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Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 207-213
Differential Cytotoxicity of Cord Blood and Bone Marrow-Derived
Natural Killer Cells
By
Clair M. Gardiner,
Anne O' Meara, and
Denis J. Reen
From The Children's Research Centre, Our Lady's Hospital for Sick
Children, Crumlin, Dublin, Ireland.
 |
ABSTRACT |
Allogeneic cord blood is now being widely used as a source of stem
cells for hematologic reconstitution after myeloablative therapy, with
reported significantly lower levels of graft-versus-host disease (GVHD)
compared with the use of allogeneic bone marrow (BM). This study was
undertaken to investigate biologic aspects of natural killer (NK) cell
activity, as recognized effector cells of the GVHD and
graft-versus-leukemia (GVL) response, from cord blood and conventional
BM. NK-cell activity levels of freshly isolated cells from cord blood
and BM against K562 targets were comparable. Lymphokine activated
killer (LAK) cells from both hematopoietic cell sources were compared
for their ability to kill target cells by necrotic or apoptotic
mechanisms using specific target cell lines. Cord blood cells had
significantly higher necrosis-mediated cytotoxic activity against Daudi
target cells compared with BM-derived cells. Cord blood LAK cells had
relatively high levels of apoptotic-mediated cytotoxicity against YAC-1
target cells, whereas BM-derived LAK cells were unable to induce
apoptosis in these cells. Interleukin-2 (IL-2) induced significant
granzyme B activity in cord cells in contrast to BM cells, in which
very little activity was measured. Western blotting confirmed these
findings, with IL-2 inducing granzyme B protein expression in cord
cells but not detectable levels in BM cells. BM cells had significantly
lower cell surface expression of IL-2R and prolonged culture in IL-2
was only partially able to restore their deficient apoptotic cytotoxic
activity. Thus, major differences exist between cord blood-derived and
BM-derived mononuclear cells with respect to their NK-cell-associated
cytotoxic behavior. This could have important implications for stem
cell transplantation phenomena, because it suggests that cord blood may
have increased potential for a GVL effect.
| |
INTRODUCTION |
NATURAL KILLER (NK) cells play a number
of recognized roles in bone marrow transplantation (BMT).1
They have been identified as an effector cell in graft-versus-host
disease (GVHD)2,3 and also as an effector cell in the
beneficial graft-versus-leukemia (GVL)4,5 effect. Cord
blood is now being used as an alternative source of stem cells to BM
for hematologic reconstitution.6 Results thus far show a
significantly reduced incidence of GVHD in cord blood transplant
patients even when unrelated donors are used.7,8 However,
the follow-up period is still too short for any impact on GVL to be
known. Cord blood cells have been reported to have reduced NK-cell
activity relative to adult peripheral blood.9,10 This may
contribute to the increased morbidity and mortality associated with
neonatal infections.11 Cord cells can be induced by various
stimuli, in particular interleukin-2 (IL-2), to develop high lymphokine
activated killer (LAK) activity.12,13
LAK cells have been shown to kill target cells by at least two
mechanisms, necrosis and apoptosis.14 Perforin, which is
found in the cyotoxic granules of NK cells, is accredited as the
protein responsible for necrosis, which primarily involves target cell
membrane damage.15 Apoptosis of target cells may be induced
by a number of mechanisms, including fas/fas-ligand
interactions16 and tumor necrosis factor- (TNF-)
secretion,17 or via granzymes.18,19 Granzymes
are a family of serine proteases found in the cytotoxic granules of
activated cytotoxic lymphocytes.20 Granzyme B has been
reported to be essential for the rapid induction of apoptosis in
susceptible target cells,21,22 although the exact mechanism
by which this happens remains to be elucidated. It is now accepted that
apoptosis represents the physiologic form of cell death in
vivo.23 Therefore, it seems reasonable to expect it to also
represent the primary mechanism involved in cell-mediated cytotoxicity,
and there is a growing body of evidence in the literature to support
this hypothesis.19,24 However, relatively little is known
about this pathway at regulatory or effector mechanism levels.
