CD38 is functionally dependent on the TCR/CD3 complex in human T cells

^a Laboratory of Cell Biology, Department of Genetics, Biology and Medical Chemistry
^b Postgraduate School of Clinical Pathology, University of Torino Medical School, 10126 Torino, Italy
^c Instituto de Parasitología y Biomedicina, Consejo Superior de Investigaciones Científicas, 18001 Granada, Spain
^d Division of Immunology, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
^e Institute of Biology and Genetics, University of Ancona Medical School, 60131 Ancona, Italy

	ABSTRACT

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One of the functions of surface CD38 is the induction of phosphorylation of discrete cytoplasmic substrates and mobilization of cytoplasmic calcium (Ca²⁺). The present work addresses the issue of whether the signaling mediated via CD38 operates through an independent pathway or, alternatively, is linked to the TCR/CD3 signaling machinery. We studied the signals elicited through CD38 by the specific agonistic IB4 monoclonal antibody (mAb) by monitoring the levels of cytoplasmic Ca²⁺ and the induced phenotypic and functional variations in T cell growth. IB4 mAb presented the unique ability to increase cytoplasmic Ca²⁺ levels, which correlated with the phosphorylation of the PLC-1. These effects were blocked by phorbol 12-myristate 13-acetate (PMA) and were dependent on the presence of a functional TCR/CD3 surface complex, no effects being recorded on mutant Jurkat cells lacking part of the CD3 structures. CD38 signaling appeared to share with TCR/CD3 the ability to induce apoptotic cell death in Jurkat T cells, an event paralleled by specific up-regulation of the Fas molecule and inhibited by cyclosporin A. CD28, a costimulatory molecule, is synergized by increasing CD38-induced apoptotic cell death. The results indicate the existence of a strong functional interdependence between CD38 and TCR/CD3.—Morra, M., Zubiaur, M., Terhorst, C., Sancho, J., Malavasi, F. CD38 is functionally dependent on the TCR/CD3 complex in human T cells. FASEB J. 12, 581–592 (1998)

Key Words: T lymphocytes • cell surface molecules • second messengers • signal transduction • apoptosis

	INTRODUCTION

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AFTER ITS IDENTIFICATION in the early 1980s (1, 2), many functions have been attributed to human CD38, a multilineage type II transmembrane glycoprotein of 45 kDa. CD38 was initially described as being prevalently expressed by premature or terminally differentiated lymphoid cells (1, 3) and used extensively as a marker in the systematic classification of hematopoietic malignancies (4). Recently, it was reported to be expressed by other tissues: pancreas (5), brain (6), muscle, and other organs (J. E. Fernandez et al., unpublished results). Enzymatic activities, a role in adhesion, and involvement in cell signaling are key properties of CD38 (7). The observation that human CD38 displays an amino acid sequence similar to the enzyme ADP-ribosyl cyclase purified from Aplysia californica (8, 9) was followed by the demonstration of enzymatic activities of CD38. Indeed, CD38 is a bifunctional ectoenzyme with ADP-ribosyl cyclase and hydrolase activities, leading to the conversion of NAD⁺ to cyclic ADP-ribose (cADPR)² and to the hydrolysis of cADPR to ADP-ribose (ADPR) (10). This enzymatic function has relevant biological implications, since cADPR has been shown to regulate Ca²⁺ levels both in sea urchin egg homogenates (11) and in detergent-permeabilized T lymphocytic cell lines (12).

A second important feature of the CD38 molecule is its role in the regulation of heterotypic adhesion, mainly between lymphocytic and endothelial cells, where it displays selectin-like features (13). Such an initial finding was central to the identification of a CD38 ligand (CD38L), a molecule prevalently expressed by endothelial cells, platelets, macrophages, and a discrete subset of T cells (14). The characteristic that rendered CD38 an activation molecule is its expression and role during activation processes (3, 15–17). These conclusions were inferred from the effects induced on ligation by agonistic monoclonal antibodies (mAb's). Transmembrane signaling via CD38 operates by regulating either proliferation or growth arrest, according to the microenviroment or different maturation stages (18, 19). Other signaling activities were identified in HL-60 cells treated with retinoic acid, where CD38 ligation is followed by tyrosine phosphorylation of cellular proteins (e.g., the c-cbl proto-oncogene) (20). CD38 ligation in B cells is followed by a rapid and transient tyrosine phosphorylation of several intracellular proteins, such as the protein kinase syk, the p85 subunit of phosphatidylinositol-3 kinase, and phospholipase C- (PLC-) (21). Recently, it was reported that CD38 ligation in T cells results in phosphorylation of the Raf-1/MAP kinase and CD3-/ZAP-70 signaling pathway (22).

