Signaling through CD38 induces NK cell activation
Roberto Mallone1,
Ada Funaro1,
Mercedes Zubiaur2,
Germano Baj1,
Clara M. Ausiello3,
Carlo Tacchetti4,
Jaime Sancho2,
Carlo Grossi4 and
Fabio Malavasi
1 Laboratory of Immunogenetics, Department of Genetics, Biology and Biochemistry, University of Torino, 10126 Torino, Italy
2 Department of Cellular Biology and Immunology, Instituto de Parasitologia y Biomedicina, CSIC, 18071 Granada, Spain
3 National Institutes of Health (ISS), 00161 Roma, Italy
4 Department of Experimental Medicine, Section of Human Anatomy, University of Genova, 16100 Genova, Italy
Correspondence to:
F. Malavasi, Laboratorio di Immunogenetica, Dipartimento di Genetica, Biologia e Biochimica, Via Santena 19, 10126 Torino, Italy
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Abstract
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Human CD38 is a signal transduction molecule, and, concurrently, an ectoenzyme catalyzing the synthesis and degradation of cyclic ADP-ribose (cADPR), a potent Ca2+ mobilizer. One facet of CD38 that has not yet been addressed is its role in NK cells. To this end, the events triggered by CD38 ligation with agonistic mAb were analyzed on freshly purified human NK cells. Ligation was followed by (i) a significant rise in the intracellular level of Ca2+, (ii) increased expression of HLA class II and CD25, and (iii) tyrosine phosphorylation of discrete cytoplasmic substrates. The phosphorylation cascade involved CD3-
and Fc
RI
chains,
-associated protein (ZAP)-70 and the proto-oncogene product c-Cbl. NK effector functions were then analyzed: CD38 signaling was able (iv) to induce release of IFN-
and, more prominently, of granulocyte macrophage colony stimulating factor, as assessed by measuring both mRNA and protein products; and, lastly, (v) to induce cytolytic effector functions on target cells after IL-2 activation, as shown both by cytotoxicity assays and ultrastructural changes. The tyrosine-phosphorylated substrates and all the effects mediated by CD38 were similar to those observed following triggering via CD16 (Fc
RIIIA); moreover, Ca2+ mobilization via CD38 no longer operated in NK-derived cell lines lacking CD16. These results suggest that the activation signals transduced by CD38 in NK cells elicit relevant cellular events. The effects are similar to those elicited via CD16 and possibly rely on common signaling pathways.
Keywords: calcium, CD16, ectoenzymes, surface receptors, tyrosine phosphorylation
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Introduction
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CD38, initially described as an activation antigen, attracted widespread interest when it was attributed the role of ADP-ribosyl cyclase and cyclic ADP-ribose (cADPR) hydrolase, two enzymatic activities involved in the conversion of nicotinamide adenine dinucleotide (NAD+) first to cADPR and then to ADPR (1,2). Consequently, CD38 has been counted among the members of the ectoenzyme family, a growing number of molecules which includes >3% of all surface receptors (3).
Some ectoenzymes are paradoxical in that they also mediate other apparently unrelated functions, mostly dealing with the regulation of cellcell contacts and transmembrane signaling. CD38 has thus moved from consideration as an orphan receptor to consideration as a pleiotropic molecule involved in adhesion (4,5) and signaling, in spite of its apparently unsuitable cytoplasmic domain. The nature of CD38 as a transducing channel was confirmed by the identification of a counter-receptor (5), identified as CD31 (6), that, following interaction with CD38, triggers the same events seen in the surface, cytoplasmic and nuclear districts when specific mimotopic mAb are used (6).
Several laboratories have undertaken the analysis of the signals implemented after CD38 ligation. The results so far obtained indicate that CD38 engagement in T cells leads to activation of the Raf-1/MAP kinase and the CD3-
/
-associated protein (ZAP)-70/phospholipase C (PLC)-
1 pathways (7), Ca2+ mobilization and induction of apoptosis (8). These events are dependent on the presence of a functional TCRCD3 complex (8,9). CD38 ligation in B lymphocytes inhibits proliferation and induces apoptosis of B precursors (10); signal transduction in these cells involves tyrosine phosphorylation of Syk, PLC-
(11), Tec (12), c-Cbl (13) and of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-K) (11). Mature B cells yield contrasting results: CD38 signaling is followed by proliferation and prevention of apoptosis (14,15); several events are dependent, at least in murine models, on the association of CD38 with BCR (16). Ligation of CD38 in myeloid cells enhances superoxide generation induced by chemotactic peptide (17), and induces tyrosine phosphorylation of c-Cbl (18) and its association with the p85 subunit of PI 3-K (19). Thus, CD38 relies upon different complexes for its signaling purposes, acting as a molecular parasite of TCR in T lymphocytes (8), BCR in B lymphocytes (16) and HLA class II in monocytoid cells (20).
This paper originates from the need to complete our understanding of the signaling properties of CD38 in human NK cells, a population which has only relatively recently begun to be explored (2123). Interest in NK cells also stems from the fact that they lack the TCR and BCR complexes, and are thus likely to offer insights into the signaling mechanism(s) driven by CD38. The results obtained indicate that CD38 delivers activation signals in NK cells through a complex molecular machinery largely shared by CD16.
