAb (50
µl of purified mAb prepared in 0.05 M
Na2CO3/NaHCO3 buffer, pH 9.6,
overnight at 4°C). mAb concentrations for coating were 5 times those
determined as optimum by immunofluorescence flow cytometry. Wells were
washed four times with PBS and then blocked for 1 h with medium
containing 5% heat-inactivated FCS before addition of cells.
Measurement of cell proliferation
NK cells were either untreated or labeled before culture with
CFSE (Molecular Probes, Eugene, OR) (19). NK cells were incubated at
room temperature in medium containing 10 µM CFSE for 10 min and then
washed before culture. NK cells were harvested on day 5 or 6 of culture
(CFSE experiments) or were maintained until day 11 or 12. When
required, cultured NK cells were treated with 0.5 mM disodium EDTA in
PBS (5 min, 37°C) to dissociate cell aggregates. Treated cells were
washed in PBS and then resuspended in PBS containing 5% FCS and
maintained on ice before analysis. NK cell growth was assessed by
counting the cells using a hemocytometer. All cells recovered from
cultures stimulated by -irradiated MM-170 cells were viable, as
assessed by trypan blue exclusion. Some apoptotic cells, identified by
their shrunken appearance, were present when NK cells were cultured
without -irradiated MM-170 cells; these cells were not included in
the cell yields. For CFSE analysis, cells were collected using a
FACScan (Becton Dickinson, San Jose, CA) and analyzed for CFSE
intensity using PC-lysis software. In some experiments, propidium
iodide (PI) (final concentration 0.5 µg/ml) was added before analysis
of the cells by flow cytometry to identify the apoptotic cells.
Cell division in CFSE-labeled NK cells is calculated based on the sequential halving of fluorescence intensity in daughter cells (19). To calculate CFSE intensity for different divisions, the geometric mean fluorescence intensity (MFI) of unlabeled cultured NK cells (autofluorescence) is subtracted from the MFI of CFSE-labeled NK cells cultured on untreated plastic (control). This value is divided by 1, 2, 4, 8, 16, and 32 to give the MFI of cells after 0, 1, 2, 3, 4, and 5 divisions, respectively. The boundaries that define the different cell divisions are the midpoint between these MFI values. To these values are added the MFI of unlabeled cultured NK cells (autofluorescence) relevant to the culture being analyzed, that is, control or CD16 ligation. The boundaries calculated define M1 (quiescent), M2 (1 division), M3 (2 divisions), M4 (3 divisions), M5 (4 divisions), and M6 (5 divisions). The percentage of cells at each division is determined and based on the yield of cells in the culture; the number of cells at each division is calculated. These numbers are divided by the progeny of each division cycle, that is, 1, 2, 4, 8, 16, or 32, to give the number of surviving, but as yet undivided cells (M1) and the number of cells that give rise to progeny (M2 through M6). The sum of these numbers is an estimate of the NK cell progenitors that survive culture. Cells in M1 are included in this estimate.
Measurement of cell death
Apoptotic cell death was measured by the binding of Annexin V-FITC to phosphatidylserine on the external plasma membrane of intact cells (22). NK cells were cultured at 50,000 in 0.1 ml of complete medium containing rIL-2 on flat 96-well plates that had been precoated with control purified CD56 mAb WV3, or with purified CD16 mAb B73.1. At 6 h, NK cells were harvested and incubated with Annexin V-FITC and PI according to the instructions provided by the manufacturer (Clontech, Palo Alto, CA).
