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IMMUNOBIOLOGY
From the Laboratory of Immunogenetics, Department
of Genetics, Biology and Biochemistry, University of Torino
Medical School, Torino, Italy; the Experimental Medicine Research
Center, Torino, Italy; the Department of Cell Biology and Immunology,
Institute of Parasitology and Biomedicine, Consejo Superior de
Investigaciones Científicas, Granada, Spain; the Institute of
Biology and Genetics, University of Ancona Medical School, Ancona,
Italy; the Department of Medical Science, A. Avogadro University of
Eastern Piedmont, Novara, Italy; and the Department of Bacteriology and
Medical Mycology, Istituto Superiore di Sanità, Rome, Italy.
CD38, a surface glycoprotein of unrestricted lineage, is an
ectoenzyme (adenosine diphosphate [ADP] ribosyl cyclase/cyclic ADP-ribose hydrolase) that regulates cytoplasmic calcium. The molecule also performs as a receptor, modulating cell-cell interactions and delivering transmembrane signals, despite showing a structural ineptitude to the scope. CD38 ligation by agonistic monoclonal antibodies induced signals leading to activation of the lytic machinery
of natural killer (NK) cells from adults; similar signals could not be
reproduced in YT and NKL, 2 CD16 Human CD38 is the prototype of a family of proteins
that share structural similarities and ectoenzymatic activities
involved in the production of calcium-mobilizing
compounds.1-3 Aside from its ectoenzymatic activities and,
apparently with independent modalities, CD38 may perform as a receptor,
ruling adhesion and signaling in T4 and B
lymphocytes,5 monocytes,6 and natural killer
(NK) cells.7,8 The receptor functions of CD38 are regulated through interaction with a counterreceptor, identified as
CD31.9 The signaling events initiated by interactions
between CD38 and CD31 (and fully mimicked by agonistic anti-CD38
monoclonal antibodies [mAbs]) were initially studied in the dynamic
context of circulating CD38+ T lymphocytes adhering to
CD31+ endothelial cells.10 Use of this model
allowed definition of some of the events that take place after the
interaction and that include calcium (Ca++) mobilization
from cytosolic stores, tyrosine phosphorylation of selected substrates,
activation of nuclear factors, and secretion of
cytokines.11
It is generally agreed that CD38 controls a specific signaling pathway
in T cells, B cells, NK cells, and monocytes. In spite of this
evidence, the modalities through which the signal is initiated remain
elusive. The molecule has neither the canonical structure of a receptor
nor the hallmark domains. Indeed, the cytoplasmic tail is short and
lacks docking sites and it is not tyrosine phosphorylated on
activation.12,13 Such negative characteristics are even more evident in CD157, the other member of the protein family, whose
signaling features are known, notwithstanding a
glycophosphatidylinositol linkage to the cell
membrane.14,15
Some clues can be extrapolated from cocapping experiments, which
show that CD38 associates on the cell membrane with professional signaling receptors such as the T-cell receptor (TCR)-CD3 complex in T
cells, the B-cell receptor (BCR) in B cells, and CD16 in NK
cells.16 A hypothesis to explain the signaling properties of CD38 is that the molecule exploits the signaling machinery of
professional receptors to deliver its own independent signals. This
idea was first supported by experiments using CD38+ T-cell
lines deficient in components of the signaling apparatus of the TCR-CD3
complex.17,18 The inability of CD38 to signal in these
cells was overcome by reconstituting a complete TCR-CD3 complex,
thereby indicating that CD38 signaling depends on the presence of a
functional TCR. These observations were recently expanded by studies
using T lymphocytes purified from the intestinal lamina propria as a
model in which the TCR complex is physiologically impaired.19 A comparative analysis of circulating versus
residential T lymphocytes from the same individuals indicated that CD38
signaling is sensitive to the operational environment and seems to
proceed through distinct pathways, even within the same cell lineage. The features of CD38 signaling in lamina propria T cells impaired in
TCR-mediated signalings were clearly different from those of circulating T cells and derived line models with a functional TCR (eg,
lack of Ca++ mobilization and phosphorylation of the
phospholipase C The question behind this paper is whether the molecular parasitism
exerted by CD38 in T cells may also occur in other cell lineages and
serves to defines a novel type of coreceptor molecule. Here, we
report results obtained in the NK lineage by using NK-like lines either
expressing or deficient in membrane CD16. The results show conclusively
that the presence of CD16 is a necessary requisite for CD38 to control
a complex signaling pathway that includes cytoplasmic and nuclear
events, release of cytokines, and activation of cytolytic functions.