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Methods
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Antibodies
CD38 stimulation was performed with the agonistic anti-CD38 mAb IB4 (IgG2a) (24), used in the form of an F(ab')2 fragment to avoid binding through Fc
RIIIA (CD16), unless otherwise specified. Other mAb used were IB6 (non-agonistic anti-CD38, IgG2b) (25), CB16 (anti-CD16, IgG2a), Moon-1 (anti-CD31, IgG1) (5), anti-CD28 (IgG2a) and JAS (anti-gp120, IgG2a irrelevant isotype-matched control), all produced in our laboratory. Anti-CD3, anti-CD20 and anti-HLA class II mAb used for NK cell purification were all locally produced. Affinity-purified goat antibody to mouse IgG (whole molecule) (G
MIgG; Cappel, Organon Teknika, Durham, NC) was used as a cross-linker in the form of an F(ab')2 preparation. 1G2 mAb coupled to agarose beads from Oncogene (Calbiochem, Cambridge, MA) was used for anti-phosphotyrosine (pTyr) immunoprecipitation. Other antibodies used for Western blotting and immunoprecipitation were: anti-pTyr mAb PY20 (Transduction Laboratories, Lexington, KY); 1D4.1 (anti-CD3-
mAb) (26) and 448 (anti-CD3-
serum), the kind gift from Dr B. Alarcòn (Centro de Biologì a Molecular, Madrid, Spain); anti-ZAP-70 rabbit antiserum, generously made available by Dr J. M. Rojo (Centro de Investigaciones Biologicas, Madrid, Spain); and anti-c-Cbl (Santa Cruz Biotechnology, Santa Cruz, CA). The anti-Fc
RI
chain C36 rabbit polyclonal antiserum was obtained by immunizing New Zealand White rabbits with the synthetic peptide CGVpYTGLSTRNQETpYETLKJHEKRRASV conjugated to soluble keyhole limpet hemocyanin (Sigma, St Louis, MO). The immunogen was a dually phosphorylated peptide corresponding to the immunoreceptor tyrosine-activation motif (ITAM) of the Fc
RI
. The peptide was chemically synthesized with an additional N-terminal Cys for coupling to affinity matrices, and with an additional C-terminal sequence, ArgArgAlaSerVal (RRASV), for quantitative measurement of the coupling efficiency of the peptide. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was from Promega (Madison, WI).
NK cell isolation and NK cell lines
NK cells were purified from peripheral blood mononuclear cells (PBMC) by negative selection with Dynabeads magnetic particles (Dynal, Oslo, Norway) conjugated with sheep anti-mouse IgG. Briefly, PBMC obtained from healthy donors by centrifugation on density gradient were deprived of monocytes by a plastic adherence step and successively incubated for 1 h at 4°C in the presence of anti-CD3, anti-CD20 and anti-HLA class II mAb (5 µg/ml/5x106 cells). After three washings, cells were incubated for 1 h at 4°C with immunobeads according to the manufacturer's instructions. The preparations obtained were
97% CD16+, CD3, HLA class II and CD14, as assessed by cytofluorimetric analysis. The continuous human NK lines YT (27) and NKL (28), both CD38+CD16, were used for comparative analyses. Lack of CD16 expression was further confirmed by PCR analysis of the specific mRNA product.
Measurement of intracellular Ca2+
Changes in intracellular Ca2+ concentrations ([Ca2+]i) were monitored by flow cytometry after loading cells with the Ca2+-sensitive fluorescent dye Fluo 3-AM (Molecular Probes, Eugene, OR), as previously described (29,30). Briefly, either freshly isolated peripheral blood NK cells or YT and NKL cells were washed twice in HBSS (pH 7.0) with 5% FCS, resuspended in the same medium at a concentration of 2x106 cells/ml and incubated for 30 min at 37°C with 5 mM Fluo 3-AM in the presence of 0.01% Pluronic F127 (Sigma). Cells were then washed twice, incubated for 10 min at room temperature with the primary mAb at different concentrations (15 µg/ml, depending on the mAb), washed again and analyzed continuously at 37°C on a FACSort flow cytometer (Becton Dickinson, Milan, Italy) with Lysys II software. Stimulation was induced by the addition of 20 µg/ml of G
MIgG mAb. Cross-linking was performed, as the application of soluble antibodies alone has little effect on [Ca2+]i according to observations reported by other authors (30,31). Standard controls included incubations with an irrelevant isotype-matched mAb, with G
MIgG mAb alone, with the non-agonistic anti-CD38 mAb IB6 and with the ionophore A23187 (Molecular Probes). Cells were gated by size and side scatter to eliminate both debris and dead cells from analysis. Dynamic changes in [Ca2+]i were monitored by continuously plotting the shift in the Fluo 3-AM fluorescence over a 540 s time-course.