Antibodies
mAbs used were: OKT3 (CD3, IgG2a), OKT11 (CD2, IgG1), HB205 (CD58, IgG1), HB202 (CD11a, IgG1), and HB203 (CD18, IgG1) from hybridoma cells obtained from the American Type Culture Collection (Manassas, VA); WV3 (CD56, IgG1) and WV2 (CD94, IgG1) produced in our laboratory; B73.1 (CD16, IgG1) from the hybridoma cell line kindly provided by Dr. G. Trinchieri (Wistar Institute, Philadelphia, PA); RR1/1 (CD54, IgG1), a gift from Dr. T. Springer (Dana-Farber Cancer Institute, Boston, MA); HuLyM9 (CD71, IgG1), a gift from Dr. I. F. C. McKenzie (Austin Research Institute, Heidelberg, Victoria, Australia); CLB-FcGran1 (CD16, IgG2a) obtained from the Red Cross Blood Transfusion Service (Amsterdam, The Netherlands); G10.3 (IgG3) kindly provided by the organizers of the Sixth International Workshop on Human Leukocyte Differentiation Antigens; and Tu69 (CD25, IgG1), purchased from Cymbus Bioscience (Southhampton, U.K.). FITC-conjugated sheep anti-mouse Ig (code DF) was obtained from AMRAD Pharmacia Biotech (Boronia, Victoria, Australia). mAbs were purified by protein A-Sepharose CL-4B chromatography (Pharmacia Biotech) using procedures described by the manufacturers. The specificity of mAb WV3 (CD56) and WV2 (CD94) was demonstrated by reactivity with transfectant cell lines kindly provided by Dr. Lewis Lanier (DNAX Research Institute).
Phenotype analysis
Procedures used for analysis of NK cell surface Ags were as described previously (16). Briefly, 2 x 104 cells were incubated for 30 min on ice in 96 V-well plates with predetermined optimum concentrations of mAbs. Cells were washed three times in PBS containing 5% heat-inactivated FCS and 0.1% sodium azide (PBS/FCS), followed by incubation with FITC-conjugated sheep anti-mouse Ig for 30 min on ice. Cells were then washed three times and resuspended in PBS/FCS, and an equal volume of 2% paraformaldehyde in PBS (pH 7.4) was added. Cells were analyzed by flow cytometry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-irradiated MM-170
malignant melanoma cells and IL-2. The activated NK cells kill the
MM-170 cells by day 5 and then proliferate vigorously until day 14,
after which cell growth slows and the cells become quiescent by day 18
(16). NK cells can be maintained for up to 2 wk in the quiescent state
and can be restimulated to proliferate in secondary culture (21). This
study used culture-generated quiescent NK cells to examine the role of
CD16 in activating NK cell proliferation. CD16 ligation stimulates NK cell death and NK cell activation
Initial experiments confirmed that CD16 ligation stimulates both
NK cell death and NK cell activation. In these experiments, quiescent
NK cells were cultured in complete medium containing rIL-2 on plastic
coated with purified CD56 mAb WV3 (control) or on plastic coated with
purified CD16 mAb B73.1 (CD16 ligation). NK cells were analyzed at
6 h for apoptosis and at 22 h for expression of activation
Ags. The results of representative experiments typical of those
obtained with five different donors are presented in Fig. 1
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A). This is illustrated by the
dot plot of granularity (side scatter, SSC) versus size (forward
scatter, FSC), and by staining with Annexin V-FITC that binds to
externalized phosphatidylserine, an early indicator of apoptosis (22).
For the experiment shown, 46.7% of NK cells cultured for 6 h on
plastic wells coated with CD16 mAb are in the early stages of
apoptosis, compared with 1% in control cultures. A proportion of NK
cells stimulated by CD16 ligation has progressed beyond the initial
stage of apoptosis and is already permeable to PI at 6 h. In the
experiment shown, 14.5% of cells in cultures stimulated by CD16
ligation are permeable to PI compared with 3.5% in control cultures.
The total number of cells binding Annexin V (both PI+ and
PI-) is 61.2% for cultures stimulated by CD16 ligation
compared with 4.5% in control cultures. This corresponds closely to
the distinct population of apoptotic cells defined on the basis of
granularity and size, which was 60% for cultures stimulated by CD16
ligation compared with 5.4% in control cultures. For experiments with
six different donors, on average the total number of cells binding
Annexin V was 50.9 ± 7.4% (SEM) in cultures stimulated by CD16
ligation, compared with 6.7 ± 1.7% in control cultures. In the same
experiments, when the number of NK cells cultured was reduced fivefold
from 50,000 to 10,000 per well (equivalent to 25 x
104/ml and 5 x 104/ml, respectively), the
total number of cells binding Annexin V was 36.5% ± 6.1% in cultures
stimulated by CD16 ligation, compared with 3.2% ± 0.8% in control
cultures. Thus, at lower NK cell concentrations, there is still a
substantial amount of apoptosis stimulated by CD16 ligation.