Cells
Cells were cultured in RPMI-1640 medium (Sigma, Milan, Italy) with 10%
heat- inactivated fetal-calf serum (FCS; Seromed, Berlin, Germany), 50 µg/mL gentamicin, 100 U/mL penicillin, and 100 µg/mL streptomycin
(all from Sigma). The NKL cells required addition of human recombinant
interleukin (IL) 2 (200 IU/mL; Chiron, Emeryville, CA).
Antibodies
Fluorescein isothiocyanate (FITC)-conjugated anti-CD25 and phycoerythrin (PE)-conjugated anti-CD69 mAbs were from BD Bioscience (Milan, Italy). FITC-conjugated goat antimouse immunoglobulin (GaMIg; Caltag, Burlingame, CA) was used in indirect immunofluorescence studies. Tetrarhodamine isothiocyanate (TRITC)-conjugated GaMIg and FITC-streptavidin were both from Dako (Glostrup, Denmark). IB4 and CB16 mAbs were conjugated to biotin (Bio-Spa, Milan, Italy). An affinity-purified F(ab')2 preparation of rabbit immunoglobulin (Ig) to mouse IgG (RaMIg; locally produced) and an F(ab')2 donkey antimouse IgG (DaMIgG; Jackson ImmunoResearch Laboratories, West Grove, PA) were used as cross-linkers. The recombinant antiphosphotyrosine (anti-pTyr) antibody coupled to RC20-horseradish peroxidase (HRP) was from BD Biosciences. E10 mAb, an anti-phospho-p44/p42 mitogen-activated protein kinase (MAPK; T202/Y204), was from New England Biolabs (Beverly, MA). The affinity-purified rabbit polyclonal anti-extracellular-regulated kinase (anti-ERK) 2 and anti-ZAP70 (LR) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-ZAP70 (Zap-4) serum was from S.C. Ley (London, United Kingdom). HRP-conjugated, affinity-purified goat antirabbit IgG (GaRIgG [Fc]) and goat antimouse IgG (GaMIgG) were from Promega (Madison, WI). GaMIgG-coated magnetic beads (Dynal, Oslo, Norway) were used to select CD16+ cells in cultures of YT and NKL lines. Constructs CD16 cDNA in a pMX plasmid (L.L. Lanier, San Francisco, CA)25 was amplified by polymerase chain reaction (PCR), with the primers designed according to the published CD16 sequence (GeneBank accession no. M24854). PCR amplification was done by using an automated DNA thermal cycler (Perkin Elmer, Boston, MA) for 30 cycles after an initial denaturation for 5 minutes at 94°C. The reaction product was visualized by electrophoresis on a 1.5% agarose gel containing Tris-borate-EDTA buffer and ethidium bromide (0.5 µg/mL).An aliquot (1 µL) of the PCR product was ligated to a pcDNA3.1 expression vector by using the TA cloning system, and transformation was carried out on Escherichia coli TOP10 cells (all from Invitrogen, Carlsbad, CA). Positive transformants were analyzed for the presence and correct orientation of CD16 cDNA both by PCR (using a combination of the T7 forward primer and a specific reverse primer that bound to the inner sequence of CD16 cDNA) and by digestion with the KpnI (10 U/µg) restriction enzymes (New England Biolabs). The selected transformant, CD16-pcDNA3.1, was analyzed by sequencing, grown in LB medium, and purified by using a Quantum Prep plasmid kit (Bio-Rad, Hercules, CA). Transfection CD16-pcDNA3.1 (20 µg) was linearized by treatment with ScaI restriction enzyme (20 IU/µg, New England Biolabs), purified with ethanol, checked for purity and concentration, and used to transfect YT cells by electroporation (250 V/0.4 cm and 960 µF). After a 2-week incubation in medium containing 1 mg/mL G418 (Sigma), neomycin-resistant colonies were isolated, recloned by using serial dilution, and referred to as YT CD16+. YT cells were similarly transfected with the empty pcDNA3.1 vector, selected by using G418, and referred to as YT mock cells.Ca++ fluxes Intracellular Ca++ concentrations were measured by flow cytometry after loading the cells with Fluo 3-acetoxymethyl (Fluo 3-AM; Sigma), a Ca++-sensitive fluorescent dye. YT CD16+ or YT mock cells and CD16+ and CD16 NKL cells were washed twice in RPMI-1640 medium with
5% FCS and incubated (106 cells/mL for 1 hour at 37°C)
with 5 µM Fluo 3-AM in the presence of 0.01% pluronic
F127 (Sigma). Cells were then washed, incubated for 10 minutes at room
temperature with the selected mAb (10 µg/mL), and washed again.