Modulation of surface molecules
Purified NK cells (2x106/ml) were cultured at 37°C for the indicated time intervals (see figures) in RPMI 1640 medium with 5% FCS in the presence of soluble IB4 F(ab')2 mAb, anti-CD16 mAb, an irrelevant IgG2a mAb or a combination of IB4 plus anti-CD16 mAb. After incubations, cells were washed and resuspended in PBS containing 0.2% BSA and 1% NaN3, and incubated with FITC- or phycoerythrin (PE)-conjugated anti-HLA class II, anti-CD25 or anti-CD69 mAb (Becton Dickinson) for 1 h at 4°C. The analysis was performed on a FACSort (Becton Dickinson). Excitation was from an argon laser at 488 nm. Background antibody binding was estimated by isotype-matched negative control mAb. Acquired data were analyzed with Lysys II software (Becton Dickinson).
Phosphorylation experiments
Cells were incubated for 10 min on ice with an F(ab')2 fraction of the IB4 mAb, anti-CD16 mAb or an F(ab')2 fraction of an isotype-matched, unreactive mAb (anti-gp120) for the not stimulated (NS) condition. Each mAb was used at a concentration of 10 µg/106 cells. The unbound mAb was eliminated by washing with cold IMDM and then the cells were incubated (10 min on ice) with an F(ab')2 preparation of a G
MIgG. The cells were subsequently reacted with the relevant mAb at 37°C for 4 min, after which lysis was performed for 20 min on ice with 1% NP-40 lysis buffer (20 mM HEPES, pH 7.6, 150 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 1mM EGTA, 50 µM phenylarsine oxide, 10 mM iodoacetamide, 1 mM PMSF, and 2 µg/ml of antipain, chymostatin, leupeptin and pepstatin), as previously reported (7). After removal of nuclei by centrifugation, an aliquot of the lysates was diluted in Laemmli sample buffer, boiled for 5 min and stored at 80°C prior to running on SDSPAGE. The remainder was used for immunoprecipitation incubating with the antibody of interest and recovering the immune complexes by means of recombinant Protein ASepharose beads (Repligen, Cambridge, MA). After washing, the beads were boiled in either reducing or non-reducing sample buffer (as specified in the figure legends) for 5 min and the elutes run on SDSPAGE. The gel was then transferred onto a PVDF membrane with a semi-dry transfer apparatus (Hoefer Pharmacia Biotech, San Francisco, CA) in Trisglycine buffer containing 20% methanol and 0.035% SDS at 0.8 mA/cm2. To ensure proper recovery of all migrated proteins, transfer efficiency was checked by Ponceau red stain. The membrane was blocked in 1% BSA, washed in TBS-T (10 mM Tris, pH 7.4, 100 mM NaCl, 0.1% Tween 20) and reacted with HRP-conjugated PY20 anti-pTyr mAb for 2 h. The filter was then washed again and developed using ECL reagents (Amersham, Little Chalfont, UK). The membrane was subsequently stripped by thorough washing in a buffer containing 150 mM NaCl and 10 mM TrisHCl (pH 2.2), blocked again in 2.57% non-fat dry milk and incubated with the primary antibody of interest diluted in 1% milk for 1 h. After washing, HRP-conjugated goat anti-rabbit IgG was added and the membrane was washed and developed again with ECL reagents.
Cytokine release
NK cells were resuspended at 2x106/ml in RPMI 1640 medium supplemented with 5% FCS and antibiotics, and cultured in the presence of anti-CD16 and IB4 mAb at predetermined optimal concentrations (20 µg/ml) for 24, 36 and 48 h respectively. Total cellular RNA was extracted according to the guanidium isothiocyanate method (32). RNA (1 µg in a 20 µl reaction volume) was transcribed using Moloney murine leukemia virus reverse transcriptase and PCR amplification was conducted starting from as low as 1 ng of the original RNA. Cytokine-specific primer pairs were synthesized according to published sequences (DNA synthesizer; Applied Biosystems, Foster City, CA). PCR was performed in a 9600 Perkin-Elmer (Foster City, CA) thermal cycler, as previously described (33). The reaction product was visualized by electrophoresis using 10 µl of the reaction mixture. Culture supernatants were collected and used to measure IFN-
and GM-CSF cytokine secretion by ELISA tests (Quantikine; R & D System, Minneapolis, MN), following the manufacturer's instructions.
Cytotoxicity assays
The ability of the agonistic IB4 mAb to trigger the cytolytic activity of NK cells was evaluated in a conventional 51Cr-release assay. The murine mastocytoma cell line P815 was used as target, and labeled for 1 h with 51Cr (100 µCi/106 cells), washed twice with medium and plated at 5x103 cells/well in 96-well U-bottom plates. In the redirected killing assay, effector cells were activated with IL-2 (100 U/ml for 5 days), plated in triplicate at various E:T in the presence of anti-CD16, anti-CD38 and a control reactive isotype-matched mAb. Labeled target cells were added to each well and 100 µl of supernatant was collected after 4-h incubation at 37°C and analyzed in a
-counter. The percent specific lysis was calculated as:
Electron microscopy
Activated NK cells were treated with anti-CD38, anti-CD16 or an irrelevant reactive isotype-matched mAb for 20 min at 4°C, and subsequently added to P815 target cells (E:T = 10:1) and incubated at 37°C. The ultrastructural analysis was performed after 10 min, 20 min and 2 h. Cells were fixed with 2.5% glutaraldehyde (Polysciences, Warrington, PA) in 0.1 M cacodylate buffer, pH 7.3 and post-fixed with 1% OsO4 (Polysciences) in the same buffer. Following en bloc staining with 1% uranyl acetate and dehydration with ethanol, samples were embedded in LX112 (Polysciences). Grey-silver sections were stained with uranyl acetate and lead citrate, and observed with Zeiss EM 10C or EM902 electron microscopes.