Data in the literature describing cytotoxic activities of BM NK cells
are varied with respect to potential and kinetics of induction. Freshly
isolated BM is generally reported to have low killing
activity.25,26 Agah et al27 have reported
rapidly induced, potent killing in BM by IL-2, whereas others have
reported slower induction with much donor variation.25,26
Similar to cord blood, nothing is known about the potential of BM LAK
cells to induce apoptosis in target cells. This study was undertaken to
compare aspects of cord blood and BM cytotoxic activities using model
systems to measure both necrotic and apoptotic death of various tumor
target cell lines.
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MATERIALS AND METHODS |
Effector cells.
Approximately 10 mL of BM was obtained from the posterior ileac crests
of anesthetized healthy adolescent and adult patients undergoing
elective orthopedic surgery. Cord blood samples were obtained from
full-term, normal delivery, healthy infants. Samples were collected
into 0.01 mol/L EDTA in phosphate-buffered saline (PBS), pH 7.4.
Mononuclear cells were isolated by lymphoprep (Nycomed, Oslo,
Norway) density-gradient centrifugation.28
Cells cultured in vitro were grown in RPMI-1640 medium supplemented
with 10% fetal calf serum (GIBCO, Paisley, UK), 20 mmol/L
L-glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin,
and 20 mmol/L HEPES, referred to as complete medium (CM). IL-2 (Cetus
Corp, Emeryville, CA) was used at 500 U/mL and was added at the start
of culture.
Phenotyping.
Mononuclear cells isolated by density-gradient
centrifugation28 were stained with monoclonal antibodies
and examined for cell surface expression of NK cell antigens by flow
cytometry. Directly conjugated anti-CD56 phycoerythrin (PE) (Becton
Dickinson, Oxford, UK), anti-CD16 fluorescein isothiocyanate (FITC)
(DAKO, High Wycombe, Bucks, UK), and anti-CD8 PE (DAKO) antibodies were
used.
NK cell purification.
CD56+ cells were purified using the Mini MACS system
(Miltenyi Biotec, Bergisch-Gladback, Germany). Briefly, isolated
mononuclear cells were incubated with anti-CD56 magnetic beads for 15
minutes at 4°C. Cells were washed, resuspended in 0.5 mL wash
buffer (PBS, 2 mmol/L EDTA, 0.5% bovine serum albumin, pH 7.2), and
passed through a magnetized column. Negative cells were eluted by
washing. The column was then removed from the magnet and
CD56+ cells were eluted using 1 mL wash buffer.
51Cr release assay.
A standard 4-hour 51Cr release assay was used to measure
necrotic death of target cells.29 Briefly, 106
target cells were labeled with 50 µCi
Na251CrO4 for 1 hour at 37°C.
Cells were washed twice with PBS and resuspended in CM, and 1 ×
104 cells were aliquoted per well in 100 µL volumes.
Effector cells in an equal volume were added, in quadruplicate, to give
the desired effector:target (E:T) cell ratios. The plate was
centrifuged at 150g for 5 minutes to initiate cell contact and
incubated at 37°C for 4 hours. The plate was then centrifuged at
350g for 10 minutes, supernatants were decanted, and samples
were counted using a  -counter (Wallac-LKB, Milton-Keynes, UK). CM
alone or HCl (1 mol/L) was added to labeled target cells for
calculation of spontaneous (spons) or maximum (max) release,
respectively. The percentage of kill (% kill) was calculated using the
following equation: % Kill = (Experimental  Spons)/(Max
Spons) × 100%.
125I-UdR release assay.
This assay was used to measure apoptosis of target cells induced by LAK
effectors.30 Target cells (5 × 105) were
labeled with 10 µCi 125I-deoxyuridine
(125I-UdR) in 200 µL for 2 hours at 37°C. Cells were
washed twice with PBS, resuspended in CM at 106 cells/mL,
and aliquoted in 0.5 mL volumes into 1.5-mL eppendorf tubes in
triplicate. Effector cells in CM were added to give the desired E:T
cell ratios. Tubes were centrifuged at 800 rpm in a minifuge for 5
minutes to inititate cell contact. After 4 hours, the tubes were
centrifuged at 5,000 rpm in a minifuge for 5 minutes. Supernatants were
collected into tubes and cell pellets were resuspended in 1 mL lysis
buffer (5 mmol/L Tris/HCl, pH 8.0; 0.1 mol/L EDTA, pH 8.0; 0.5% Triton
X-100). After a brief vortex, tubes were centrifuged at full speed in a
minifuge for 20 minutes to separate fragmented from bulk DNA.