When analyzed at 22 h following CD16 ligation, a proportion of NK
cells expresses activation Ags (Fig. 1B). The results show
the expression of CD25 (the 
-chain of the high affinity IL-2R) and
CD71 (the transferrin receptor) on viable cells from a typical
experiment. For NK cells stimulated by CD16 ligation, on average 24.8
± 6% (n = 5) of viable cells express CD25, and 29.6
± 5.4% (n = 5) express CD71. By contrast in control
cultures, on average only 2.3 ± 1.1% (n = 5) of
viable cells express CD25, and 4.9 ± 1% (n = 5)
express CD71. Collectively, these results confirm that both NK cell
death and NK cell activation are consequences of CD16 ligation.
CD16 ligation stimulates NK cell division
To show that NK cells divide following CD16 ligation, quiescent NK
cells were labeled with the intracytoplasmic dye CFSE before culture
(19). Incorporation of CFSE is stable. When cells divide, the intensity
of fluorescence in daughter cells is one-half that of the parent cell.
In initial experiments, NK cells were analyzed after 12 days of
culture. At this time, NK cells recovered from control cultures were
brightly stained, whereas NK cells recovered from culture wells coated
with purified CD16 mAb B73.1 showed only weak CFSE staining that was
close to the autofluorescence level of cultured unlabeled cells (data
not shown). In the next experiments, cultures were analyzed at 6 days,
an early time point in the course of NK cell proliferation (16). At 6
days, the level of fluorescence of proliferating CFSE-labeled NK cells
falls within a range that is above the level of autofluorescence of
unlabeled cultured NK cells. In these cultures, measurement of CFSE
intensity permits quantitation of cell division and estimation of
progenitor cell numbers, as outlined in Materials and
Methods. Progenitor cells are defined as the cells giving rise to
the proliferating population. Calculation of progenitor cell numbers
includes cells that are still undivided at day 6. This is justified
since NK cell proliferation is asynchronous (see Fig. 2), and day 6 is early in the course of
NK cell proliferation (16). Furthermore, as stated above, CFSE
intensity of NK cells cultured on plastic coated with CD16 mAb is
reduced to background by day 12, implying that all cells that survive
have the potential to eventually divide.
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are from a representative experiment (1 of
18 experiments using NK cells from five different donors) comparing
CFSE-labeled NK cells cultured in complete medium for 6 days on plastic
coated with control CD56 mAb, with NK cells cultured on plastic coated
with CD16 mAb. Dot plots of granularity (SSC) versus size (FSC)
demonstrate the populations of viable cells (R1) and apoptotic cells
(R2) (Fig. 2A). The settings on the flow cytometer differed
from those in Fig. 1A. Control cultures contain few
apoptotic cells, and the viable cells are small in size. By contrast,
cultures stimulated by CD16 ligation contain a significant number of
apoptotic cells, and the viable cell population has many blast cells.
Dot plots of PI staining (to detect apoptotic cells) versus CFSE
intensity are presented in Fig. 2B for cells in gate R1 and
gate R2. In gate R1, only 1% of cells stain with PI compared with gate
R2, in which 89% stain with PI. The 11% of cells that do not stain
with PI in gate R2 may be cells at an early stage of apoptosis,
although this was not tested by Annexin V binding. Viable cells in
control cultures have a uniformly high CFSE intensity, whereas viable
cells in cultures stimulated by CD16 ligation show discreet patterns of
CFSE intensity, indicative of cell division. This pattern is seen also
for apoptotic cells in cultures stimulated by CD16 ligation, indicating
that NK cell death occurs following cell division. Dot plots of size
(FSC) versus CFSE intensity (Fig. 2C) show that in cultures
stimulated by CD16 ligation, cells with CFSE intensity lower than
undivided cells are the blast cells. The histogram presentation of the
data in Fig. 2D shows the CFSE intensity of cultured cells
compared with the autofluorescence of unlabeled cultured cells (line
histogram), and the calculated boundaries for different divisions, M1
being the undivided (quiescent) cells and M2 through M6 being cell
divisions 1 through 5. Quantitative analysis of the CFSE data in Fig. 2D is presented in Table I.