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Results
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Ca2+ mobilization
Previous reports showed that the interaction of CD16 with ligands (either immune complexes or anti-CD16 antibodies) induces a rapid rise in [Ca2+]i (31). We investigated whether CD38 binding by agonistic mAb could mediate similar effects in purified NK cells (Fig. 1A
, panel 1). The rise in [Ca2+]i after CD38 ligation with the agonistic mAb IB4 became apparent only after cross-linking with G
MIgG. The profile is characterized by an ascending slope for the first 2 min, followed by a plateau which is maintained for the rest of the reading time (9 min). CD16 signaling also needed cross-linking of the specific mAb in order to yield results recordable with the system adopted. The Ca2+ profile (Fig. 1A
, panel 2) was slightly different from that observed with CD38: a faster rise with an earlier (~1.5 min) and higher peak was followed by a rapid decline, reaching the basal level at the end of the reading time. Thus, the Ca2+ fluxes recorded followed different kinetics, i.e. marked by lower but constant levels in the CD38 pathway and higher, although transient, spikes obtained after CD16 ligation. Triggering of the NK cells via CD31 (Fig. 1A
, panel 3) also induced some Ca2+ movements, although of lower intensity, as previously reported in other cellular models (5). None of the mAb induced Ca2+ mobilization when not cross-linked with G
MIgG, suggesting that engagement of more than two receptor molecules is required. Similar experiments performed using IB6 (a non-agonistic anti-CD38 mAb) (Fig. 1A
, panel 4), or with an isotype-matched irrelevant IgG2a mAb (Fig. 1A
, panel 5) or with G
MIgG alone (not shown), did not yield any recordable effect. As a further positive control, NK cells were treated with the ionophore A23187 (Fig. 1A
, panel 6).

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Fig. 1. [Ca2+]i modifications in purified NK cells (A) and CD16 NK cell lines (B) upon triggering with different mAb. Cells were preincubated with 5 µg/ml IB4 F(ab')2 mAb (A, panel 1 and B, panels 1 and 2), 1 µg/ml anti-CD16 mAb (A, panel 2), 10 µg/ml Moon-1 (A, panel 3), 10 µg/ml IB6 (A, panel 4) and analyzed continuously on a FACSort flow cytometer. G MIgG (20 µg/ml) was added 10 s after the start of reading. Standard controls included the exposure to an irrelevant isotype-matched mAb (A, panel 5) and the A23187 ionophore (A, panel 6). Data are presented as density plots (color code, ranging from red and yellow to blue, green and orange for progressively higher [Ca2+]i of the shift in the Fluo 3-AM fluorescence (y-axis) over a 540 s time-course (x-axis). Results refer to a representative experiment performed in triplicate.
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To investigate the potential interconnection between the CD38 and CD16 transduction pathways involved in Ca2+ release, the same experiments were performed on YT (27) (Fig. 1B
, panel 1) and NKL (28) (Fig. 1B
, panel 2) cell lines, which are reported to display an NK phenotype, but lack CD16 expression, although they are CD38+. As shown in Fig. 1
(B), ligation of CD38 on these cells did not give rise to any Ca2+ movement. Stimulation with an irrelevant IgG2a mAb and via CD28 were included as negative and positive controls respectively (not shown).
Modulation of HLA class II, CD25 and CD69 surface expression
To further study the activation signals driven by CD38, we examined a selected panel of surface receptors. As shown in Fig. 2
, signaling via CD38 as well as via CD16 is followed by an increased epitope density of HLA class II. The combination of these two mAb did not show additive effects. A similar up-modulation was observed for CD25, an effect previously reported to be induced via CD16 (34); in this instance, simultaneous ligation of CD16 and CD38 was followed by enhanced expression. CD69, an early activation molecule, was not significantly influenced by CD38 nor by CD16 signaling alone, while combination of the two mAb induced a modest shift in the CD69 fluorescence. No modulation of any of these receptors was observed after incubation of the NK cells with an irrelevant isotype-matched mAb (not shown).

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Fig. 2. Modulation of HLA class II, CD25 and CD69 surface expression following CD38 and CD16 ligation. One-parameter flow cytometric analysis of HLA class II, CD25 and CD69 expression in basal conditions (grey profiles) and following treatment for either 36 h (HLA class II and CD25) or 4 h (CD69) with anti-CD38, anti-CD16 or the combination of the two mAb (white profiles). x-axis = fluorescence intensity/cells; y-axis = number of cells registered/channel. Number of cells tested = 5000. Results refer to a representative experiment performed in triplicate.