The idea behind this work was to determine whether CD38 operates through an independent pathway or shares some or many steps with the T cell receptor (TCR)/CD3 pathway, as hinted at by the physical association reported for the two molecules in T cells (23). The working hypothesis is that CD38 is a component of a multireceptorial complex that includes TCR/CD3 in human T cells. The strategy adopted relied on an evaluation of the ability of CD38 to modulate Ca²⁺ levels and interfere with the effects implemented via TCR/CD3 and one of the most-studied coreceptors, CD28. We were able to assess the relative contributions of CD38 signaling to Ca²⁺ mobilization, phenotypic variations, activation/proliferation, and apoptosis. The conclusions of this work indicate that CD38 signaling is dependent on expression of the TCR/CD3 complex, is mediated by downstream components shared with this activation pathway, and triggers apoptotic cell death.

	MATERIALS AND METHODS

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Monoclonal antibodies and reagents
IB4 [anti-CD38, immunoglobulin 2a (IgG2a)] (24), IB6 (anti-CD38, IgG2b) (25), SUN-4B7 (anti-CD38, IgG1), CBT3-G (anti-CD3, IgG1) (26), CB19 (anti-CD19, IgG1), and Vß8 (anti-TCR ß8 chain, IgG1) are mAb's produced in the laboratory. The F(ab')₂ fragmentation of the IB4 mAb was obtained as previously described (3). OKT10 (anti-CD38, IgG1) is from the American Type Culture Collection (Rockville, Md.). CD28.2 (anti-CD28, IgG) was kindly provided by Dr. B. Malissen (Marseille, France). OKT3 (anti-CD3, IgG1) was purchased from Ortho-Pharmaceuticals (Raritan, N.J.) and Leu¹⁷ (anti-CD38, IgG1) is from Becton Dickinson (Milano, Italy). All mAb's used for functional tests were prepared and purified by affinity chromatography on protein A-Sepharose and high-performance liquid chromatography on hydroxylapatite, as described (26). The anti-phosphotyrosine mAb (anti-pTyr, IgG2a) was obtained from Oncogene Research (Calbiochem, Cambridge, Mass.), and the anti-PLC-1 is a rabbit polyclonal antibody from Sigma (Milano, Italy). Anti-CD38 Leu¹⁷, CD3, CD5, CD28, CD69, and CD95 mAb's (directly conjugated with FITC or PE) were purchased from Becton Dickinson; HIT2, another anti-CD38 mAb, was kindly provided by Mr. B. Johnson (Caltag Laboratories, San Francisco, Calif.).

Cell lines
Jurkat cells were used as a wild-type line or as clones derived by limiting dilution: the selection procedure was repeated at least three times to obtain phenotypically homogeneous cells expressing high amounts of surface CD38. Other clones used were Jurkat 31–13 (TCR ß chain-defective and consequently CD3^-, CD38⁺) and Jurkat B7 (a clone derived from 31–13 after transfection with a TCR ß chain construct, and consequently CD3⁺, CD38⁺) (27, 28). The clones were made available by Dr. A. Alcover (Institut Pasteur, Paris, France) and Dr. B. Alarcón (Centro de Biologia Molecular, Madrid, Spain). Cells were cultured in RPMI-1640 medium (Sigma) with 10% heat-inactivated fetal calf serum (FCS) (Hyclone, Logan, Utah), L-glutamine, 100 units/ml penicillin, and 100 units/ml streptomycin (hereafter referred to as complete medium) in a humidified 5% CO₂ incubator at 37°C.