Supernatants were combined with those collected previously and counted
on a -counter for 1 minute each. Spons count was obtained from tubes
in which CM alone was added to the radiolabeled target cells and max
release was calculated by adding the radioactivity remaining in the
spons pellets to the supernant counts: % Kill = (Experimental
Spons)/(Max Spons) × 100%.
DNA extraction, electrophoresis, and autoradiography.
YAC-1 cells (5 × 105) were labeled with 10 µCi
125I-UdR for 2 hours at 37°C. Cells were washed and
incubated with 5 × 106 cord blood LAK effectors to
give a final E:T of 10:1. Cells were centrifuged at 500 rpm for 5
minutes in a minifuge to inititate cell contact. After 4 hours,
pelleted cells were subjected to protein and RNAse digests and two
phenol-chloroform-amyl alcohol DNA extractions.31 Purified
DNA was diluted in TE buffer (10 mmol/L Tris/HCl, 1 mmol/L EDTA, pH
7.4) and centrifuged at full speed in the minifuge to separate
fragmented from intact DNA. Supernatants were precipitated in 2 vol of
100% ethanol overnight. Precipitated DNA was pelleted by
centrifugation and, after removal of the ethanol, air-dried for 30
minutes. Each DNA sample was resuspended in 50 µL TE buffer with 5
µL loading buffer and electrophoresed through a 100 mL, 1.5% agarose
minigel at 35 V for 4 hours, using TAE as running buffer (0.04 mol/L
Tris-acetate, 0.001 mol/L EDTA, pH 8.0).32 One microgram of
 HindIII molecular weight markers was used and ethidium
bromide (3 µL of 10 mg/mL stock) was added directly to the gel to
visualize DNA. The resulting gel was dried using a Hoefer Drygel Jr
(San Francisco, CA) onto a sheet of Whatmann No.1 filter paper
(Maidstone, UK). This was exposed to x-ray film
overnight and an autoradiograph was developed using
standard techniques.33
Asp-ase assay for granzyme B enzyme activity.
Granzyme B has a rare enzyme substrate specificity for aspartic
acid.18 Effector cell lysates were prepared by resuspending
cultured cells at 5 × 106/mL or freshly isolated
cells at 1 × 107/mL in 1 mL lysis buffer (0.1 mol/L
HEPES, 0.05 mol/L CaCl2, 0.5% NP40, pH 7.5). Cells were
freeze-thawed twice and centrifuged in a minifuge at 10,000 rpm for 15
minutes. The resulting supernatants were diluted (0.1 mol/L HEPES, 0.05
mol/L CaCl2, pH 7.5) and aliquoted into a 96-well
flat-bottomed plate in 50 µL volumes. The substrate used was
n-boc-ala-ala-asp-pNA (Bachem, Bubendorf, Switzerland).
Fifty microliters of 2 mmol/L substrate was added per well. Control
samples consisted of substrate and diluent alone. Substrate conversion
was calculated by measuring the change in optical density
(OD) at 405 nm after 3 days of incubation at 37°C.
Western blotting.34
Cells were extracted at 5 × 107/mL into lysis buffer
(10 mmol/L Tris/HCl, pH 8.0, 100 mmol/L NaCl, 1% NP40, 1 mmol/L
phenylmethyl sulfonyl fluoride, 1 µg/mL aprotinin,
0.001 mol/L EDTA, 50 µmol/L leupeptin, 1 µg/mL pepstatin, and 1
µg/mL antipain). Thirty-microliter cell extracts were diluted with
nonreducing loading buffer (62.5 mmol/L Tris, 3% wt/vol sodium dodecyl
sulfate [SDS], 10% wt/vol glycerol, and 0.01% wt/vol bromophenol
blue) and electrophoresed on a 15% nonreducing SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) gel. Samples were electroblotted onto
nitrocellulose paper and probed with an anti-granzyme B monoclonal
antibody (Pharmacell, France). 3-Amino-9-ethyl carbazole
(AEC) was used to visualize band staining.