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Phosphorylation of CD3-
, Fc
RI
, ZAP-70 and c-Cbl
After analyzing early events triggered either via CD38 or CD16 (i.e. Ca2+ mobilization, expression of CD69), the transduction pathways activated by these molecules were then defined by Western blot analysis of the proteins tyrosine phosphorylated after short-term stimulation at 37°C. Anti-pTyr Western blot on NK immunoprecipitates enriched with tyrosine-phosphorylated proteins clearly demonstrated a differential phosphorylation of discrete cytoplasmic substrates in cells incubated with IB4 or control anti-CD16 mAb (Fig. 3A
). Similar results were obtained using the F(ab')2 preparation of the IB4 mAb. The most relevant bands featuring increased intensity compared to the basal condition were at 120, 7075 and 23 kDa (as indicated by arrows); their mol. wt are compatible with the proto-oncogene product c-Cbl, ZAP-70 and the phosphorylated form of monomeric CD3-
respectively. All these molecules are reported to be recruited following CD16 triggering (3539). Figure 3
(B) reports the results obtained by increasing exposure time of the lower part of the membrane shown in Fig. 3
(A): signaling via CD38 (as well as via CD16) is followed by a marked phosphorylation of a protein migrating as a doublet at 2123 kDa, as indicated by arrows. The identity of these bands as phosphorylated forms of monomeric
chains was confirmed by stripping the same membrane and reacting it with an anti-
serum (Fig. 3E
, left). This specific antibody recognized the same bands highlighted by the anti-pTyr mAb in Fig. 3
(B); furthermore, analysis on whole lysates with the same antiserum displayed a relative abundance of unphosphorylated
species of lower mol. wt (Fig. 3E
, right). The finding of unphosphorylated
bands detected in almost identical fractions in all the conditions considered (Fig. 3E
, right) confirmed that the same amount of total proteins was present at each experimental point. The upper part of the membrane shown in Fig. 3
(A) was then reprobed with an anti-c-Cbl mAb and the results demonstrated that the 120 kDa band seen in anti-pTyr Western blot is c-Cbl (Fig. 3C
, left). Also in this instance, the same amounts of unphosphorylated proteins were detected on whole lysates under all the experimental conditions considered (Fig. 3C
, right). Reprobing of the middle part of the membrane shown in Fig. 3
(A) with the specific antiserum also identified the bands highlighted ~7075 kDa as ZAP-70 species (Fig. 3D
, left) and equal loading was confirmed on whole lysates (Fig. 3D
, right).

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Fig. 3. Western blot analysis of tyrosine-phosphorylated substrates upon CD38 ligation on purified NK cells. (A) Anti-pTyr immunoprecipitates were probed with anti-pTyr mAb. (B) Lower part of (A), as indicated by the asterisk, highlights additional bands after prolonged exposure. (C) Top part of (A), stripped and reprobed with anti-c-Cbl mAb. (D) Middle section (A), stripped and reprobed with anti-ZAP-70 serum. (E) Bottom part of (A), stripped and reprobed with anti- serum. Probing on whole lysates is also shown on the right side of (C)(E). Gels were run under reducing conditions. Results refer to a representative experiment performed in duplicate. NS, not stimulated.
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To further document CD3-
tyrosine phosphorylation, a second set of experiments was designed where
chain immunoprecipitation was performed in order to obtain a part of the blot enriched with this substrate. Additionally, samples were run under non-reducing conditions to visualize the
chain as part of
homodimers or, possibly,
heterodimers, as previously described in both T and NK cells (38). Whole lysates from NK cells stimulated via CD38 or CD16 and probed with anti-pTyr mAb highlighted the phosphorylation of multiple proteins whose migration is compatible with
-
homodimers, as indicated by the bracket in Fig. 4
(A); this is in line with the characteristic migratory pattern of the tyrosine-phosphorylated species of CD3-
, reported as multiple bands resulting from the combination of hyperphosphorylation and multiubiquitination (36,40). On the contrary, the phosphorylated
forms appeared as a single band of ~46 kDa on the CD3-
immunoprecipitate (Fig. 4B
, left). This finding is probably due to preferential binding by the 1D4.1 mAb used for immunoprecipitation to selected p
p
forms. Another doublet of lower mol. wt was visible on the whole-cell lysate in Fig. 4
(A): its ~36 kDa electrophoretic mobility is compatible with phosphorylated
heterodimers. Reprobing the anti-CD3-
immunoprecipitates with an anti-
serum (Fig. 4B
, right) confirmed the identity of the phosphorylated bands observed in the left half of Fig. 4
(B) as p
p
forms. Excess unphosphorylated
homodimers at ~3234 kDa and other
-containing species at ~25 kDa (likely corresponding to
heterodimers) were also observed (Fig. 4B
, right).