Modulation of surface molecules by soluble or immobilized mAb's
Cells were cultured at 37°C for the selected time intervals in complete medium in the presence of soluble or immobilized mAb's. The carrier with immobilized mAb's was prepared by coating 24-well culture plates (Costar, Milano, Italy) with polyclonal goat anti-mouse IgG Fc antibodies (50 µg/ml) (Cappel, West Chester, Pa.) for 1 h at 37°C. Stimulating mAb's or isotype-matched control mAb's were added successively, followed by incubation for 3 h at 4°C. Antibodies were cross-linked by incubation with 0.1 mM dimethylpimelidate (Sigma) for 1 h at 20°C. Finally, plates with immobilized antibodies were treated with 0.1 M glycine in phosphate-buffered saline (PBS) (pH 7.2) to quench residual reactive groups. The stimulated cells were collected, washed twice, and used for the selected tests.

Flow cytometry analysis for cell phenotyping
Cells were resuspended in PBS containing 0.2% bovine serum albumin (BSA) and 0.1% sodium azide (radioimmunoassay buffer) and incubated with mAb for 1 h at 4°C. After washing twice with radioimmunoassay buffer, cells were incubated (30 min at 4°C) with FITC-conjugated F(ab')₂ goat anti-mouse Ig (Silenus, Hawthorn, Australia). 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 Lysis II (Becton Dickinson) software. Conversion of acquired data to channel linear values was performed according to Schmidt et al. (29).

Measurement of cytoplasmic Ca²⁺
Cytoplasmic Ca²⁺ was measured by using the Fluo-3/AM (Sigma) fluorescent calcium marker. Cultured cells exposed to mAb's or other treatments were collected, washed twice with PBS, and incubated for 30 min at 37°C in RPMI-1640 medium containing Fluo-3/AM (final concentration: 4 µM). Cells were then washed three times and kept at 4°C in the dark. Before assay, cells were incubated 5 min at 37°C and successively analyzed on a FACSort. After measuring the basal level of fluorescence, mAb's were added to samples and cell events acquired continuously. The median channel value of fluorescence was calculated with the Lysis II software collecting 2000 events acquired every 20 s (30). The calcium ionophore A23187 (Sigma) was used as an indicator of cell loading by the fluorescent calcium marker. In extracellular Ca²⁺ chelation experiments, Jurkat cells loaded with Fluo-3/AM were incubated (5 min, room temperature, in the dark) with 5 mM EGTA (Sigma) and subsequently analyzed for Ca²⁺ mobilization.

Electrophoresis, Western blotting, and PLC-1 analysis
Cells were grown to a density of up to 10⁶/ml, centrifuged and serum starved for 4 h in RPMI-1640 medium + 0.5% FCS, washed in PBS, and resuspended (1–2x10⁷ cells/sample) in serum-free RPMI-1640 medium at 4°C. Relevant mAb's were added for 10 min on ice, followed by incubation at 37°C for an additional 1 min, as reported in detail in ref 22. Cells were then lysed in 1% NP-40 lysis buffer [20 mM Hepes (pH 7.6), 150 mM NaCl, 50 mM NaF, 1 mM Na₃VO₄, 1 mM EGTA, 50 µM C₆H₅AsO, 10 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride and 2 µg/ml each of the protease inhibitors antipain, chymostatin, leupeptin, and pepstatin] for 15 min on ice. Nuclei were removed by centrifugation at 12,000 g for 15 min at 4°C. Laemmli sample buffer was added to aliquots of whole-cell lysates or immunoprecipitates and mixtures were heated at 90°C for 5 min. Samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a mini-gel (Hoefer, San Francisco, Calif.). Proteins were electrophoretically transferred to Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, Mass.), using a semi-dry transfer apparatus (Hoefer) in a continuous buffer system (39 mM glycine, 48 mM Tris, 20% methanol) for 1–2 h at a constant current of 0.8 mA/cm². The membranes were blocked by incubation for 1 h at room temperature in 1% BSA (washing buffer). All primary antibodies were added to the washing buffer for 1–3 h at room temperature. The polyclonal anti-PLC-1 antibody was used at 1:7,000 dilution. The filters were washed extensively between incubations with washing buffer. Blots were developed by chemiluminescence using the ECL (Amersham, Little Chalfont, U.K.) detection system and then exposed to Hyperfilm-ECL (Amersham). For reprobing, PVDF membranes were incubated for 10 min at room temperature in stripping buffer [0.2 M glycine (pH 2.2), 0.1% SDS, 1.0% Tween-20], followed by cycles of washing buffer.