IL-2R detection.
IL-2R expression was measured using a fluorokine receptor assay (R&D
Systems, Abingdon, UK), as per the manufacturer's instructions. The
primary reagent consisted of a human IL-2 biotin conjugate. This
reagent did not discriminate between the various IL-2R subunits. An
avidin-fluorescein conjugate was added to detect bound primary reagent
and flow cytometry was used to quantify cells staining positive for the
reagent.
Statistics.
The Mann Whitney statistical test was used to compare cord blood and BM
data.
| |
RESULTS |
NK cell activity of freshly isolated cord and BM cells against K562
target cells.
The NK activity of freshly isolated cells was measured against K562
target cells using a 51Cr release assay and the results are
shown in Fig 1. Cord cells had higher
cytotoxicity at all E:T cell ratios tested, but the differences were
not statistically significant.
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| Fig 1.
NK activity of freshly isolated cord blood and BM
mononuclear cells. ( ) Cord blood (n = 9) and ( ) BM (n = 7)
mononuclear cells were isolated by lymphoprep gradient centrifugation.
Cells were washed and set up in a 51Cr release assay at the
E:T cell ratios indicated. Bars show the % kill + 1 standard
deviation (SD). Data were compared using the Mann Whitney statistical
test.
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Cord blood and BM contain similar percentages of NK cells.
A total of 12.2% ± 10.0% and 13.6% ± 10.6% of cord blood
lymphocytes (n = 6) and 10.6% ± 5.1% and 8.1% ± 4.7% of BM
lymphocytes (n = 6) expressed CD56 and CD16 antigens, respectively. CD8
was expressed on 16.0% ± 3.8% (n = 5) of cord blood lymphocytes
and on 22.0% ± 11.9% (n = 5) BM lymphocytes.
Cord LAK cells can kill target cells by apoptosis.
Cord LAK cells have been shown to kill by necrotic means,10
but their potential to kill by inducing apoptotic death of target cells
has not been demonstrated. Cord LAK cells cultured in IL-2 (500 U/mL)
for 3 days were incubated with 125I-UdR-labeled YAC-1
cells for a period of 4 hours. After this time, cell mixture DNA was
extracted, electrophoresed, and autoradiographed. The resulting
autoradiograph is shown in Fig 2. It
clearly shows that DNA extracted from target cells alone remains
intact, whereas target cell DNA from the cell mixture has a
characteristic DNA ladder pattern indicative of apoptosis. Thus, cord
LAK cells are capable of killing their target cells by apoptosis.
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| Fig 2.
Cord blood LAK cells can kill YAC-1 target cells by
apoptosis. Cord blood mononuclear cells were cultured in IL-2 (500
U/mL) at 37°C. After 3 days, the cells were washed and 5 ×
106 cells were incubated with 5 × 105 YAC-1
target cells that had been prelabeled with 125I-UdR. After
4 hours, DNA was extracted from the cell mixture, electrophoresed, and
autoradiographed. The left-hand lane shows DNA extracted from YAC-1
cells in isolation. The right-hand lane shows YAC-1 DNA extracted from
the cell mixture preparation. The DNA ladder is indicative of
apoptosis. This figure is representative of three experiments.
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LAK activity of cord blood and BM mononuclear cells against Daudi and
YAC-1 target cells.
Cord blood and BM mononuclear cells were cultured in IL-2 for 3 days
and their LAK activity against both Daudi and YAC-1 target cell lines
was quantified. LAK cells kill the Daudi cell line by necrotic means,
as measured by the 51Cr release assay. In contrast, they
kill YAC-1 cells by inducing them to undergo apoptosis. DNA
fragmentation, which is indicative of apoptosis, was measured using an
125I-UdR release assay. Table 1shows the data obtained. Cord LAK cells had high cytotoxicity against
both Daudi and YAC-1 target cells. In contrast, BM had significantly
lower killing of Daudi cells (P < .001) and a virtual absence
of any killing of YAC-1 target cells. This absence of killing of
YAC-1 cells by unfractionated BM-derived NK cells was confirmed
using purified NK-cell preparations. Three of four purified BM-derived
NK-cell preparations displayed minimal apoptotic killing of YAC-1
targets compared with purified cord blood-derived NK cells at similar
E:T cell ratios (Fig 3; P < .05 at E:T of
3.13:1).