To verify the possible involvement of Fc
RI
in CD38 signaling, the same lysates deprived of CD3-
in the experiments described in Fig. 4
underwent a second immunoprecipitation with an anti-
chain serum. The results, shown in Fig. 5
(A), indicate the presence of a tyrosine-phosphorylated protein of ~25 kDa with a migratory pattern compatible with p
p
homodimers (36), visualized by running the samples under non-reducing conditions. Subsequent recognition of the band by an anti-
chain serum confirmed that the substrate engaged by CD38 ligation is indeed Fc
RI
(Fig. 5B
). Similar results were obtained after CD16 signaling. Longer exposure of the bottom part of the filter (Fig. 5C
) led to the identification of unphosphorylated
migrating at ~14 kDa. Fc
RI
phosphorylation was less prominent than that of CD3-
, a finding which is probably due to preferential physical associations of the homodimeric
species with CD16, as already reported (41), or with CD38. Indeed, CD38 and CD16 molecules do display marked physical lateral associations on the membrane of NK cells (42).
Cytokine release: mRNA expression and production of IFN-
, GM-CSF and tumor necrosis factor (TNF)-
CD16 is reported to mediate transcription and release of IFN-
and GM-CSF (34,43). We performed comparative tests after CD38 ligation. Freshly purified NK cells cultured for 24, 36 or 48 h in the presence of F(ab')2 preparations of the IB4 mAb were characterized by a consistent accumulation of mRNA for IFN-
and GM-CSF, whereas expression of mRNA for TNF-
was only slightly increased (Fig. 6
). The initial increments of IFN-
mRNA levels over the basal levels observed after 24 and 36 h became significantly marked at 48 h (Fig. 6
, top panel). Such effects were faster and higher in amplitude upon ligation of CD16. Further, the simultaneous addition of mAb specific for CD38 and CD16 was not apparently followed by significant additive or synergistic effects. GM-CSF mRNA was apparent also in basal conditions (Fig. 6
, second panel). However, CD38 ligation was paralleled by increased levels of the specific transcripts, quantitatively similar to those elicited by anti-CD16 mAb. A possible additive effect was observed for GM-CSF, although the qualitative nature of the PCR assay and the high levels of transcript obtained with the two mAb separately make comparison undependable. The accumulation of TNF-
mRNA after exposure to the IB4 anti-CD38 mAb was less significant; the same was true for anti-CD16 mAb, with no synergies between CD38 and CD16 (Fig. 6
, third panel). The expression of ß-actin mRNA used as control was similar in all samples (Fig. 6
, bottom panel).

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Fig. 6. Kinetics and profile of cytokine mRNA expression in purified NK cells cultured in the presence of anti-CD38, anti-CD16 or both. After 24, 36 and 48 h, mRNA was extracted and reverse transcribed. The cDNA obtained was used to assay for the presence of specific cytokine mRNA by PCR. Results refer to a representative experiment performed in triplicate.
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Cytokine induction was also quantitatively evaluated by assaying the protein product release (Fig. 7
). Time-course experiments in NK cells cultured in the presence of either anti-CD38 or anti-CD16 mAb indicate that the levels of IFN-
secreted become significant after 36 h (not shown). Thus, this time-point was selected for these experiments. CD38 engagement induced a modest increase in IFN-
secretion as compared to basal levels (P < 0.01); on the contrary, CD16 signaling was followed by a marked response (Fig. 7A
). Moreover, IFN-
release was not significantly increased when NK cells were simultaneously incubated with anti-CD38 and anti-CD16 mAb (Fig. 7A
). Additional effects were seen in the regulation of GM-CSF secretion (Fig. 7B
). Both CD38 and CD16, when independently engaged, give rise to increased levels of the cytokine (P < 0.01 for both stimulations as compared with control). The simultaneous signaling was paralleled by levels of GM-CSF secreted in the supernatant higher than those obtained when signals were given independently.

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Fig. 7. ELISA analysis of cytokine secretion in purified NK cells cultured in the presence of anti-CD38, anti-CD16 or both. After 36 h, culture supernatants were collected and assayed for IFN- (A) and GM-CSF (B) contents. Results are expressed as mean ± SD of three experiments performed.
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Cytotoxicity
The involvement of CD38 in the cytolytic function of NK cells was evaluated in a redirected cytotoxicity assay against target P815 cells (Fig. 8
). The assay was performed using NK treated with IL-2, since the lysis observed after ligating CD38 on resting NK was negligible. The results obtained indicate that CD38 signaling in IL-2-treated NK cells is followed by a triggering of cytolytic programs and the amount of specific lysis parallels E:T. CD16 signaling included as positive control also yielded a relevant lysis of target cells; in contrast, exposure of the same cells to an irrelevant isotype-matched binding mAb did not give rise to significant effects. The simultaneous addition of anti-CD16 and anti-CD38 mAb in the assay did not display additive or synergistic effects (not shown).

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Fig. 8. NK cytotoxicity via CD38. Purified NK cells were activated for 6 days with IL-2 (100 U/ml) and analyzed for their ability to lyse target murine P815 cells in a redirected cytotoxicity assay in the presence of anti-CD38, anti-CD16 or an irrelevant reactive isotype-matched mAb at different E:T. Results are expressed as mean ± SD of three experiments performed.