Assessment of apoptosis
The percentage of apoptotic cells was determined by flow cytometry analysis, as previously described (31), by evaluating: 1) the modification of scatter parameters in freshly isolated cultured cells incubated with or without the relevant mAb's, and 2) the percentage of hypodiploid nuclei after gating on DNA content in cells treated with the same mAb's or left untreated. Briefly, cultured cells were collected, washed twice in PBS, and resuspended in hypotonic fluorochrome solution [50 µg/ml of propidium iodide (Sigma), 0.1% sodium citrate and 0.1% Triton X-100] for 4–8 h at 4°C in the dark (31). Cell samples were acquired and analyzed with Lysis II and CellFit software (Becton Dickinson).

	RESULTS

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Induction of increased intracellular Ca²⁺ levels by agonistic anti-CD38 mAb
Mobilization of cytoplasmic Ca²⁺ was studied in wild-type Jurkat cells (CD38⁺, CD3⁺) after ligation of CD38 by mAb's IB4, IB6, SUN-4B7, and the reference OKT10, respectively. As shown in Fig. 1A, IB4 was the only mAb able to induce increased levels of cytoplasmic Ca²⁺. This anti-CD38 mAb-induced Ca²⁺ mobilization was dose dependent and clearly detectable at mAb concentrations of 500 ng/ml (data not shown). Higher concentrations yielded increased effects, as inferred from the fluorescence channel intensity, and reduced the latency of the response. The optimal concentration was 5 µg/ml, used throughout this study. No significant differences were seen between the intact IgG and F(ab')₂ fragments of the IB4 mAb. Figure 1B (left side) shows the results of the phosphorylation of PLC-1 after ligation of CD38 and CD3, respectively. Treatment with IB4 mAb is followed by marked effects on tyrosine phosphorylation of a 150 kDa protein in whole-cell lysates (upper left panel, lane 3). This protein comigrated with PLC-1 (upper right panel, lane 3). Similar effects were obtained by using OKT3 (upper right and left panel, lanes 2). The control OKT10 (an mAb ineffective on Ca²⁺ mobilization) has no apparent effect on PLC-1 tyrosine phosphorylation (bottom, lanes 3).

View larger version (63K): [in this window] [in a new window]   Figure 1. The unique ability of the CD38-specific mAb IB4 to induce cytoplasmic Ca2+ mobilization and tyrosine phosphorylation of a 150 kDa protein in Jurkat T cells. A) Mobilization of cytoplasmic Ca2+ in wild-type Jurkat cells (CD38+, CD3+) after CD38 ligation by IB4 (), IB6 (), SUN-4B7 (), and OKT10 () mAb or after CD3 ligation by CBT3-G () mAb. Jurkat cells were labeled with Fluo-3/AM and analyzed on a FACSort cytofluoremeter. Y axis: median channel value of fluorescence. X axis: time in seconds (s). Each point represents 2000 events acquired at 20 s intervals, adding the relevant mAb at time 0. The results represented are derived from one of three independently performed experiments. B) Left: tyrosine phosphorylation of a 150 kDa protein by CD38 or CD3 ligation in wild-type Jurkat cells (CD38+, CD3+). Cells were serum-starved for 4 h in RPMI-1640 medium + 0.5% FCS, washed in PBS, resuspended at 1–4 x 107 cells/sample in serum-free RPMI-1640 medium, warmed at 37°C for 10 min, and then treated for 3 min at 37°C with IB4 mAb (lane 3, top), OKT10 (lane 3, bottom), or OKT3 (lane 2, top and bottom) or were nonstimulated (NS; lane 1, top and bottom). This was followed by cross-linking with the F(ab')2 fraction of a goat anti-mouse IgG antibody for 2 min at 37°C. 1% NP-40 lysates were resolved by 10% SDS-PAGE under reducing conditions and transferred to a PVDF membrane. The filter was probed with an anti-phosphotyrosine mAb. The position of the molecular mass markers is indicated on the right. Right: the same filters as portrayed on the left side were stripped and reprobed with a polyclonal anti-PLC-1 antibody.