Cross-linking RaMIg (20 µg/mL) was added 10 seconds after starting a
continuous fluorescence-activated cell-sorter scanning (FACSort; BD
Biosciences) analysis at 37°C. Changes in Ca++
concentrations were monitored by plotting the shift in the Fluo 3-AM fluorescence during 540 seconds and presented as
changes in Fluo 3-AM intensity over time.19 An
irrelevant isotype-matched mAb was included as the control, and
efficient loading of the cells was verified by adding the A23187
ionophore (Sigma).
Tyrosine phosphorylation YT CD16+ and YT mock cells were starved for 12 hours in RPMI-1640 medium with 0.5% FCS at 37°C in a 5% carbon dioxide (CO2) incubator, collected, and incubated for 10 minutes on ice with an F(ab')2 preparation of the IB4 mAb (10 µg/106 cells), anti-CD16 mAb (5 µg/106 cells). An F(ab')2 preparation of the isotype-matched irrelevant JAS mAb (10 µg/106 cells) was used for the control condition. The unbound mAb was eliminated by washing with cold medium, and the cells were then incubated for 10 minutes on ice with an F(ab')2 fragment of DaMIgG at a concentration of 4 µg/106 cells. The cells were subsequently allowed to react with the relevant mAb at 37°C for 1 minute, and lysis was obtained by using 1% NP-40 lysis buffer (20 mM HEPES [pH 7.6], 150 mM sodium chloride [NaCl], 50 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM ethyleneglycotetraacetic acid, 50 µM phenylarsine oxide, 10 mM iodoacetamide, 1 mM phenylmethyl sulfonyl fluoride, and 2 µg/mL each of antipain, chymostatin, leupeptin, and pepstatin) for 20 minutes on ice.26 After removal of nuclei by centrifugation, an aliquot of the lysates was diluted in Laemmli sample buffer, boiled for 5 minutes, and stored at 80°C before being
subjected to sodium dodecyl sulfate-polyacrylamide electrophoresis
(SDS-PAGE). Immunoprecipitation and Western blotting were done as
described previously.26,27
The gel was then transferred to a polyvinylidene difluoride (PVDF) membrane with a semidry transfer apparatus (Hoefer, Pharmacia Biotech, San Francisco, CA) in Tris-glycine buffer containing 20% methanol and 0.035% SDS at 0.8 mA/cm.2 To ensure proper recovery of all migrated proteins, transfer efficiency was confirmed by Ponceau red staining. The membrane was blocked in 1% bovine serum albumin (BSA); washed in 10 mM Tris (pH 7.4), 100 mM NaCl, and 0.1% Tween 20; and allowed to react with HRP-conjugated RC20 anti-pTyr mAb for 2 hours. For reblotting, the filter was subsequently stripped by washing in a buffer containing 150 mM NaCl and 10 mM Tris-hydrochloric acid (pH 2.2), blocked again in 2.5% nonfat dry milk, and incubated for 1 hour with the primary antibody of interest diluted in 1% milk. After washing, HRP-conjugated GaRIgG was added and the membrane was washed and developed again by using electrogenerated chemiluminescence reagents (Amersham, Little Chalfont, United Kingdom). Cytokine release YT CD16+ and YT mock cells were plated (106 cells/well) in 24-well plates (BD Biosciences) coated with RaMIg (20 µg/mL) in RPMI-1640 medium with 5% FCS in the presence of anti-CD38, anti-CD16 (both at a concentration of 10 µg/mL), or both.4 To determine whether CD31-CD38 cognate interactions were inducing interferon (IFN-) release, YT
CD16+ and YT mock cells were cocultured for 24 hours at
37°C in a 5% CO2 incubator with CD31+
transfectants, with the mock-transfected cells used as controls. The
amounts of IFN- produced were determined by using an immunoenzymatic kit (R&D Systems, Minneapolis, MN).
