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Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3864-3871
CD38 Triggers Cytotoxic Responses in Activated Human Natural Killer
Cells
By
Giuseppe Sconocchia,
Julie A. Titus,
Alessandra Mazzoni,
Alberto Visintin,
Federica Pericle,
Stuart W. Hicks,
Fabio Malavasi, and
David M. Segal
From the Experimental Immunology Branch, National Cancer Institute,
National Institutes of Health, Bethesda, MD; and the Institute of
Biology and Genetics, University of Ancona School of Medicine, Ancona,
Italy.
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ABSTRACT |
Receptors used by natural killer (NK) cells to mediate natural
cytotoxicity are poorly defined, although it is now clear that a number
of adhesion molecules can serve this function. CD38 transduces signals
on T- and B-cell lines, and we asked whether it could trigger lytic and
secretory responses in human NK cells. By using an anti-CD38 monoclonal
antibody in reverse antibody-dependent cellular cytotoxicity
experiments, it is shown that CD38 engagement triggers cytotoxic
responses by activated NK cells, but not by cytotoxic T
lymphocytes or fresh NK cells. Cross-linking with anti-CD38 F(ab')2 caused activated NK cells to
release granzymes and cytokines, but did not trigger an increase in
intracellular Ca2+. Fresh NK cells acquired
CD38-dependent lytic function during activation with interleukin-2
(IL-2), and inhibitor studies suggested that IL-2 stimulated the de
novo expression of proteins that act between CD38 and the lytic
machinery in NK cells. The induction of proteins that link commonly
expressed adhesion molecules to effector mechanisms could provide a
paradigm for pathogen recognition by the innate immune system.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
NATURAL KILLER (NK) CELLS, the primary
lymphoid mediators of natural cytotoxicity and antibody-dependent
cellular cytotoxicity (ADCC), are controlled by positive and negative
cytolytic signals.1,2 Negative signals are transduced by 2 classes of major histocompatibility complex (MHC)
I-binding molecules: by receptors containing Ig-like domains known as
KIRs in humans and receptors, including the Ly-49 family of proteins in
mice and CD94/NKG2A in humans that have extracellular C-lectin
domains,3,4 and by molecules with unknown
specificity.5 The best-characterized positive triggering molecule on NK cells is CD16 (Fc RIIIA), the receptor
responsible for mediating ADCC.1,6 CD16 associates
noncovalently with  and  homodimers and heterodimers that contain
tyrosine-based activation motifs (ITAMs) in their cytoplasmic domains.
Cross-linking of CD16 leads to phosphorylation of these ITAMs and of
syk, ZAP-70, and phospholipase C- (PLC-),
followed by a number of downstream events, including the release of
intracellular Ca2+, that ultimately result in the
exocytosis of cytolytic granules at the effector-target interface. It
has been recently shown that activating forms of KIRs and Ly-49 follow
a similar positive signaling pathway but use DAP-12, a homologue of
, as a signal transducer.3,7,8
Receptors mediating natural cytotoxicity, the innate ability of NK
cells to lyse tumor or virally infected target cells in the absence of
antibody, have been more difficult to characterize because multiple
different receptors can trigger cytolysis. Thus, a monoclonal antibody
(MoAb) to any 1 triggering molecule would only marginally inhibit
lysis. However, it has been possible to bypass the normal
receptor-ligand interaction and to study the capacity of individual
molecules to transduce cytotoxic signals by using receptor-specific IgG
MoAbs to redirect lysis of FcR+ target
cells.9,10 By using this approach, a number of cytotoxic triggering molecules have been identified, including
CD2,11-13 CD44,14-16 CD69,17
NKRP-1,18 CD40,19 B7.2,19 and
NK-TR,20 suggesting that these molecules might be involved
in natural cytotoxicity. Another molecule expressed by NK cells is
CD38, a type II integral membrane receptor that binds to CD31 (PECAM-1)
and has adenosine diphosphate (ADP)-ribosyl cyclase
ectoenzymatic activity.21-23 CD38 is expressed early in the
differentiation of CD34+ stem cells to lymphocytes and
remains in mature, CD56+, CD16+ NK
cells24,25 but not in resting B or T cells, although it is
re-expressed in activated T cells. Ligation of CD38 on Jurkat T
cells,26,27 immature B cells,28 and
differentiated HL-60 cells29 by anti-CD38 MoAbs induces
protein tyrosine phosphorylation. In Jurkat, but not in immature B
cells or MHC-nonrestricted T cells,30 CD38 cross-linking
also induces increases in intracellular Ca2+. A number of
important functional consequences of CD38 engagement include
upregulation of receptors such as B7.231 and
CD73,32 apoptosis,27
proliferation,31 and cytokine release.30
The potent signaling properties of CD38 in T-cell, B-cell, and
granulocytic cell lines prompted us to ask whether CD38 might also
serve as a cytotoxic triggering molecule on NK cells. We show here that
an agonistic anti-CD38 MoAb redirects cell-mediated cytolysis and
induces cytokine and granzyme release in activated NK cells. We
conclude that the CD38 adhesion molecule is indeed a cytotoxic trigger
on NK cells that could contribute significantly to the positive
triggering events leading to natural cytotoxicity.