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Ultrastructural features of NK cells activated via CD38
The redirected cytotoxicity assay against target P815 cells was also used to study the ultrastructural changes occurring after CD38 triggering (Fig. 9
). Figure 9
(A) shows the ultrastructure of the target P815 cells. The mast cell lineage of the P815 cells is indicated by the presence of granules containing characteristic `scrolls'. Figure 9
(B) shows the structure of the IL-2-activated NK cells: granules containing abundant electron-dense matrix and tubular structures are present, as shown to better advantage in Fig. 9
(C). Morphological equivalent of activation are induced when NK cells were treated with IB4 (anti-CD38) mAb. Figure 9
(D) shows an effector to target contact: the activated NK cells lack electron-dense granules, which are replaced by vacuoles that are empty or contain scanty tubular structures. Simultaneously, apoptotic changes of target cells became apparent, indicating the ongoing cytotoxicity. Similar effects were observed following incubation with anti-CD16 mAb (not shown), as previously reported (44). P815 cells incubated with NK cells pre-treated with an irrelevant reactive isotype-matched mAb as negative control did not show any significant ultrastructural change on either side (not shown).

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Fig. 9. (A) Ultrastructure of the P815 murine mastocytoma cells used as targets (T) in the redirected killing assay (bar = 0.3 µm). (B) Ultrastructure of NK cells used as effectors (E) in the redirected killing assay (bar = 0.71 µm); cells were kept under the same experimental conditions as those of the assay. (C) Granules containing electron-dense matrix and tubular structures at higher magnification (bar = 0.26 µm). (D) An effector (E) to target (T) contact is shown in the redirected killing assay after treating NK cells with anti-CD38 mAb. Large, empty vacuoles are predominant in the effector cell (E) (arrows), some of which contain small tubular structures. No electron-dense granules are detected. The target cell (T) shows evidence of advanced apoptosis, as indicated by dispersed masses of strongly electron-dense chromatin (bar = 0.64 µm). The results shown are those obtained after incubating for 20 min the effector cells with the target P815 cells.
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Discussion
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The characteristic ability of human CD38 to operate as an ectoenzyme and also as an adhesion molecule involved in immune regulation was primarily described in T cell models, and was later observed also in the B and myeloid lineages. The role played by CD38 in NK cells has only been marginally studied and exclusively in terms of surface expression (42,45). More recently, CD38 has been shown to trigger lytic and secretory responses in IL-2-activated human NK cells, thus entering the list of receptors able to mediate natural cytotoxicity (23).
This paper reports on the results of concerted efforts to determine the CD38 functions as a receptor and a signaling molecule in NK cells. The capability of CD38 to deliver regulatory signals for key cellular functions in NK cells was our first working hypothesis. Such behavior is quite unexpected for two reasons: (i) because of its role as an ectoenzyme involved in the demolition of NAD+ and (ii) because of the apparent unsuitability of its cytoplasmic domain for transduction purposes. To investigate this issue, we monitored early (e.g. Ca2+ currents, phosphorylation of cytoplasmic substrates, expression of surface CD69 marker), intermediate (cytokine messages) and late events (expression of surface HLA class II and CD25, cytokine release, cytotoxicity).
Inferences derived from studies of T and B cells clearly indicate that CD38 exploits the signaling machinery of the TCR and, at least in murine models, the BCR respectively (8,9,16). Consequently, our second hypothesis postulated the existence of a surface signaling molecule in NK cells which co-operates with CD38. Our attention was focused on CD16, the only surface IgG-binding molecule expressed by NK cells (46). CD16 is involved in antibody-dependent cell cytotoxicity and is a signaling molecule which shares several structural and functional homologies with TCR (47). Further evidence for CD16 as a candidate for this task was the report on the existence of lateral associations with CD38 (42).
Results obtained in the present work confirm our first working hypothesis. Indeed, the CD38 molecule engaged by agonistic mAb operates as a receptor involved in the regulation of Ca2+ currents. Analysis of the Ca2+ profiles in resting NK cells indicates that CD38 ligation is followed by a longer-lasting signal than that elicited by CD16, in contrast with observations reported in IL-2-activated NK cells (23). Up-modulation of late surface activation antigens (i.e. HLA class II and CD25) was also efficiently induced through both CD38 and CD16, with additive effects in the case of CD25.
Analysis of the phosphorylation of selected cytoplasmic substrates showed that CD38 ligation was followed by phosphorylation of the CD3-
and Fc
RI
chains, ZAP-70 and of the proto-oncogene product c-Cbl. The transduction pathways followed were apparently similar to those described for CD16. CD16 signaling abilities are reported to rely upon non-covalent associations with disulfide-linked
and
chain homo- and heterodimers (35,36,38,41,4851), which are polypeptide subunits specialized in coupling to the intracytoplasmic transduction machinery (47). These subunits express ITAMs in their intracytoplasmic domain which are phosphorylated by Lck upon CD16 triggering (52); thus, they are capable of recruiting and activating the SH2 domains of ZAP-70/Syk (39,5356) and shc (36). Following the multiple cascades initiated via these early phosphorylation steps, CD16 stimulation leads to proximal responses such as increases in intracellular Ca2+ concentration (31) and release of intracytoplasmic NK granuli as well as distal responses, such as gene transcription and expression of activation molecules (i.e. CD25) and lymphokines (i.e. IFN-
) (34,43). Further evidence of CD38 signaling was provided by the analysis of the effects on cytokines selected from among those playing a role in the NK cell economy. The messages for IFN-
and GM-CSF were clearly influenced by the signals delivered by CD38 ligation, even if to a lesser extent than that triggered via CD16. Further, CD38 signaling was followed by the release of appreciable amounts of IFN-
, while it was more efficient in enhancing GM-CSF release; in the latter case, the combined effects of CD38 and CD16 ligations were additive.