This analysis shows that for control cultures, the 28,000 NK cells
recovered originate from an estimated 27,664 progenitor cells, and that
only 2.4% of these progenitor cells divide (27,328 remain in M1). By
contrast, the 38,700 NK cells recovered in cultures stimulated by CD16
ligation originate from an estimated 12,813 progenitor cells, of which
52.6% divide (6,076 remain in M1). When cell yields at day 6 are
compared with the estimate of progenitor cells in the 18 different
experiments, there is on average a 2.14 ± 0.27 (SEM)-fold
increase in cell number in cultures stimulated by CD16 ligation,
compared with 1.07 ± 0.02-fold in control cultures. This increase
in cell number is reflected in the number of NK cells that enter
division (M2 to M6) at day 6 of culture, which was on average 30.5 ±
4% (range 8.4–52.7%). For the experiment shown in Table I,
comparison of progenitor cell numbers estimated in control cultures
(27,664) and in cultures stimulated by CD16 ligation (12,813) shows
that 46.3% of NK progenitor cells survive CD16 ligation. The number of
progenitor cells that survive CD16 ligation varied in different
experiments and is not donor dependent. For the 18 different
experiments, on average 43.4 ± 4.4% (range 11.9–71.3%) of NK cell
progenitors survive CD16 ligation compared with controls. A consequence
of progenitor cell loss in cultures stimulated by CD16 ligation is the
poor yield of NK cells at day 6 of culture, which was on average 73.4
± 5.3% (range 36.1–138.2%, n = 18) of that in
control cultures.
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B). Weak growth after day 6 contrasts with the vigorous
growth that occurs when quiescent NK cells are stimulated by
-irradiated MM-170 cells (21).
NK cell division is enhanced when NK cells are stimulated by CD16
ligation during coculture with -irradiated MM-170 stimulator cells
and rIL-2
NK cells proliferate when restimulated in secondary culture with
-irradiated MM-170 cells and rIL-2 (21). Analysis of NK cell
proliferation using CFSE-labeled NK cells shows that CD16 ligation
enhances NK cell proliferation in these cultures. This result is
illustrated by the experiment shown in Fig. 3, and is representative of six
experiments with NK cells from four donors. In these experiments,
cultures were usually analyzed on day 5, rather than day 6, to ensure
that the CFSE staining of proliferating cells was in a range above the
autofluorescence of unlabeled cultured NK cells. NK cells cultured
without stimulation are uniformly brightly stained at day 5 of culture
(data not shown). When NK cells are cultured with -irradiated MM-170
cells, only a minority have divided at this early time point in
culture. When NK cells are cultured with -irradiated MM-170 cells
and stimulated by CD16 ligation, virtually all NK cells divide by day
5. Quantitative analysis of the data in Fig. 3 is presented in Table II. For NK cells stimulated by
-irradiated MM-170 cells, 10,000 NK cells were cultured and 3,500
were recovered on day 5. These 3,500 NK cells originate from an
estimated 2,446 progenitor cells, a 1.4-fold increase in cell number.
Of the 2,446 progenitor cells, 24.6% divide (1,845 remain in M1). For
NK cells cultured with -irradiated MM-170 cells and stimulated by
CD16 ligation, 10,000 cells were cultured and 13,000 were recovered on
day 5, a net increase in NK cell numbers. These 13,000 NK cells
originate from an estimated 2,529 progenitors, demonstrating a 5.1-fold
increase in NK cell numbers during the 5 days of culture. Of the 2,529
progenitor cells in cultures stimulated by CD16 ligation, 85.6% divide
(364 remain in M1). A comparison of progenitor cell numbers for NK
cells cultured with -irradiated MM-170 cells reveals that these are
virtually the same in control cultures (2,446) and cultures stimulated
by CD16 ligation (2,529). For six different experiments, on average the
number of progenitor cells in cultures stimulated by CD16 ligation was
100.5% ± 5.5% of that in control cultures. Comparison of cell yields
and estimates of progenitor cell numbers reveals that there is on
average a 2.2 ± 0.4-fold increase in cell number when NK cells
cultured with -irradiated MM-170 cells, compared with on average a
6.3 ± 0.8-fold when NK cells also are stimulated by CD16
ligation, that is, CD16 ligation enhances MM-170 cell-stimulated NK
cell division.