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MATERIALS AND METHODS |
Antibodies and reagents.
Phycoerythrin (PE)-anti-CD38 (H1T2), PE-anti-CD16 (3G8), and
unconjugated anti-CD11a (G43-25B, IgG2b and HI111, IgG1) and anti-CD56
(B159, IgG1) were from Pharmingen (San Diego, CA). 3G8 (IgG1,
anti-CD1633), OKT3 (IgG2a anti-CD334), W6/32
(IgG2a, anti-HLA35), and IB4 [IgG2a and
F(ab')2, anti-CD3836] have been
described. OKT10 (IgG1 anti-CD38) and M300 (IgG1 myeloma protein, MOPC
300) were from the American Type Culture Collection (Manassas, VA).
Actinomycin D was from Sigma Chemical Co (St Louis, MO).
Effector and target cells.
Peripheral blood mononuclear cells (PBMCs) were
isolated from buffy coats from normal National Institutes of
Health Blood Bank (Bethesda, MD) donors by
Ficoll-Hypaque density gradient separation. Peripheral blood
lymphocytes (PBLs) were prepared from PBMCs by monocyte
depletion using plastic adherence, followed in some experiments by
nylon wool adsorption.15 PBLs were activated for 6 days
(unless stated otherwise) in culture medium (RPMI 1640 plus 10% fetal
calf serum [FCS], glutamine, penicillin, and streptomycin) containing
200 to 400 Cetus Units/mL recombinant human interleukin-2 (rhIL-2;
Cetus-Chiron, Emeryville, CA) and, in some experiments, 5% Lymphocult-T (conditioned medium from phytohemagglutinin
[PHA]-activated PBMCs; Biotest Diagnostics, Denville,
NJ). This procedure induces cytotoxic activity in NK and
CD56+ T cells but does not activate the great majority of T
cells, which lack CD56.37 NK cells were purified from
unactivated or activated PBLs by negative selection using a Vario MACS
system with an NK isolation kit (Miltenyi Biotec, Auburn, CA) and were always greater than 95% CD16+ and less than 1%
CD3+. Cytotoxic T lymphocytes (CTLs) were
obtained from high buoyant density PBLs38 that had been
activated for 4 days with culture medium containing 200 to 400 Cetus
Units/mL rhIL-2 and 5% Lymphocult-T and then negatively selected for
CD56 using the Vario MACS system. P815 (mouse
FcR+ mastocytoma) target cells (American
Type Culture Collection) were labeled with 51Cr as
described.39
Cytotoxicity assay.
Effector cells were incubated for 20 to 30 minutes at room temperature
with or without MoAb (2.5 to 10 µg/mL) and diluted serially. Ten
thousand 51Cr-labeled P815 target cells in 100 µL culture
medium were then added to 100 µL of diluted effector cells, and
cytotoxicity was measured in triplicate samples using a 4-hour
51Cr-release assay.39 The percentage of
specific lysis was calculated as follows: 100 × (CPMexperimental  CPMspontaneous)/(CPMmaximum CPMspontaneous). Spontaneous release was 10% or less of
the maximum in all experiments reported.
Surface expression.
Cells were incubated on ice for 30 minutes with either a fluorescein
isothiocyanate (FITC)- or PE-conjugated MoAb, followed by a second
incubation with an MoAb conjugated with the other fluorophore. They
were washed twice and analyzed with a FACScan flow cytometer (Becton
Dickinson Immunocytometry, San Jose, CA). Propidium iodide staining and
forward and side light scatter were used to gate on viable cells, and a
total of 5,000 to 10,000 cells were analyzed per measurement.