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| Fig 3.
LAK activity of purified cord blood and BM NK cells
against YAC-1 target cells. NK cells were purified and cultured in IL-2
(500 U/mL). CD56+ cells comprised 86.7% ± 7.0% and
82.8% ± 9.0% of purified populations in cord blood (n = 5) and BM
(n = 4), respectively. After 3 days, the cells were washed and their
cytotoxicity against YAC-1 cells measured using the
125I-UdR release assay and at three E:T ratios. The points
represent individual samples. The average spons/max ratio was 8.0%.
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Measurement of granzyme B enzyme activity.
Granzyme B activity levels were measured in cord blood and BM LAK cell
extracts. The results obtained are shown in Fig
4. Freshly isolated cord cells had low
granzyme B enzyme activity. Incubation with IL-2 induced granzyme B
activity in cord LAK cells. This was significantly higher than that of
BM LAK cell extracts, supporting the cytotoxicity data obtained.
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| Fig 4.
Granzyme B enzyme activity data. () Cord blood (n =
8) and () BM (n = 4) mononuclear cell preparations were incubated
in IL-2 (500 U/mL). After 3 days, the cells were washed and lysed and
the resulting cell extracts were incubated with a substrate for
granzyme B. Freshly isolated cord blood mononuclear cell extracts (n
= 8) were also analyzed. Enzyme activity was followed
colorimetrically at 405 nm and is expressed as a change in OD + 1 SD
with time. Readings were taken after 3 days of incubation at 37°C.
The Mann Whitney statistical test was used to compare data. *P
< .02.
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Granzyme B expression in cord blood and BM LAK cells.
Cord blood and BM LAK cells were examined for granzyme B protein
expression by Western blotting. Figure 5shows that all cord blood samples that had been cultured in IL-2 had
strong granzyme B bands present. In contrast, none of the BM samples
tested had detectable levels of the protein.
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| Fig 5.
Expression of granzyme B in IL-2-stimulated cord blood
and BM cells. Cord blood (n = 3) and BM (n = 4) mononuclear cells
were cultured in IL-2 (500 U/mL) for 3 days. Samples were extracted,
electrophoresed on 15% nonreducing SDS-PAGE gel, and immunoblotted
using anti-granzyme B monoclonal antibody. AEC was used to visualize
immunostaining.
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IL-12 does not induce BM cells to kill YAC-1 target cells by
apoptosis.
In an effort to induce apoptotic killing activity, BM mononuclear cells
were cocultured with IL-2 and IL-12. IL-12 has been reported to induce
NK activity and to synergize with IL-2 in the induction of LAK
activity.35,36 BM cells were cultured for 3 days at 500
U/mL IL-2 and 1 ng/mL IL-12. The % kill for the cocultured BM
cells at E:T 25:1 was 1.3% ± 2.0%, compared with 4.0% ±
4.4% for cells cultured in IL-2 alone (n = 3).
BM cells have variable kinetics of induction of LAK activity.
In further experiments to try to induce BM cells to kill YAC-1 targets,
BM cells were incubated for longer time periods in IL-2. LAK activity
against Daudi target cells reached a consistent level of killing after
7 days of incubation in IL-2 (Fig 6).
However, the data for the YAC-1 target cells were variable: one sample
had adequate killing, two samples developed low killing activity, and
one sample failed to induce any apoptosis in YAC-1 cells.
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| Fig 6.
BM cytotoxicity after prolonged exposure to IL-2. BM
mononuclear cells were isolated and set up in culture with IL-2 (500
U/mL) for 7 days. After this time, the cells were washed and their
cytotoxicity against Daudi and YAC-1 cells measured in cytotoxicity
assays. Individual results are shown. A standard E:T of 25:1 was used.