Cell-mediated cytotoxicity Tumor cell lines were labeled with chromium 51 (51Cr; NEN, Cologno Monzese, Italy; 100 µCi (3700 Bq)/106 cells) for 1 hour at 37°C, washed, and used as targets. The cytotoxic activity of the YT and NKL cell lines was measured in standard 4-hour 51Cr release assays.28 For redirected cytotoxicity studies, P815 and K562 targets were labeled with 51Cr, washed, and incubated with a rabbit antimouse IgG (RaMIgG; 5 µg/mL) for 20 minutes at 4°C. The unbound mAb was removed by washing, and the cells (5 × 104 in 100 µl) were added to each well. The effector cells were incubated for 20 minutes at room temperature with the selected mAbs (5 µg/mL), washed, and serially diluted (final volume, 100 µL). After 4 hours of incubation at 37°C, 100 µL was collected from each sample and the radioactivity measured with a-counter.8
The percentage of specific lysis was calculated as follows:
[(experimental counts per minute Fluorescence resonance energy transfer studies The OKT10 mAb was conjugated to Cy3 dye with a FluoroLink-Ab Cy3 labeling kit (Amersham), and conjugations were verified by spectrophotometric and spectrofluorometric measurements. Cells were washed with ice-cold phosphate-buffered saline (PBS) with 5% FCS and 0.1% sodium azide, incubated on ice for 1 hour with the FITC-conjugated mAb (the donor fluorophore) and the Cy3-conjugated OKT10 (the accepting fluorophore), washed, and analyzed immediately with a FACSort instrument to determine energy transfer between FITC and Cy3-labeled proteins on the cell surface. Fluorescence resonance energy transfer (FRET) to Cy3 was detected by using standard methods.29 FITC was excited at 488 nm and Cy3 emissions were collected at greater than 600 nm. The median linear channel of fluorescence was used as an indicator of the presence (a positive shift over background level) or absence (no shift or negative shift) of energy transfer. The Wilcoxon matched pair, signed rank test was used to determine the significance of results.Cocapping experiments YT CD16+ cells (0.5 × 106) were incubated with the selected biotinylated primary mAb for 30 minutes on ice, washed, and allowed to react with FITC-conjugated streptavidin for 20 minutes on ice. Samples were then moved to 37°C for 40 minutes to induce capping, and ice-cold PBS with 0.5% BSA and 0.1% azide were added.30 Counterstaining was done with unlabeled mAbs and TRITC-conjugated GaMIg. After washing, cells were fixed, placed on poly-L-lysine-coated (Sigma) coverslips, and analyzed with a C-VIEW-12-BUND camera fitted to an Olympus 1 × 70 microscope (Milan, Italy). The images were collected by using ANALYSIS software.
Establishment of a CD16+ YT line and selection of
CD16+ and CD16 YT cells, which represent an
accepted model of continuous in vitro NK lineage. Transfection by
electroporation of a pcDNA3.1 expression vector enclosing the
full-length human CD16 gene in YT cells resulted in selection of clones
expressing CD16 (Figure 1A). Transfection
with the empty plasmid had no effect (Figure 1B). The
CD16+ cells were further subcloned by limiting dilution,
and selection was maintained by use of immunomagnetic beads and CD16
mAb separation. The CD38+ NKL line, which originally
expressed CD16 at a low density in a 40% subset of cells, was split
into CD16+ and CD16 subsets by extensive
cloning using limiting dilution (Figure 1E,F). The CD16+
NKL subline was maintained under positive selection by using the same
methods employed for the YT CD16+ transfectants. The
homogeneity and stability of the CD16 NKL subline was
ensured by cycles of cloning. These procedures did not affect
CD38 expression, which remained at comparable levels in the YT
CD16+ cells and the YT mock cells (Figure 1C,D) and in the
CD16+ and CD16 NKL cells (Figure 1G,H).
Ca++ mobilization Although expressed by wild-type YT cells, CD38 cannot by itself mobilize Ca++. Experiments were devised to determine whether the presence of surface CD16 confers on (or restores to) CD38 the ability to signal. The first observation was that the transfected CD16 molecule was an efficient receptor and that engagement with an agonistic mAb was followed by Ca++ mobilization (Figure 2, panel 3). The second observation was that CD38 ligation in YT CD16+ cells induced cytoplasmic Ca++ currents, thereby indicating that the presence of CD16 was a necessary and sufficient requisite responsible for the newly acquired feature of CD38 (Figure 2, panel 1). The profile was characterized by a rapid rise in intracytoplasmic Ca++ levels, stably maintained for more than 200 seconds and then declining slowly during ~ 100 to 150 seconds. The wave was similar in amplitude and kinetics to that obtained with CD16, although the latter was slightly more sustained (Figure 2, panel 3). None of the mAbs induced Ca++ mobilization without cross-linking by RaMIg, suggesting that engagement of more than 2 receptor molecules is required. Use of an F(ab')2 preparation of the IB4 mAb yielded the same Ca++ profiles, thereby ruling out the presence of Fc receptor (FcR)-mediated or background-mediated effects (data not shown). Furthermore, addition of the nonagonistic anti-CD38 mAbs OKT10, SUN-4B7, and IB6 (data not shown) as well as of an isotype-matched irrelevant IgG2a mAb (Figure 2, panel 5), or RaMIg alone (Figure 2, panel 6, left), did not yield detectable effects. The absence of Ca++ fluxes in the YT mock cells could not be referred to impaired cell labeling, since the ionophore A23187 induced the expected maximal Ca++ mobilization (Figure 2, panel 6, right).