Effector-target conjugates.
Effector-target conjugates were detected by flow cytometry using a
modification of a previously published procedure.40
Activated NK cells were suspended in 5 mL phosphate-buffered saline
(PBS) at a concentration of 2 × 106 cells/mL and 50 µL of 1 mmol/L FITC (Sigma) in ethanol was added. The cells were
incubated for 5 minutes at 37°C, washed, centrifuged, and incubated
on ice for 30 minutes with or without 2.5 µg of MoAb in a volume of
0.1 mL. After washing, Ficoll-Hypaque-fractionated P815 cells in a
total volume of 0.1 mL RPMI 1640 were added at a target:effector ratio
of 1.5:1. The cells were pelletted, incubated at room temperature for 1 hour, and gently resuspended in 0.5 mL PBS. NK cells could be
distinguished from the P815 cells by flow cytometry on the basis of
their lower side light scatter. Conjugates were calculated as the
percentage of FITC+ cells having the same or higher side
light scatter as the P815 cells. At least 10,000 FITC+ (NK)
cells were analyzed, yielding 1,500 as the minimum number of conjugates
detected in any experiment.
N- -benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) thioesterase
assay.
Serine esterase release from activated, purified NK cells was measured
using a modification of a previously described method.41 Briefly, 96-well Immulon microtiter immunoassay plates (Dynatech Laboratories, Chantilly, VA) were incubated overnight with 50 µL/well
of 15 µg/mL MoAb in borate-buffered saline, pH 8.5. After 3 washes
with PBS, 1 to 4 × 105 NK cells in 200 µL RPMI 1640 without phenol red and without serum but containing 400 U/mL IL-2 were
added to each well. Plates were centrifuged for 5 minutes at
300g and incubated for 3 hours at 37°C in 5%
CO2. After incubation, 50-µL aliquots of
supernatants were transferred to a flat-bottom microtiter plate and 50 µL of 1 mmol/L 5',5'-Dithio-bis-(2-nitrobenzoic acid)
(DTNB; Sigma) in Hanks' balanced salt solution (HBSS) was added to
each well. Immediately after the addition of DTNB, plates were read at
414 nm using a Titertech (Huntsville, AL) Multiskan
enzyme-linked immunosorbent assay (ELISA) reader to obtain blank
values. One hundred microliters of 1 mmol/L BLT (Calbiochem, San Diego,
CA) in HBSS was added to each well, and the plates were incubated at
room temperature until a visible yellow color developed. The plates
were again read in the ELISA reader, and blank values were subtracted.
Cytokine release.
Immulon 96-well microtiter immunoassay plates were incubated overnight
with 50 µL/well of 25 µg/mL MoAb in borate-buffered saline, pH 8.5. After 3 washes with PBS, 3 × 105 IL-2-activated,
purified NK cells in 200 µL complete medium were added to each well.
After 20 hours of incubation at 37°C, cell-free supernatants were
harvested and either tested immediately or stored at 20°C.
Tumor necrosis factor- (TNF-) and interferon- (IFN-) levels
were quantitated with Biosource International (Camarillo, CA) ELISA
kits, following the manufacturer's instructions. Limits of detection
were 1 pg/mL for TNF- and 4 pg/mL for IFN-.
Cytoplasmic Ca2+.
Cytoplasmic Ca2+ was measured by using the Fluo-3/AM
fluorescent calcium marker, as described.42 Briefly,
purified, IL-2-activated NK cells were washed with HBSS and loaded for
30 minutes at room temperature with 4 µmol/L Fluo-3/AM (Molecular
Probes, Eugene, OR) in the presence of 0.06% Pluronic F-127 (Molecular
Probes). Cells were diluted 5-fold in HBSS containing 1% FCS,
incubated for 30 minutes at room temperature, washed 3 times, and kept
at 4°C in HEPES-buffered saline (137 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L Na2HPO4, 5 mmol/L glucose, 1 mmol/L
CaCl2, 0.5 mmol/L MgCl2, 1 mg/mL bovine serum
albumin [BSA], and 10 mmol/L HEPES, pH 7.4). Five minutes before
fluorescence-activated cell sorting (FACS) analysis, the
Fluo-3-labeled cells were warmed to 37°C. They were then
stimulated with 10 µg/mL of IB4 F(ab')2, W6/32, or
3G8 MoAb, followed by the addition of 10 µg/mL of rabbit
F(ab')2 antimouse IgG (Southern Biotechnology,
Birmingham, AL). Proper loading was checked by treating cells with 2 µg/mL of ionomycin.