The average spons/max ratios were 11.1% and 7.0% for Daudi and YAC-1
cells, respectively.
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A reduced number of BM cells express the IL-2R.
Cord blood and BM mononuclear cells were phenotyped for their
expression of IL-2R. A total of 18.0% ± 9.8% of cord blood gated
lymphocytes (n = 7) expressed IL-2R. On double label analysis, 13.4%
± 5.2% CD56+ cord cells and 17.6% ± 4.7%
CD16+ cord cells (n = 4) coexpressed IL-2R. Only 2.5%
± 2.6% of lymphocyte gated BM cells (n = 4) expressed IL-2R
(P < .01).
| |
DISCUSSION |
NK and LAK cells can induce both necrotic and apoptotic death of target
cells.14 Apoptosis of target cells can be induced by a
number of mechanisms, including fas/fas-ligand
interactions,16 triggering by TNF-17
(secreted by effector cell), or triggering by granzyme
proteins.18,19 Perforin, the key molecule involved in
necrosis, exerts its effect upon insertion into the target cell
membrane, where it polymerizes to form a pore, causing cell death by
osmotic lysis.15 Research on cytotoxicity mechanisms to
date has focused on necrosis. However, the distinction between the two
processes may not be as clear-cut as was first assumed. Recent
literature suggests that perforin may represent part of the apoptosis
mechanism of killing,19,24 where it may function to
facilitate transfer of cytotoxic granule contents into a target cell
where they can trigger apoptosis. Whereas there is no doubt that
perforin can cause cell lysis,15 it is possible that this
may be restricted to particular situations, eg, in target cells, where
necrosis may be the only option available, due to inactivation or
disruption of the indigenous apoptosis biochemical pathway. This
situation occurs in many cell lines. The K562 cell line, which has long
served as the target cell line for measurement of NK
activity,29 has been reported to be relatively resistant to
induction of apoptosis due to the presence of the bcr-abl
complex.37 Therefore, one can conclude that, in vitro, or
indeed in vivo, these cells will invariably die by necrosis, as
detected in a 51Cr release assay. In this study, the YAC-1
cell line was chosen for the apoptosis assay, because DNA fragmentation
is readily measured and the cells are recognized by LAK effectors.
Daudi cells, which do not undergo measurable DNA fragmentation (data
not shown), were used as targets to measure necrosis.
The NK-cell-mediated killing potential of freshly isolated cord blood
and BM cells was examined using the K562 target cell line, and,
although cord cells had higher killing at all E:T cell ratios tested,
the differences seen were not significant. Data in the literature
regarding cytotoxicity of BM cells are somewhat variable, with
low,25 no,26 or variable38 killing
figures reported. Reported kinetics of LAK induction also vary, ranging
from 139 to 626 days. In this study, BM LAK
cells (3-day cultures) had variable killing against Daudi cells, as
indicated by the high standard deviation figure. Cord LAK activity,
generally reported to be higher than that of adult peripheral blood
cells,12,13 was also significantly higher than in BM cells.
Cord blood LAK cells were shown qualitatively to induce apoptosis in
target cells by agarose electrophoresis. This positive finding was
paralleled in the 125I-UdR release assay, which is used to
quantify DNA fragmentation as an indicator of apoptosis. In contrast,
BM-derived cells had a complete inability to induce apoptosis of YAC-1
target cells, a finding confirmed using purified NK-cell preparations.
The significance of one purified NK-cell BM sample mediating apoptotic
killing activity is not immediately apparent and may reflect peripheral
blood NK-cell contamination of the BM aspirate. The contrasting finding
of high cord LAK apoptotic activity versus absent BM LAK activity was
confirmed at the effector molecule level. It is unlikely that cytotoxic
T cells contributed to this difference because the percentages of CD8
cells in cord blood and BM were similar. BM cells were also relatively
resistant to efforts to stimulate their apoptotic mechanism: culture in
IL-12 did not, and prolonged culture in IL-2 was only partially
successful in overcoming this nonresponsiveness. The basis for the
absence of apoptotic cytotoxicity in BM cells remains unclear. BM NK
cells may represent a functionally immature cell type, compared with
cord cells, with acquisition of apoptotic cytotoxic potential
reflecting a more mature effector phenotype. Alternatively, BM NK cells
might be fully mature cells that have been potently downregulated, at a
functional level, by the immunosuppressive environment of the BM. High
concentrations of TGF- in BM, previously reported by this
group40 and others,41 may play a key role in
the observed IL-2 nonresponsiveness. IL-2 only partially restored
cytotoxicity of adult42 and cord mononuclear cells that had
been preincubated in TGF- (data not shown). It seems likely that a
combination of altered effector cell maturation status and
environmental influences contribute to the cytotoxicity characteristics
of BM NK cells that distinguish them from their cord blood
counterparts.