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-irradiated MM-170 cells, there is no loss of NK progenitor
cells following CD16 ligation. This contrasts with the substantial loss
of progenitor cells when NK cells are cultured alone and stimulated by
CD16 ligation (Table I). This result is not simply a consequence of the
lower NK cell numbers in these cultures (10,000 instead of 50,000),
since as stated above, CD16-stimulated apoptosis measured in a 6-h
assay was substantial at both cell concentrations. The role of MM-170
cells in preventing progenitor cell loss remains to be established.
Adding a mixture of mAb to adhesion molecules (CD11a, CD18, CD54, CD2,
and CD58) did not prevent progenitor cell survival in cultures
containing MM-170 cells (data not shown).
CD16 ligation enhances NK cell division stimulated by -irradiated
MM-170 cells and rIL-2, with increased cell yields at day 5 of culture.
However, when NK cell growth is measured later in culture, there is no
difference in cell yields as a consequence of CD16 ligation. In nine
different experiments using NK cells from three donors, the growth of
NK cells to day 12 was on average 42.7 ± 7.1 (SEM)-fold in
control cultures and on average 41.3 ± 11.9-fold with CD16
ligation. It should be pointed out that after day 6 or 7, NK cells are
no longer being stimulated by CD16 ligation since proliferating cells
are subcultured onto untreated plastic. All cells recovered on day 12
are entirely CD16+.
Other experiments (not shown) established that when NK cells are
activated by CD16 ligation, there is still the requirement for
-irradiated MM-170 stimulator cells (metabolically inactive MM-170
cells are ineffective), and the requirement for rIL-2 (rIL-15 is
ineffective) for stimulating sustained NK cell growth (21, 23).
CD16 ligation facilitates proliferation and sustained growth of NK
cells that proliferate weakly when stimulated by -irradiated MM-170
cells and rIL-2
NK cells from three donors proliferate weakly, if at all, when
restimulated in secondary culture with -irradiated MM-170 cells and
rIL-2. For NK cells from these donors, CD16 ligation facilitates
MM-170-stimulated NK cell proliferation measured on day 6, and NK cell
growth is sustained. For nine experiments with NK cells from these
donors, the NK cell numbers in control cultures increased by on average
only 3.1 ± 0.7-fold during 11 days of culture. By comparison,
with CD16 ligation, NK cell numbers increased by on average 19.8
± 5.9-fold. NK cell growth in these cultures continued until day 18,
after which cells became quiescent. These quiescent NK cells again
required CD16 ligation, rIL-2, and costimulation with -irradiated
MM-170 cells to proliferate, demonstrating that the inability of these
NK cells to proliferate without concomitant CD16 ligation was a stable
property of NK cells from these donors.
The experiment in Fig. 4 shows the effect
of CD16 ligation on proliferation of NK cells during 6 days of culture
using CFSE-labeled NK cells, and is representative of nine experiments
with NK cells from these donors. When stimulated by -irradiated
MM-170 cells, the NK cells remain uniformly brightly stained. However,
when NK cells are cultured with -irradiated MM-170 cells and
stimulated by CD16 ligation, NK cells show decreased CFSE intensity
indicating cell division. Quantitative analysis of this data is
presented in Table III. For NK cells
stimulated with -irradiated MM-170 cells, 10,000 NK cells were
cultured, and 5,000 NK cells were recovered at day 6. These 5,000 NK
cells originate from 4,900 progenitor cells, of which 4% divide (4,800
remain in M1). By contrast for NK cells stimulated by CD16 ligation and
-irradiated MM-170 cells, 10,000 NK cells were cultured and 12,200
NK cells were recovered at day 6. These 12,200 NK cells originate from
4,782 progenitor cells, and 49.4% divide (2,420 remain in M1). As for
NK cells from other donors (Table II), under these culture conditions
in the presence of -irradiated MM-170 cells, there is no loss of NK
cell progenitors when NK cells are stimulated by CD16 ligation. For the
nine different experiments, on average the number of NK cell
progenitors that survive CD16 ligation compared with control cultures
is 147 ± 20.5% (SEM). Comparison of cell yields and estimates of
progenitor cells shows that there is on average a 3.5 ± 0.7-fold
increase in NK cell number when NK cells are stimulated by CD16
ligation, compared with a 1.2 ± 0.1-fold increase in NK cell
number in control cultures. NK cells that proliferate are entirely
CD16+. The results show that when NK cell proliferation
stimulated by -irradiated MM-170 cells and rIL-2 is weak, CD16
ligation enhances NK cell division early in culture and this results in
strong and sustained NK cell growth.