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RESULTS |
CD38 is a cytotoxic triggering molecule in activated NK cells.
Although CD38 transduces a variety of signals in B-cell, T-cell, and
granulocytic cell lines26-29 and induces a cytotoxic
response in an MHC-unrestricted T-cell line,30 it is not
known whether it has the capacity to trigger cytolysis in isolated
subsets of PBLs. Therefore, PBLs from normal donors were incubated in
reverse ADCC10 experiments with
FcR+ P815 target cells in the presence of
either anti-CD38 MoAb or MoAbs against CD3 and CD16, known cytotoxic
triggers in CTLs and NK cells, respectively. In these experiments, IgG
MoAbs redirect lysis by simultaneously binding to FcR on
the target cells and to triggering molecules on the effector cells.
P815 cells are of mouse origin and are not specifically recognized by
antihuman CD38 or other mouse antihuman MoAbs used in this study.
Figure 1A shows that IL-2-activated PBLs
do in fact contain cells that mediate CD38-directed cytolysis. Purified
NK cells and CTLs were then used to determine which types of cells
mediate this activity. The purified NK cell preparation (Fig 1B)
mediated moderate amounts of lysis of P815 cells, a relatively
NK-resistant target, in the absence of MoAb. Lysis was not affected by
anti-CD3 MoAb, indicating that levels of CTL contamination were
negligible, but was markedly enhanced by the anti-CD38 and anti-CD16
MoAbs. By contrast, conventional CTL, depleted of NK cells and a small
subset of T cells by CD56 negative selection, exhibited potent CTL
activity, but failed to mediate CD38- or CD16-directed lysis. Removal
of the Fc portion of IB4, the anti-CD38 MoAb used above, abrogated its
capacity to mediated reverse ADCC, as expected, and OKT10, a less
agonistic anti-CD38 MoAb than IB4,43 consistently mediated
lysis at a lower level than IB4 (Fig 1D). FACS analysis
(Fig 2) showed that all NK
(CD16+) cells and the great majority of CTL expressed CD38.
Thus, the inability of the anti-CD38 MoAb to trigger lysis by CTLs was
not due to the lack of expression of CD38 on these cells. We conclude that CD38 is a cytotoxic trigger on activated NK cells but not on
conventional CTLs.
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| Fig 1.
CD38 is a cytotoxic triggering molecule for activated NK
cells but not CTL. IL-2-activated PBLs (A), purified IL-2-activated
NK cells (B), or CD56-depleted CTL (C) from separate donors were tested
in reverse ADCC assays using anti-CD38 (IB4), anti-CD3 (OKT3), or
anti-CD16 (3G8) MoAbs to redirect the lysis of P815 target cells.
Effector:target (E:T) ratios are indicated in the legends. In (D), the
indicated concentrations of intact IB4, IB4 F(ab')2,
intact OKT10 (a less agonistic anti-CD38 MoAb), and M300 (nonspecific
IgG control) were used with PBLs at an E:T ratio of 15:1 to redirect
lysis. All effector cells were preactivated as described in Materials
and Methods. *P < .05, **P < .01 when compared with
the no antibody control (A, B, and C) or the M300 control (D) at the
same E:T ratio.
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| Fig 2.
Expression of CD38 on NK cells and CTLs. Contour diagrams
of PBL incubated for 3 days in (A) medium alone, (B) medium containing
IL-2, and (C) medium containing IL-2 plus 10 ng/mL actinomycin D. (D)
Single-parameter histogram showing CD38 expression on CTLs. In (A)
through (C), the NK (CD16+) cells were 95%, 93%, and
99% CD38+, respectively, and the mean CD38 fluorescence
intensities on the CD16+CD38+ cells were
147, 134, and 232. In (D), cells were 90% positive for CD3 (not shown)
and 80% positive for CD38.
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Comparison of triggering functions of NK surface molecules.