Identification of the 150 kDa phosphoprotein as PLC-1
To formally identify the 150 kDa phosphoprotein as PLC-1, Jurkat cells were first treated with IB4 and control OKT3 mAb's, and successively immunoprecipitated with a polyclonal antibody specific for PLC-1. The samples underwent Western blot analysis with an mAb specific for pTyr. Figure 2 (upper panel) shows a marked increase of the phosphorylated protein upon mAb triggering, either in lysates or in immunoprecipitates. The same samples used for the above experiment were stripped and reprobed with the anti-PLC-1 antibody. Results ( Fig. 2, lower panel) clearly show that the phosphorylated protein is PLC-1, in either lysates or immunoprecipitates.

View larger version (70K): [in this window] [in a new window]   Figure 2. Tyrosine phosphorylation of PLC-1 induced by CD38 or CD3 ligation in wild-type Jurkat cells (CD38+, CD3+). Cells were stimulated for 1 min at 37°C with anti-CD38 mAb IB4+F(ab')2 GmIg (lanes 3 and 5), anti-CD3 mAb OKT3+F(ab')2 GmIg (lanes 2 and 5), or were unstimulated (NS) (lanes 1 and 4), as described (22). Whole-cell lysates (lanes 1–3) or anti-PLC-1 immunoprecipitates (IP) (lanes 4–6) were separated on 10% SDS-PAGE and probed by Western blotting (WB) with an anti-phosphotyrosine (pTyr) antibody (upper panel), followed by stripping and reprobing with an anti-PLC-1 peptide antiserum (lower panel). Immunoblotting was performed with the ECL system (Amersham). Position of PLC-1 is indicated.

Characterization of Ca²⁺ mobilization induced after CD38 ligation
EGTA, a chelator of extracellular Ca²⁺, was used to determine whether the Ca²⁺ mobilized after CD38 ligation derives from intracellular or extracellular compartments (32). Figure 3 shows that in the presence of 5 mM EGTA, there was still a significant increase in the concentration of intracellular Ca²⁺ upon CD38 ligation by IB4 mAb (panel a) or CD3 ligation by CB3T-G mAb (panel b). This initial increase in intracellular Ca²⁺ was followed by a faster drop to basal levels vs. cells stimulated in the absence of EGTA. This suggests that the initial Ca²⁺ response to CD38 ligation derives from intracellular Ca²⁺ stores, whereas the later and more substained Ca²⁺ release comes from an influx from the extracellular compartment. EGTA did not influence the binding of mAb's to CD38 (data not shown).

View larger version (24K): [in this window] [in a new window]   Figure 3. Effect of EGTA or PMA on the CD38-mediated increase in intracellular Ca2+. a, b) Substained Ca2+ mobilization induced via CD38 derives from an extracellular influx. Increases in intracellular Ca2+ after ligation of CD38 (a) or CD3 (b) molecules in Jurkat cells in the presence () or absence () of EGTA (5 mM). Y axis: median channel value of fluorescence. X axis: time in s. Each point represents 2000 events acquired at 20 s intervals, adding the relevant mAb at time 0. The results represented are derived from one of three independently performed experiments. c, e) Blocking of CD38-induced Ca2+ fluxes by PMA. Ca2+ mobilization after ligation of CD38 (c) or CD3 (e) molecules in Jurkat cells treated () or not treated () with PMA (10 ng/ml, 2 h). Cultured Jurkat cells were labeled with Fluo-3/AM and analyzed on a FACSort cytofluoremeter. Y axis: median channel value of fluorescence. X axis: time in s. Each point represents 2000 events acquired at 20 s intervals, adding the relevant mAb at time 0. Overlaid histograms represent CD38 (d) or CD3 (f) surface expression profiles in cells treated with PMA (solid histograms) vs. untreated cells (open histograms) (Y axis, number of events; X axis, fluorescence intensity). The results represented are derived from one of three independently performed experiments.