Similar experiments were done with NKL sublines to confirm that the
observed effects were not restricted to a genetically modified line.
When ligated with an anti-CD38 mAb and then cross-linked by RaMIg, NKL
CD16+ cells gave rise to prominent Ca++ fluxes
(Figure 2, panel 7). The resulting wave was different from that
observed with the YT CD16+ cells: the spike was steeper and
higher, with a declining phase beginning after ~ 50 seconds, similar
to the profile observed in normal human NK cells or T lymphocytes
freshly purified from blood.8,19 CD16 behavior in these
cells paralleled that observed in the YT model (Figure 2, panel 9),
with Ca++ mobilization starting ~ 50 seconds after
addition of RaMIg and peaking after ~ 200 seconds, with stable
levels maintained until the end of the recording time. The
isotype-matched mAb was ineffective, as was RaMIg alone (Figure 2,
panels 11 and 12, left). No Ca++ mobilization was observed
after exposure of the NKL CD16 Tyrosine phosphorylation of cytoplasmic substrates After it was determined that the YT CD16+ cells became responsive to CD38 signaling, the main steps in the pathway were identified by conducting the following experiments. The cytoplasmic substrates acquiring tyrosine phosphorylation on signaling by CD38 and CD16 were analyzed in YT CD16+ cells and control YT mock cells. Both cell populations were treated for 1 minute at 37°C with the F(ab')2 fragment of the anti-CD38 mAb IB4 or the anti-CD16 mAb CB16 and further cross-linked by addition of the F(ab')2 preparation of a donkey antimouse Ig (DaMIg). The F(ab')2 fragment of an irrelevant isotype-matched mAb was used as the control.Anti-pTyr Western blot assays done on cell lysates clearly showed a
differential phosphorylation of discrete cytoplasmic substrates in YT
CD16+ cells incubated with F(ab')2 IB4 mAb or
CB16 (Figure 3, lanes 2 and 3). The most
relevant bands with increased intensity compared with that of
control-stimulated cells (Figure 3, lane 1) were at about 72, 70, 52, 44, 42, 38, and 36 kd. In contrast, neither CD38 nor CD16 engagement by
specific mAbs induced any significant increase in protein tyrosine
phosphorylation in YT mock cells (Figure 3, lanes 5 and 6). These
results indicate that CD16 expression is required for efficient
CD38-mediated protein tyrosine kinase activation.
ZAP70 was selected as a target because it is tyrosine phosphorylated on
CD38 activation in normal human NK cells. CD38 ligation in YT
CD16+ cells resulted in a significant increase in ZAP70
(Figure 4A), as determined by
immunoprecipitation and immunoblotting with anti-pTyr. No effects were
observed in YT mock cells (Figure 4, upper panel, lane 2 versus lane
5). CD16 triggering induced a prominent tyrosine phosphorylation in YT
CD16+ cells but not in YT mock cells, as expected (Figure
4, upper panel, lane 3 versus lane 6). All samples had comparable
amounts of protein (Figure 4A, lower panel).
CD38-mediated ERK activation in T cells depends on surface expression
of a functional TCR-CD3 complex.27 The requirements for
CD38-mediated ERK activation were tested by immunoblotting total cell
lysates from both YT CD16+ and YT mock cells with
anti-diphospho-ERK antibodies before and after CD38 or CD16 ligation
(Figure 4B). The blots were stripped and reprobed with total
ERK-specific antibodies to ensure that the individual lanes were
loaded with an equivalent amount of proteins. CD38 or CD16 triggering
in YT CD16+ cells caused a significant activation of ERK-1
and ERK-2 (Figure 4B, upper panel, lanes 2 [IB4 F(ab')2]
and 3 [CB16]) compared with results in control-treated cells (Figure
4B, lane 1). In contrast, anti-CD38 or anti-CD16 ligation failed to
induce ERK activation in CD16 CD38 signaling in YT CD16+ cells triggers secretion
of IFN- secretion is one of the events controlled
by MAPK activation in NK cells and a sensitive indicator of activation of YT cells.31 We found that CD16+ YT cells
cultured for 20 hours in the presence of the IB4 mAb used as either a
full molecule or an F(ab')2 fragmentand cross-linked with
RaMIg secreted IFN- in the culture medium in amounts consistently greater than those secreted by controls. Treatment with anti-CD16 mAb
and cross-linking with RaMIg also promoted IFN- secretion. No
synergy was observed when the cells were treated with both mAbs under
the experimental conditions used (Figure
5).