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-irradiated MM-170 cells and rIL-2. Similar results were obtained
when CD16 was ligated using soluble mAb of murine IgG3 class, that is,
the MM-170 cell-reactive mAb G10.3. In these experiments, there also
was no progenitor cell loss in cultures stimulated by CD16 ligation.
Growth using G10.3 mAb was assessed over a 13-day culture period using
NK cells that were entirely dependent upon CD16 ligation and MM-170
cells for proliferation. The growth of 50-fold was inhibited by more
than 95% in the presence of soluble CD16 mAb CLB-FcGran1, which
recognizes the Fc binding region on CD16 (24) (data not shown). Thus,
ligating CD16 at the Fc binding site (with G10.3 mAb) or at the B73.1
epitope that is distinct from the Fc binding site (24, 25) results in
equivalent effects on NK cell proliferation. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study uses polyclonal quiescent NK cells generated in primary culture and analyzes the effect of CD16 ligation on stimulating NK cell proliferation in secondary culture. When NK cells are stimulated by CD16 ligation and cultured in medium containing rIL-2 for 6 days, the recovered cells show a heterogeneous pattern of fluorescence indicative of asynchronous cell division. By contrast, NK cells in control cultures remain brightly stained, demonstrating that these cells are quiescent. These data demonstrate unequivocally that CD16 ligation stimulates NK cell division. Earlier reports showed that following CD16 ligation, NK cells express activation Ags (6, 18) and incorporate [3H]thymidine (28, 29), consistent with NK cell proliferation. However, NK cell growth measured after several days of culture was poor (18). The data we obtained using CFSE-labeled NK cells not only establish that CD16 ligation stimulates NK cell division, but also confirm that considerable cell death occurs in these cultures. A substantial amount of CD16-stimulated cell death occurs within hours of CD16 ligation (11, 12, 13, 14), a result confirmed in the present study in which early apoptosis was detected by the binding of Annexin V-FITC to phosphatidylserine on the external membrane of intact NK cells (22). Some NK cells also die following CD16-stimulated cell division, as shown by analysis of the small number of PI-stained CFSE-labeled NK cells at day 6 of culture. This cell death probably accounts for the poor yield when CD16-stimulated NK cell cultures are maintained until day 12.
The present studies suggest that costimulation is important in
preventing NK cell death following initiation of NK cell division by
CD16 ligation, since when CD16 is ligated in the presence of
-irradiated MM-170 cells, there is no loss of NK progenitor cells.
This is analogous to the role of costimulation in T cell and B cell
responses. In the case of T cells activated by TCR ligation,
costimulation through CD28 engaging CD80 or CD86 on APCs results in
sustained growth that is for the most part due to induction of the
survival gene Bcl-xL (30, 31, 32). When Ag is presented to T
cells in the absence of these CD28 ligands, such as on hepatocytes,
early Ag-specific T cell proliferation is followed by cell death,
rather than sustained growth (33). In the case of B cells, apoptosis
following B cell receptor ligation is prevented by CD40 engaging CD40
ligand on activated T cells (34), and this also is likely to involve
induction of Bcl-xL (35, 36). In the case of NK cells,
receptors involved in costimulation are not known, and indeed human NK
cells do not express CD28 (37) (Warren, unpublished). The identity of
the physiologic equivalent of the MM-170 cells and the necessary
ligands for interaction with NK cell receptors to prevent apoptosis
also needs to be determined. Further studies are required to define the
mechanism by which MM-170 cells promote NK cell survival and whether
induction of survival genes such as Bcl-xL is involved.