The last issue considered was the influence of CD38 signaling on cytolytic functions, the most relevant biological effects driven by NK cells. The signals elicited by CD38 ligation were followed by significant lysis of the target cells. Such effects were visible on IL-2-activated NK cells and paralleled the effects induced via CD16, although to a lesser extent. These events were also documented at ultrastructural levels by electron microscopy. The CD38-driven lytic functions required IL-2 activation of the effector cells, a feature shared by other receptors (57). The IL-2 requirement is likely due to the de novo synthesis of proteins that link selected surface molecules to the lytic machinery in NK cells (23).
The data obtained allow us to conclude that CD38 acts as a receptor capable of delivering potent signals in NK cells which influence many aspects of the cell economy. These range from molecular modifications decoding the transmission of signals to the inside of the cell (i.e. Ca2+ release and protein tyrosine phosphorylation) to complex cellular events (i.e. modulation of surface molecules, cytokine release and cytotoxicity) indicating successful delivery of the triggered signals to their final targets. An open issue is whether the effects triggered by means of anti-CD38 agonistic mAb can also be elicited by the natural ligand(s) of CD38, either CD31 or NAD+, as demonstrated for CD31 in other cellular models (6).
The present findings also give preliminary support to our second hypothesis of a possible interplay between CD38 and CD16, but conclusive inferences cannot yet be drawn. It is possible that CD38 in its million year evolution from a cytoplasmic enzyme to a surface molecule developed a simple and utilitarian strategy based on symbiosis with other molecules specialized in signaling (58). The resulting behavior of CD38 demonstrates a clever ability to change its partner according to cell lineage. Indeed, the inefficiency of CD38-mediated Ca2+ release in CD16 NK cell lines and the similarities in the cellular events and phosphorylation cascades elicited by the two receptors suggest that the signaling companion adopted by CD38 in NK cells may be CD16. The two signaling pathways might merge either at the level of CD16 itself or downstream of CD16. A more precise definition of this issue will be the immediate follow-up of the present investigation: experiments are currently in progress in our laboratory to assess whether CD16 transfection in these CD16 NK cell lines is able to reconstitute the signaling properties.
A likely scenario is where CD38 has the ability to take part in the reorganization of the membrane structure, leading to an enrichment in microdomains which are rich in kinases and adaptor molecules on the inner side, and in the molecules involved in signaling on the outer side (59). A structure similar to immunological synapses could be envisaged for NK cells (60,61); this might include active participation of the cytoskeleton (62), as already demonstrated in the case of CD38 (63). The attribution of a precise co-localization of CD38 in such structures, which are gaining relevance in co-stimulatory signals, will constitute further follow-up of this research.
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Acknowledgments
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Work supported by grants from AIRC (Milano, Italy), Telethon (Roma, Italy), Special Projects `AIDS' (Istituto Superiore di Sanità, Roma, Italy) and `Biotechnology' (CNR/MURST, Roma, Italy) (to F. M.) and MURST (Roma, Italy); CICYT Grants SAF96-0117 and SAF99-0024 (to J. S.); Bilateral projects CSIC/CNR 97/98 (to J. S. and F. M.); and CSIC/CNR 99 (to M. Z.). R. M. was a Telethon Fellow and now is at the Postgraduate School of Internal Medicine, University of Torino, Torino, Italy. G. B. was a Fellow of the `G. Ghirotti' Foundation. M. Z. is supported by a Contract of Incorporation from the Ministry of Education and Culture, Spain. The Regione Piemonte, the Cariverona (Verona, Italy) and Compagnia SanPaolo (Torino, Italy) provided valuable financial contributions.
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Abbreviations
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cADPR cyclic ADP ribose |
[Ca2+]i intracellular Ca2+ concentration |
G MIgG goat anti-mouse IgG |
GM-CSF granulocyte macrophage colony stimulating factor |
HRP horseradish peroxidase |
ITAM immunoreceptor tyrosine-activation motif |
NAD+ nicotinamide adenine dinucleotide |
PBMC peripheral blood mononuclear cells |
PI 3-K phosphatidyl inositol 3-kinase |
PLC phospholipase C |
pTyr phosphotyrosine |
TNF tumor necrosis factor |
ZAP-70 -associated protein 70 |
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Notes
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Transmitting editor: C. Terhorst
Received 11 September 2000,
accepted 7 December 2000.
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