PMA, an activator of protein kinase C, induces a decrease of inositol-3-phosphate, leading to a significant inhibition of mobilization of cytoplasmic Ca²⁺ via CD3 and CD2 in Jurkat cells (33, 34). Treatment of Jurkat cells for 2 h in the presence of PMA (10 ng/ml) is followed by a complete suppression of the CD38-induced Ca²⁺ response ( Fig. 3c). Under the same conditions, the CD3-induced Ca²⁺ response is indeed only partially diminished ( Fig. 3e). Control experiments show that the addition of PMA does not significantly affect CD38 expression ( Fig. 3d), whereas surface CD3 is strongly reduced ( Fig. 3f). These results rule out the possibility that the refractoriness to CD38 signaling observed after PMA is secondary to a decreased density of surface CD38 molecules.

Regulation of cell surface expression of membrane proteins after CD38 ligation
To assess whether the surface lateral associations related to CD38 are relevant for the signaling machinery, we tried to correlate CD38 and TCR/CD3 comodulation with Ca²⁺ mobilization ability by using a panel of mAb's specific for different epitopes of the CD38 molecule. The ability of the IB4 mAb to down-regulate the CD3 molecule ( Fig. 4, left) correlated with its potential to increase cytoplasmic Ca²⁺. The relationship of CD38 with TCR/CD3 was further investigated by analyzing its ability to regulate other surface molecules functionally related to TCR/CD3. CD69, a molecule inducible after prolonged Ca²⁺ mobilization via TCR/CD3, becomes detectable after Jurkat cells are treated for 4 h in the presence of IB4 mAb ( Fig. 4, right, full bars). The induction of CD69 was a unique feature of IB4 mAb: its expression was comparable in intensity to that induced by the anti-CD3 treatment. Chelation of extracellular Ca²⁺ by EGTA ( Fig. 4, right, striped bars) inhibited the effects induced by IB4 mAb, as did CBT3-G mAb. No significant differences were seen when using F(ab')₂ fragments of the IB4 mAb.

View larger version (21K): [in this window] [in a new window]   Figure 4. Mobilization of intracellular Ca2+ by the anti-CD38 mAb IB4 correlates with down-modulation of CD3 and up-regulation of CD69 surface expression. Histograms represent the median fluorescence values of CD3 or CD69 surface expression upon cell treatment with various anti-CD38 mAb's (IB4, IB6, SUN-4B7, and OKT10). The results were compared with the levels obtained by treating Jurkat cells with an irrelevant anti-CD19 (CB19) mAb or an anti-CD3 (CBT3-G) mAb. Solid bars refer to cells treated with no EGTA in the medium; striped bars refer to cells treated with EGTA (2 mM) in the medium. The results represented are derived from one of three independently performed experiments.

CD38 signaling and expression of the TCR/CD3 complex
The next issue was to analyze the effects of CD3 modulation on the Ca²⁺ mobilization observed after CD38 ligation. Jurkat cells were cultured for 12 h on plates coated with anti-CD3 mAb, washed, and then labeled with Fluo-3/AM. CD3-modulated cells failed to mobilize Ca²⁺ after CD38 ligation (data not shown). CD38 expression in the same cells was only slightly reduced, although not in proportion to the complete ablation of the Ca²⁺ response observed. Ca²⁺ mobilization induced after CD3 ligation was maintained in the reciprocal experiments (CD38-modulated cells), even if reduced quantitatively (data not shown).

The initial observations of a linkage between CD38 signaling and the TCR/CD3 complex was investigated in depth using Jurkat clones with selected characteristics. The Jurkat clone 31–13, which does not express CD3 on the cell surface due to a defective expression of the TCR ß chain and CD38⁺ (see phenotype in the insets of Fig. 5A), was refractory to Ca²⁺ mobilization inducible via CD38 ( Fig. 5A). Figure 4B reports that anti-CD38 or anti-CD3 treatments of this clone failed to induce CD69 expression. In contrast, Ca²⁺ mobilization induced via CD38 (and via CD3) and CD38-mediated CD69 surface expression were restored in the Jurkat clone B7, which is derived from the clone 31–13 after reconstitution of a functional TCR/CD3 complex on the cell surface upon appropriate cDNA transfer of the TCR ß chain ( Fig. 6A, B). Moreover, the increase of intracellular Ca²⁺ in B7 cells after CD38 ligation was followed by a marked down-modulation of surface CD3 molecules, as observed in wild-type Jurkat cells ( Fig. 6B, bottom histogram).