For NK cells from most donors, proliferation and sustained growth in
secondary culture are stimulated by -irradiated MM-170 cells and
rIL-2 (21). In these cultures, CD16 ligation provides an additional
signal that contributes to MM-170 cell-initiated NK cell proliferation,
enhancing entry of cells into division (Fig. 3). For NK cells from some
donors, CD16 ligation is essential for NK cell proliferation and
sustained growth stimulated by -irradiated MM-170 cells and rIL-2
(Fig. 4). The inability of NK cells from these donors to proliferate is
seen in secondary and subsequent cultures. In primary culture, NK cells
from these donors proliferate without the need for CD16 stimulation. It
is possible that during primary culture, receptors that activate
proliferation are lost or become nonfunctional, or alternatively, that
inhibitory receptors are acquired or become functional, thereby
preventing restimulation in secondary culture. The possibility that the
killer-inhibitory receptors (KIR), which inhibit NK cell cytotoxicity
and cytokine secretion by recognizing particular HLA alleles on target
cells (reviewed by Long et al. (38)), also regulate NK cell
proliferation was addressed in a recent study (39). Blocking KIR does
not permit proliferation stimulated by -irradiated MM-170 cells and
rIL-2 unless NK cells are suboptimally stimulated through CD16,
suggesting that mechanisms in addition to KIR regulate NK cell
proliferation. In a physiologic context, we suggest that mechanisms
that prevent NK cell proliferation can be overcome by activating NK
cells through CD16.
We showed previously that compared with resting NK cells, proliferating
NK cells produce up to 50 times higher levels of IFN-,
granulocyte-macrophage CSF, and TNF-, and acquire the ability to
produce IL-5 (40). These observations emphasize the potential
importance of NK cell proliferation in immunoregulation and
hemopoiesis. NK cell proliferation occurring following engagement of
CD16 by Ag-Ab complexes, even though this may be limited in the absence
of appropriate costimulation, could be sufficient to enhance cytokine
secretion. We recently proposed (23) that NK cell proliferation occurs
during both the innate immune response and following development of an
Ag-specific response, because both monocyte-derived cytokines (IL-15
with IL-10 or IL-15 with IL-12) and T cell-derived IL-2 are effective
in stimulator cell-initiated NK cell proliferation. We recently
reviewed evidence that NK cell proliferation occurs in vivo (41). In
particular, NK cells from patients with an NK lymphocytosis express
class II MHC and/or CD45R0 that indicate a history of proliferation
(42). Interestingly, for one patient, the NK lymphocytosis resolved
over a 2-yr period, suggesting that continued stimulation was required
to maintain the NK cell proliferative disorder. Conceivably, chronic
CD16 ligation by Ag-Ab complexes in the presence of appropriate
costimulation (such as provided experimentally by MM-170 cells) and T
cell-derived IL-2 could contribute to an NK cell proliferative
disorder.
In summary, these results demonstrate a role for CD16 as an activation receptor for NK cell proliferation. Hitherto, CD16 ligation has been shown to stimulate NK cell apoptosis, and this is proposed as a mechanism for down-regulating NK cell responses. Our results support the conclusion that both NK cell proliferation and death are consequences of CD16 ligation, and that just as with ligation of T cell and B cell Ag receptors, cellular costimulation is important in altering the balance in favor of proliferation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. H. S. Warren, Cancer Research Unit, Canberra Hospital, P.O. Box 11 Woden ACT 2606, Australia.
3 Abbreviations used in this paper: CFSE, 5- and 6-carboxyfluorescein diacetate succinimidyl ester; FSC, forward scatter; KIR, killer-inhibitory receptor; MFI, mean fluorescence intensity; PI, propidium iodide; SSC, side scatter.
Received for publication February 20, 1998. Accepted for publication September 30, 1998.
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