To strengthen the biologic relevance of the signal highlighted by using
agonistic mAbs, cytokine assays were reproduced with CD31 used as a
physiologic trigger for CD38. CD31+ transfectants were
cocultivated with YT CD16+ cells for 24 hours and
mock-transfected cells served as controls. The amounts of IFN- CD38 becomes a receptor modulating cytotoxicity in YT CD16+ cells Although lacking CD16, YT cells were reported to kill target tumor cells in conventional cytotoxicity assays, a key element of their representability of the NK lineage. The CD11a, CD28, and 2B4 molecule are some of the active killer receptors identified so far.32-34 Transfection of CD16 into these cells produces profound changes in the lytic potential of YT cells. Indeed, the presence of CD16 resulted in an increased ability to kill selected B (Raji, Daudi, RPMI 8226, Karpas 707, ARH 77, and LP-1) and myeloid targets (NB-4 and U937), but it had no effect in other cell lines of the same lineages (Namalwa, NALM-6, WT18, Kasumi-1, HL-60, Mono-Mac-6, and K562) or any of the T-cell targets assayed (Figure 6). These results could be interpreted in the light of recent findings pointing to the existence of a cell-bound ligand for CD16.35 On the other hand, they could indicate a direct contribution of the CD38-CD31 system, since U937 and NB-4 (CD31+ myeloid cells) show increased death on incubation with YT CD16+ cells.
Formal proof of the involvement of CD38 in activation of lytic programs
was provided by the results of redirected cytotoxicity assays. The
conclusions of these experiments, performed using the murine P815 and
the human K562 FcR
CD38 is laterally associated with CD16 The unique ability of CD38 to associate functionally with TCR and BCR relies on close proximity as an indispensable requisite for CD38 to exploit the signaling machinery of professional surface receptors. This need was confirmed by analyzing the lateral associations between CD38 and CD16 in YT CD16+ cells as evaluated by FRET. The results obtained indicate that CD38 and the de novo expressed CD16 are physically associated in YT cells. The association is significant and specific, as shown by the lack of energy transfer with CD71, the transferrin receptor, which was expressed at a similar density by these cells (Figure 8). Confirmation of these findings was provided by the cocapping experiments. Antibody-mediated capping is an energy-dependent redistribution of cell-surface molecules to a single pole of the cell. In general, only molecules bound by the antibody will be redistributed to the area of the cap unless they have a particular association with other structures that are in turn induced to undergo cocapping to the same area. We found that CD38 capping was followed by CD16 cocapping and vice versa (Figure 8C). No cocapping was observed with anti-HLA class I mAb, used as an isotype-matched control. In addition, a summary of data acquired from a large number of cells is presented in Figure 8C.
Cytotoxicity not restricted by major histocompatibility complex
(MHC) results from a cooperative interaction among an array of
monomorphic adhesion receptors.36,37 It was previously
reported that engagement of CD38 on normal human NK cells elicits
cytoplasmic Ca++ currents, phosphorylation of the CD3- The uniqueness of CD38 relies on its unsuitable intracellular tail and
consequent need to depend on other functional receptors to elicit
signals, as previously demonstrated in T- and B-lymphocyte models.5,17,18 The working hypothesis behind this paper is that CD38 relies on CD16 to deliver signals in NK cells. Preliminary indirect evidence comes from the analysis of the NK-like
CD16 The approach selected was to transfect the human CD16 gene into YT cells and compare the effects resulting from CD38 ligation in the 2 cell populations. The first finding was that transfection of CD16 into YT cells produces expression of a functional receptor able to mobilize Ca++ and induces tyrosine phosphorylation of several substrates. This was not unexpected, since YT cells have the necessary intracellular mediators and adaptor proteins. The second finding was that CD16 transfection confers signaling properties on CD38, as witnessed by the appearance of clear-cut cytoplasmic Ca++ currents and a complex profile of phosphorylated proteins. Some of the substrates were identified and included ZAP70 and ERK 1/2. The pathway depicted so far apparently overlaps, in many instances, that elicited by CD38 in normal NK cells and envisages sequential activation of the ZAP70 and of ERK1/2 proteins, serine-threonine kinases belonging to the MAPK family. Such a signaling cascade ultimately converges on the nucleus, resulting in changes in gene expression that control activation of the NK lytic machinery. The downstream events include production and secretion of IFN- Standard and redirected cytotoxicity studies found that CD16