View larger version (19K): [in this window] [in a new window]   Figure 5. Absence of CD38-mediated signaling in TCR/CD3 defective Jurkat cells. A) Ca2+ mobilization in the Jurkat clone 31–13 (CD38+CD3-) after ligation of CD38 () or CD3 () molecules. Y axis: median channel value of fluorescence. X axis: time in s. Each point represents 2000 events acquired at 20 s intervals, adding the relevant mAb at time 0. A23187 (1 mM) was used as ionophore. Histograms on the right show the surface expression levels of CD38 and CD3 in 31–13 cells (Y axis: number of events; X axis: fluorescence intensity). The results represented are derived from one of three independently performed experiments. B) Lack of phenotypic variation in the Jurkat clone 31–13 (CD38+, CD3-) after ligation of CD38 (top row) or CD3 (middle row) molecules with IB4 or CB3T-G mAb's, respectively. The bottom histogram represents the CD3 surface expression before and after anti-CD38 treatment. Overlaid histograms represent the cell surface expression profiles of Jurkat cells cultured with the specific mAb (open histograms) vs. cells cultured with an irrelevant anti-CD19 (CB19) (full histograms). Results are derived from one of three independently performed experiments.

View larger version (20K): [in this window] [in a new window]   Figure 6. Restoration of CD38-mediated signaling after transfection of TCR ß chain cDNA into the TCR/CD3 defective Jurkat cell clone. A) Ca2+ mobilization in the Jurkat clone B7 (CD38+CD3+) after ligation of CD38 () or CD3 () molecules. Y axis: median channel value of fluorescence. X axis: time in s. Each point represents 2000 events acquired at 20 s intervals, adding the relevant mAb at time 0. A23187 (1 mM) was used as ionophore. Histograms on the right show the surface expression levels of CD38 and CD3 in B7 cells (Y axis: number of events; X axis: fluorescence intensity). The results are derived from one of three independently performed experiments. B) Selective induction of CD69 surface expression and down-modulation of CD3 in the Jurkat clone B7 (CD38+, CD3+) after ligation of CD38 (top row) or CD3 (middle row) molecules. The bottom histogram shows down-modulation of CD3 surface expression upon anti-CD38 treatment. Overlaid histograms represent the profiles of Jurkat cells cultured with the specific mAb's (open histograms) vs. cells cultured with an isotype-matched anti-CD19 (CB19) (full histograms). Results are derived from one of three independently performed experiments.

Apoptosis and CD38 ligation in Jurkat cells
The next step was a comparative analysis of the biological effects implemented via CD38 vs. those reported via CD3 (35, 36); one of these effects includes the apoptotic signals evaluated in wild-type Jurkat cells or in selected clones. In Jurkat cells, CD38 ligation with some specific mAb's induced a significant increase in the rate of apoptotic cell death ( Fig. 7). The effect of these mAb's on apoptosis correlated with their ability to mobilize cytoplasmic Ca²⁺; indeed, cell death was observed only after incubation with IB4 mAb ( Fig. 7A). No significant differences were seen when using F(ab')₂ fragments of the IB4 mAb (data not shown). Figure 7B shows the results of the cytofluoremeter analysis of Jurkat cells cultured 20 h in the presence of IB4 mAb. A consistent fraction of these cells (~15 to 30% in the different experiments) presented morphological parameters typical of apoptosis ( Fig. 7B, R1 region in the inset) and DNA fragmentation, as assessed by the percentage of hypodiploid nuclei ( Fig. 7B, M1 area in the histogram). Experimental spontaneous cell death was < 7%, a score similar to that obtained in cultures treated with isotype-matched control mAb (data not shown).