The high concentration of TGF- in BM may also contribute to the
observation that relatively few BM cells expressed any IL-2R, compared
with almost 20% of cord blood lymphocytes. Low CD25 (IL-2R chain)
has previously been reported on lymphocyte-gated BM
cells.43 TGF- reduces IL-2R expression and inhibits
protein phosphorylation induced by IL-2.42 It is known that
culture of lymphocytes in IL-2 results in increased IL-2R
expression.44 This fact, combined with observations from
the extended IL-2 incubation experiments performed here, suggests that
longer time incubations may possibly restore the apoptotic pathway of
killing in BM cells.
It is known that NK cells play a role in the regulation of
hematopoiesis,45,46 and it is possible that they may do
this by a number of mechanisms, including direct cytotoxic
activity47,48 and/or cytokine
secretion.49,50 If apoptosis is the physiologic form of
cell death, the resistance of BM NK cells to IL-2 induction of
cytotoxicity, as reported here, suggests that cytotoxicity is not a key
effector mechanism. Other recent reports support this
finding51 and even suggest that graft rejection mediated by
NK cells is not at the level of cytotoxicity but rather by NK-cell
regulation of hematopoiesis.52-54 Given the cytokine
secretion profile of NK cells that include IL-3,55,56
TNF-,57 interferon-,58
TGF-,59 and granulocyte-macrophage colony-stimulating
factor,55,56 it seems probable that they will function as
regulatory cells via the cytokines they produce.
Despite not being able to kill YAC-1 cells by inducing them to undergo
apoptosis, IL-2-stimulated BM cells were still capable of killing
Daudi target cells. This indicates a tighter regulation of molecules,
eg, granzyme B, involved in apoptosis versus necrosis. This, in turn,
suggests that apoptosis is the more important mechanism in vivo,
thereby further supporting earlier presented arguments.
It is known that BM NK cells mediate a GVL effect in BMT
patients.4,5 The potential GVL effect of cord blood is as
yet unknown, but the data presented here suggest that cord blood may
have an increased potential for GVL compared with BM. This is indicated
by the consistently higher LAK activity of cord blood cells. Freshly
isolated cells from both cord blood and BM had comparable killing, and
it may therefore be necessary to activate cells with cytokines for
their potential to be realized. In human studies, IL-2 infusion into
patients suffering from metastatic melanoma resulted in increased
expression of both perforin and granzyme B genes by circulating
cells.60 Therefore, in vivo activation of cytotoxic cells
could potentially be used in a transplant situation to boost GVL. In a
murine transplantation model system, it has been found that in vitro
activation of BM cells and subsequent IL-2 infusion were both required
for an optimal GVL effect.39 However, the potential
beneficial effects of the high responsiveness of cord cells to IL-2, in
mediating increased GVL, are as yet unknown and deserve further
investigation.
| |
FOOTNOTES |
Submitted January 9, 1997;
accepted September 2, 1997.
Address reprint requests to Denis J. Reen, PhD, The Children's
Research Centre, Our Lady's Hospital for Sick Children, Crumlin,
Dublin 12, Ireland.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank the staff of the Coombe Women's Hospital, Dublin 8,
and the orthopedic and theatre staff of Our Lady's Hospital for Sick
Children, Dublin 12, and St. Vincent's Hospital, Dublin 4, for their
cooperation in supplying cord blood and BM samples, respectively.
| |
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Abstract
Introduction
Abstract