expression was itself sufficient to change the lytic propensities of
the cells. CD16 transfection was followed by an increased ability to
kill selected B and myeloid targets, whereas it had no effect in T-cell
lines. These results are in line with published data from independent
groups and point to the possibility of a cell-surface ligand for
CD16.35 Formal confirmation was provided by redirected lysis experiments using P815 (a murine mastocytoma cell line) and K562
(a human erythroleukemia line) to highlight the contribution of the
CD38 pathway to lysis. The results show conclusively that CD38 is an
active killing receptor, provided that CD16 is expressed on the surface
of YT cells. Because cytotoxicity is the most relevant biologic
characteristic of NK cells, these data were confirmed by using the NKL
cell line, in which CD16+ and CD16 The functional association between CD16 and CD38 is likely to take place at the plasma membrane level, since YT cells lack CD16 but have its intracellular signaling machinery. Functional synergy in leukocytes is usually indicated by close contiguity or location in specialized areas of the cell surface.43 In this study, the existence of a physical proximity between the 2 structures was confirmed by FRET and cocapping experiments done in the YT CD16+ cell line. These findings provide formal and instrumental backup results to those of cocapping experiments done using NK cells from peripheral blood.16 Furthermore, the size, adhesive properties, and localization in membrane rafts44 indicate CD38 as a candidate member of the immunologic synapse. The results concur to portray a molecule that is unable by itself to signal but that is rescued as a receptor by association with a professional signaling structure. This molecule is CD16 in NK cells. In this case, CD38 would be a unique coreceptor molecule, enabled to function by the dominant receptor itself. Crucial questions that remain to be answered concern induction of the system. Is it a ligand-induced or a substrate-induced conformational change? What is the role of the interaction between CD16 and its putative cell-surface-bound ligand in the activation of CD38? Finally, YT cells are not representative of the majority of mature NK cells in the peripheral blood of adults; for one thing, NK cells in adults do not express CD28, which is detectable on YT cells at a substantial density. In this respect, and also because the YT line was established from samples from a child with ALL and thymoma, YT cells are more likely representative of fetal NK cells, which lack CD16 and express CD28. Analysis of CD38 expression and function in fetal NK cells is likely to add insight into the biologic features of this unique receptor molecule.
We thank Lewis L. Lanier (San Francisco, CA) for providing reagents and suggestions, R. Galandrini (Rome, Italy) for providing cells, and Francesca Urbani (Rome, Italy) for assistance.
Submitted August 8, 2001; accepted November 20, 2001.
Supported by grants from AIRC, Special Projects AIDS (Istituto Superiore di Sanità), Biotechnology (CNR/MURST), and Cofinanziamento (MURST) to F.M.; from Comisión Interministerial de Ciencia y Tecnología SAF99-0024 to J.S.; and from Instituto de Salud Carlos III-FIS, Ministerio de Sanidad y Consumo, Programa Nacional de Salud (01/1073) to M.Z. Financial contributions were provided by the Compagnia di SanPaolo, Cariverona, and Ghirotti Foundations, and Regione Piemonte. S.D. is a student of the Postgraduate School of Medical Oncology, University of Torino Medical School, Torino, Italy. M.Z. is supported by Comitato de Investigadores, Programa Nacional de Salud, Ministerio de Sanidad y Consumo, Spain.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Fabio Malavasi, Laboratory of Immunogenetics, University of Torino Medical School, Via Santena, 19, 10126 Torino, Italy; e-mail: fabio.malavasi{at}unito.it.
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  F. Malavasi, S. Deaglio, A. Funaro, E. Ferrero, A. L. Horenstein, E. Ortolan, T. Vaisitti, and S. Aydin Evolution and Function of the ADP Ribosyl Cyclase/CD38 Gene Family in Physiology and Pathology Physiol Rev, July 1, 2008; 88(3): 841 - 886. [Abstract] [Full Text] [PDF]
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 A. Iannello, O. Debbeche, S. Samarani, and A. Ahmad Antiviral NK cell responses in HIV infection: II. viral strategies for evasion and lessons for immunotherapy and vaccination J. Leukoc. Biol., July 1, 2008; 84(1): 27 - 49. [Abstract] [Full Text] [PDF]
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 N. Gallay, L. Anani, A. Lopez, P. Colombat, C. Binet, J. Domenech, B. B. Weksler, F. Malavasi, and O. Herault The Role of Platelet/Endothelial Cell Adhesion Molecule 1 (CD31) and CD38 Antigens in Marrow Microenvironmental Retention of Acute Myelogenous Leukemia Cells Cancer Res., September 15, 2007; 67(18): 8624 - 8632. [Abstract] [Full Text] [PDF]