Not all MoAbs against NK surface molecules consistently induced lytic
responses. Table 1 shows that activated
PBLs from most donors mediate CD38- and CD16-dependent lysis of P815
target cells at levels significantly above the antibody-independent
background. By contrast, anti-MHC I and anti-CD11a MoAbs induced
positive responses in only 18% and 0% of donors, respectively,
although these same MoAbs bound to essentially all NK cells by FACS
analysis (data not shown). Moreover, both triggering and nontriggering MoAbs were fully capable of inducing NK cell-target cell conjugate formation (Fig 3), indicating that
conjugate formation, per se, is not a lytic signal in NK cells.
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| Fig 3.
Conjugates between P815 and NK cells. Purified
FITC-labeled activated NK cells were coated with MoAb of the indicated
specificity, washed, and mixed with P815 cells at a ratio of 1 NK cell
to 1.5 P815. The cells were pelletted, incubated for 1 hour at room
temperature, resuspended, and analyzed for light scatter and green
fluorescence. Conjugates were detected as FITC+ particles
with side scatter greater or equal to that of P815 cells.
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Induction of CD38-dependent lysis.
Unstimulated NK cells mediated little if any CD38-dependent lysis, but
culture with IL-2 induced both CD38-dependent lysis and Ab-independent
cytotoxicity (Fig 4). By contrast,
unstimulated NK cells did mediate CD16-directed lysis, indicating that
they were competent killer cells before activation (Fig 4). CD38
expression was similar on both activated and unstimulated cells (Fig 2B
and A), indicating that the acquisition of CD38 triggering function was
not due to enhanced CD38 surface expression. To determine whether de
novo gene expression was required for the induction of CD38-directed
lysis, PBLs were activated in the presence of varying concentrations of
the transcription inhibitor, actinomycin D. As shown in
Fig 5, actinomycin D, even at 10 ng/mL, had
little effect on the acquisition or maintenance of lysis mediated by CD16, indicating that it did not impair the cytolytic function of NK
cells in general. By contrast, actinomycin D strongly inhibited the
induction of CD38-dependent lysis and Ab-independent cytotoxicity (Fig
5), but had a negligible effect on CD38 expression (Fig 2C). These data
suggest that IL-2 induced the expression of at least 1 protein,
distinct from CD38 itself, that was required for CD38 lytic function.
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| Fig 4.
IL-2 activation induces CD38 triggering function. Cells
from 2 normal donors were cultured with 200 U/mL IL-2 for the indicated
times and tested for the ability to lyse P815 target cells in the
presence of medium alone (no Ab), anti-CD38, or anti-CD16 MoAbs. Lytic
units were calculated from the E:T ratio giving 25% (donor 1) or 20%
(donor 2) lysis (lytic units = 100/E:T). Asterisks indicate that
lysis was less than 25% and 20% for donors 1 and 2, respectively, at
all E:T. This experiment has been repeated 4 times with similar
results. CD38-directed lysis appeared in different donors as early as
16 hours and as late as 4 days.
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| Fig 5.
Gene transcription is required for the induction of
natural cytotoxicity and CD38-dependent lysis. PBLs from 2 separate
donors were activated for 3.5 days in medium containing 200 U/mL IL-2
and the indicated concentrations of actinomycin D. Cells were then
tested for the ability to mediate natural cytotoxicity (No Ab) or
cytotoxicity directed by anti-CD38 or anti-CD16 MoAbs.
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CD38 cross-linking triggers degranulation and cytokine release.
Cell-mediated cytolysis requires the formation of multicellular
conjugates between effector and target cells and could involve ligation
of several different receptors on the killer cells. To determine
whether the cross-linking of CD38 alone could trigger functional
responses, activated, purified NK cells were incubated with immobilized
MoAbs, and the supernatants were tested for esterase or cytokine
release (Fig 6). Immobilized anti-CD38
F(ab')2 promoted esterase, IFN-, and TNF-
release at levels that were clearly above the medium control, but that
were usually lower than those induced by anti-CD16 MoAb. By contrast,
MoAbs against CD56 and MHC I, 2 molecules expressed on NK cell
surfaces, failed to trigger cytokine or esterase release. Esterase
release results from the exocytosis of stored cytolytic substances such
as perforin and granzymes, whereas cytokine release requires de novo
protein synthesis. Thus, the cross-linking of CD38 with immobilized
F(ab')2 triggered 2 fundamentally different processes
used in NK effector responses.
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| Fig 6.
Triggered degranulation and cytokine release. Purified
IL-2-activated NK cells were incubated in microtiter wells coated with
no Ab, anti-CD16 MoAb, anti-CD38 F(ab')2, anti-CD56
MoAb, or anti-MHC-1 MoAb for 3 (esterase release) or 20 (cytokine
release) hours. Supernatants were removed and tested for BLT-esterase
(upper panel), TNF- (middle panel), or IFN- (lower panel). Cells
from different donors were used for measuring esterase and cytokine
release. *P < .05. **P < .01 as compared with the
no antibody control.
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Lack of CD38-induced Ca2+ mobilization in NK cells.
Cross-linking of CD38 induces an increase in intracellular
Ca2+ in Jurkat,27 but not in immature B
cells28 or MHC-nonrestricted CTLs.30 We
therefore asked whether CD38 cross-linking would trigger
Ca2+ mobilization in NK cells. Fluo-3-loaded NK cells,
isolated from PBLs that mediated high levels of CD38-dependent lysis
(Fig 1A) were treated with either anti-CD38 F(ab')2,
anti-CD16 MoAb (positive control), or anti-MHC I MoAb (negative
control), followed by further cross-linking (right arrow) with
antimouse Ig. As seen in Fig 7, only the
anti-CD16 MoAb induced an increase in intracellular Ca2+.
Similar results were obtained using biotin-MoAbs and streptavidin as a
cross-linker (data not shown). We conclude that CD38 cross-linking in
NK cells does not trigger Ca2+ mobilization and that the
CD38 and CD16 signaling pathways differ in this regard.
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| Fig 7.
Cross-linking of CD16 but not CD38 induces an increase in
intracellular Ca2+. Purified, IL-2-activated NK cells
were loaded with Fluo-3, warmed to 37°C, and, at the time indicated
by the left arrow, treated with anti-CD38 (IB4)
F(ab')2 ( ) anti-MHC-1 (W6/32) MoAb ( ), or
anti-CD16 (3G8) MoAb ( ). At the time indicated by the right arrow,
antibodies were further cross-linked by the addition of rabbit
F(ab')2 antimouse IgG. Cytolysis mediated by PBLs
from this donor is shown in Fig 1A. This experiment has been repeated 4 times, with similar results.
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DISCUSSION |
It is well known that NK cells lyse tumor and virally infected cells in
the absence of Ab, but the receptors used in target cell recognition
are poorly defined. In this report, we show that CD38 is a receptor
that could potentially contribute to natural cytotoxicity mediated by
IL-2-activated NK cells. The signaling capacity of CD38 was
demonstrated by using an anti-CD38 MoAb to mimic its interaction with
CD31, a ligand for CD38 that is present in especially high amounts on
endothelial cells. The choice of target cell is particularly important
in these studies. P815 was chosen not only because it expresses
FcR and is of mouse origin (and therefore does not
interact with the mouse antihuman receptor antibodies used here), but
also because it is relatively resistant to natural cytotoxicity.
Natural cytotoxicity is the result of several positive signaling
interactions being balanced by negative signals. An NK target such as
K562 that expresses low levels of MHC I molecules is lysed so
efficiently that no further increases can be seen on the addition of
antibody. On the other hand, very high engagement of inhibitory
molecules could totally block redirected lysis (eg, see Meyaard et
al5). P815 falls between these 2 extremes. In fact, we did
attempt to use an endothelial cell line that expresses high levels of
CD31 as a target cell (data not shown). Natural cytotoxicity of this
target was quite high, and anti-CD38 or anti-CD31 were unable to
significantly inhibit lysis, presumably because other triggering
molecules contributed to cytotoxic signaling.
The killing capacity of CD38 appeared to be mediated primarily by
activated NK cells. Conventional CTLs and unactivated NK cells
expressed CD38 on their surfaces and mediated lysis through CD3 and
CD16, respectively, but failed to kill through CD38. Because of our
separation procedures, we could not establish whether the CD56+ T cells were also triggered by CD38 ligation. This
NK-like subset of T cells comprises 2% to 3% of total PBLs and is the
primary mediator of CD3 directed lysis in IL-2-activated PBLs. By
contrast, the more abundant conventional CTL are
CD3+, CD56 and require
T-cell receptor (TCR) cross-linking in addition to cytokine for activation.9 Recent studies by Cesano et
al30 provide evidence on the role of CD38 in the activation
of CD56+, CD3+ T cells. In their experiments,
the IB4 anti-CD38 MoAb triggered low levels of cytolysis by the
CD56+, CD3+ TALL line and no lysis by
unfractionated LAK cells that were 90% T cells. In addition, anti-CD38
failed to induce an increase in intracellular Ca2+ levels
in both cell types. However, IB4 did induce low, but significant levels
of esterase release by both cell types, and gave robust cytokine
responses. Thus, to the extent that the TALL cells and unfractionated
LAK represent CD56+ T cells, it appears that this subset of
cells behaves similar to NK cells in response to anti-CD38, except that
they are less cytotoxic.