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S. Deaglio, T. Vaisitti, R. Billington, L. Bergui, P. Omede', A. A. Genazzani, and F. Malavasi CD38/CD19: a lipid raft-dependent signaling complex in human B cells Blood, June 15, 2007; 109(12): 5390 - 5398. [Abstract] [Full Text] [PDF]
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S. Deaglio, T. Vaisitti, S. Aydin, E. Ferrero, and F. Malavasi In-tandem insight from basic science combined with clinical research: CD38 as both marker and key component of the pathogenetic network underlying chronic lymphocytic leukemia Blood, August 15, 2006; 108(4): 1135 - 1144. [Abstract] [Full Text] [PDF]
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L. Frasca, G. Fedele, S. Deaglio, C. Capuano, R. Palazzo, T. Vaisitti, F. Malavasi, and C. M. Ausiello CD38 orchestrates migration, survival, and Th1 immune response of human mature dendritic cells Blood, March 15, 2006; 107(6): 2392 - 2399. [Abstract] [Full Text] [PDF]
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M.-T. Zilber, N. Setterblad, T. Vasselon, C. Doliger, D. Charron, N. Mooney, and C. Gelin MHC class II/CD38/CD9: a lipid-raft-dependent signaling complex in human monocytes Blood, November 1, 2005; 106(9): 3074 - 3081. [Abstract] [Full Text] [PDF]
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S. Deaglio, T. Vaisitti, L. Bergui, L. Bonello, A. L. Horenstein, L. Tamagnone, L. Boumsell, and F. Malavasi CD38 and CD100 lead a network of surface receptors relaying positive signals for B-CLL growth and survival Blood, April 15, 2005; 105(8): 3042 - 3050. [Abstract] [Full Text] [PDF]
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 S. Deaglio, T. Vaisitti, and F. Malavasi CD38 Ligation in B-Chronic Lymphocytic Leukemia Cells Induces Sequential Tyrosine Phosphorylation of ZAP70, PLC- {gamma}2 and ERK1/2 Proteins. Blood (ASH Annual Meeting Abstracts), November 16, 2004; 104(11): 959 - 959. [Abstract] [Full Text]
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T. D. Shanafelt, S. M. Geyer, and N. E. Kay Prognosis at diagnosis: integrating molecular biologic insights into clinical practice for patients with CLL Blood, February 15, 2004; 103(4): 1202 - 1210. [Abstract] [Full Text] [PDF]
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S. Deaglio, A. Capobianco, L. Bergui, J. Durig, F. Morabito, U. Duhrsen, and F. Malavasi CD38 is a signaling molecule in B-cell chronic lymphocytic leukemia cells Blood, September 15, 2003; 102(6): 2146 - 2155. [Abstract] [Full Text] [PDF]
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 C. Tenca, A. Merlo, D. Zarcone, D. Saverino, S. Bruno, A. De Santanna, D. Ramarli, M. Fabbi, C. Pesce, S. Deaglio, et al. Death of T cell precursors in the human thymus: a role for CD38 Int. Immunol., September 1, 2003; 15(9): 1105 - 1116. [Abstract] [Full Text] [PDF]
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A. Wiestner, A. Rosenwald, T. S. Barry, G. Wright, R. E. Davis, S. E. Henrickson, H. Zhao, R. E. Ibbotson, J. A. Orchard, Z. Davis, et al. ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile Blood, June 15, 2003; 101(12): 4944 - 4951. [Abstract] [Full Text] [PDF]
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 J. D. French, D. K. Walters, and D. F. Jelinek Transactivation of gp130 in Myeloma Cells J. Immunol., April 1, 2003; 170(7): 3717 - 3723. [Abstract] [Full Text] [PDF]
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 L. Righi, S. Deaglio, C. Pecchioni, A. Gregorini, A. L. Horenstein, G. Bussolati, A. Sapino, and F. Malavasi Role of CD31/Platelet Endothelial Cell Adhesion Molecule-1 Expression in in Vitro and in Vivo Growth and Differentiation of Human Breast Cancer Cells Am. J. Pathol., April 1, 2003; 162(4): 1163 - 1174. [Abstract] [Full Text] [PDF]
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J. Durig, H. Nuckel, A. Huttmann, E. Kruse, T. Holter, K. Halfmeyer, A. Fuhrer, R. Rudolph, N. Kalhori, A. Nusch, et al. Expression of ribosomal and translation-associated genes is correlated with a favorable clinical course in chronic lymphocytic leukemia Blood, April 1, 2003; 101(7): 2748 - 2755. [Abstract] [Full Text] [PDF]
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S. Deaglio, A. Capobianco, A. Cali, F. Bellora, F. Alberti, L. Righi, A. Sapino, C. Camaschella, and F. Malavasi Structural, functional, and tissue distribution analysis of human transferrin receptor-2 by murine monoclonal antibodies and a polyclonal antiserum Blood, November 15, 2002; 100(10): 3782 - 3789. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
10% of the maximum release for all valid assays.
and FcRIII-
but not CD3
.
polypeptides and intracellular associations of CD8 with p56(lck).
Eur J Immunol.
1998;28:745-754
/