The acquisition of CD38 triggering capacity by NK cells during
activation with IL-2 was blocked by actinomycin D, an inhibitor of
transcription, suggesting that CD38 lytic function depends on the de
novo synthesis of 1 or more proteins that are essential components of
the lytic pathway. Because actinomycin D had no significant effect on
CD38 expression, the newly synthesized proteins must have acted
downstream of CD38. There are 2 known pathways by which lymphocytes
lyse target cells in 4 hours (the time used in the current study):
granule exocytosis and cross-linking of death receptors such as Fas on
target cells.44 We demonstrate here that both anti-CD38 and
anti-CD16 MoAbs induce degranulation (BLT-esterase release) of NK
cells; in addition, the target cells used in this study did not express
Fas (CD95) either by functional or FACS analysis (data not shown).
Thus, CD16 and CD38 most likely killed through the granule exocytosis
pathway. Because actinomycin D had little effect on CD16-mediated lysis
on either activated or fresh NK cells, it is unlikely that the
putative, IL-2-induced proteins were involved in the degranulation
process itself. Thus, it is likely that IL-2 induced the synthesis of
proteins that act between CD38 and degranulation. Previously, we
demonstrated that CD44, like CD38, acquired cytotoxic triggering
capacity during NK cell activation and that this gain in function was
actinomycin inhibitable but not dependent on receptor
expression.45 Thus, the de novo synthesis of proteins that
link adhesion molecules to the killing machinery may be an important
component of NK cell-mediated lysis in general.
In the Jurkat T-cell line, CD38 ligation induces the tyrosine
phosphorylation of several proteins used in TCR signaling, including , ZAP-70, and PLC-1, as well as proteins in the Raf-1/MAP kinase pathway.26 Cross-linking of CD38 on Jurkat cells also
induces an increase in intracellular Ca2+ and apoptosis,
both of which require an intact TCR.27 CD16 ligation on NK
cells initiates a signaling pathway that mirrors that of the TCR in
that it induces phosphorylation of and homologous subunits,
phosphorylation of ZAP-70 and syk,1 and an increase in intracellular Ca2+.46 In addition, it has
been reported that CD16 associates with CD38 on NK cell
surfaces.36 Thus, it seemed possible that the CD38 and CD16
triggering pathways on NK cells might merge either at the level of CD16
itself or downstream of CD16. However, the fact that CD38 cross-linking
does not induce an increase in intracellular Ca2+, whereas
CD16 ligation does, suggests that the 2 triggering molecules use
different signaling pathways.
The number of adhesion molecules that can serve as cytotoxic triggers
on NK cells, which now include CD2, CD38, CD44, and CD69, continues to
increase, suggesting that the process of natural cytotoxicity may not
be triggered by specialized NK receptors, but may instead reflect the
level of expression of downstream proteins that confer triggering
capacity on commonly expressed receptors. This mechanism of triggering
might be fundamental to the cellular arm of innate immunity, but would
be avoided by cellular mediators of acquired immunity, in which it
would override a highly specific interaction with one that was less
specific. Thus, it is not surprising that CD38 and CD44 trigger lysis
on NK cells, but fail to do so when expressed on bulk CTLs generated
from PBLs15 (although some CTL clones mediate CD44-directed
lysis47,48). It has been shown that CD44 is a cytotoxic
trigger in neutrophils,49 but it remains to be established
how extensively cellular mediators of innate immunity use adhesion
molecules to initiate cytolytic responses.
| |
FOOTNOTES |
Submitted February 1, 1999; accepted July 26, 1999.
F.M. was supported by AIRC (Milan, Italy), TELETHON (Rome, Italy), and
by the AIDS and TB Projects (Higher Institute of Medicine, Rome, Italy).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to David M. Segal, PhD, Bldg 10, Room 4B36,
NIH, Bethesda, MD 20892-1360.
| |
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INTRODUCTION