View larger version (14K): [in this window] [in a new window]   Figure 7. CD38-mediated signaling induces apoptotic cell death in Jurkat cells. A) Comparative analysis of the ability of different anti-CD38 mAb's to induce apoptosis in Jurkat cells. Bars refer to the percentage of specific cell death after substructing the percentage of spontaneous cell death. Spontaneous or specific cell death was defined as the percentage of hypodiploid nuclei in Jurkat cells treated for 20 h at 37°C with the irrelevant anti-CD19 mAb or the specific anti-CD38 mAb, respectively. The results are derived from one of three independently performed experiments. B) Apoptotic effects observed in Jurkat cells after CD38 ligation with the IB4 mAb. Histogram shows the DNA content of nuclei of cells stained with propidium iodide upon IB4 treatment (X axis: FL2-H fluorescence intensity; Y axis: number of events). M1 depicts the area of hypodiploid nuclei. Contour plot (inset) shows the scatter parameters of the IB4-treated cells before DNA staining (X axis: forward scatter; Y axis: side scatter). R1 and R2 represent dead and alive cells, respectively. The percentage of cells with the scatter parameters of R1 (dead cells) closely correlate with the percentage of hypodiploid nuclei in M1 (see refs 31 and 35). 10,000 events were acquired and analyzed for each figure. Results are derived from one of three independently performed experiments.

Cyclosporin A (CsA) and CD38-mediated apoptosis
CsA is an immunosuppressive drug that interferes with a Ca²⁺-sensitive signal transduction pathway involved in apoptosis induced via TCR/CD3 or CD2 in T cells (35, 37). Likewise, treatment with CsA (100 ng/ml) in Jurkat cells resulted in a strong inhibition in the percentage of dead cells induced upon stimulation with the anti-CD38 mAb IB4 mAb for 20 h (10% of dead cells in CsA-treated cells vs. 24% in untreated cells, Fig. 8A vs. Fig. 8B). The inhibitory effect of CsA on IB4-mediated apoptosis was dose dependent, being detectable at concentrations as low as 10 ng/ml ( Fig. 8D). As expected, in Jurkat cells incubated with OKT10 mAb, an mAb that did not induce increases in the intracellular concentration of Ca²⁺, there were no changes in the presence of CsA in the percentage of dead cells (only 5% of dead cells, as measured by modification of the scatter parameters; see insert in Fig. 8C).

View larger version (39K): [in this window] [in a new window]   Figure 8. Inhibition of CD38-mediated apoptosis by cyclosporin A (CsA). Jurkat cells were cultured for 20 h in the presence (A, C) or absence (B) of 100 ng/ml of CsA after CD38 ligation by the IB4 mAb (A, B, D) or the OKT10 mAb. Histograms show the DNA content of the cell nuclei upon mAb treatment (X axis: FL2-H fluorescence intensity; Y axis: number of events). Dot plot (inset) shows the scatter parameters of the mAb-treated cells before DNA staining (X axis: forward scatter; Y axis: side scatter). 10,000 events were acquired and analyzed for each figure. The percentage numbers in the insets represent the percentage of dead cells as defined in Fig. 7. D) A dose-response experiment on the influence of CsA concentrations on IB4-induced apoptosis in Jurkat cells (X axis: CsA concentration in ng/ml; Y axis: percentage of inhibition of specific cell death in the presence of CsA, calculated with the formula: The results are derived from one of three independently performed experiments.

Influence of CD28 on CD38 signaling
Previous experiments showed that a culture of Jurkat T cells for 48 h in the presence of the IB4 mAb induce a marked up-regulation of CD28, similar to that observed after CD3 signaling. The hint of a functional linkage between the molecules prompted us to evaluate the influence of CD28 on apoptosis mediated by CD38. CD28 is a costimulatory molecule (38) whose action increases either the proliferation (39) or apoptosis (40, 41) observed after mAb ligation of TCR/CD3. Figure 9a shows the results of apoptosis (quantitatively assessed as percent of cell death over background deaths) induced after anti-CD38 (lane 1), anti-CD3 (lane 2), anti-TCR (lane 3), and anti-CD28 (lane 4) mAb treatment of Jurkat cells. An isometric contour plot (bottom) indicates the scatter parameters. Figure 9b shows that CD28 ligation amplified the effects on apoptosis via CD38 (lane 5). CD28 ligation per se failed to induce significant apoptosis in Jurkat cells (lane 4). As expected, the increased apoptosis seen after CD38 ligation was also observed when the cells were stimulated via TCR or CD3 and simultaneously via CD28 ( Fig. 9b, lanes 6 and 7, respectively).

View larger version (19K): [in this window] [in a new window]   Figure 9. CD28-mediated signaling potentiates in Jurkat cells the CD38-induced apoptosis. Upper panel: Bars depict the percentage of cell death over background as defined in Fig. 7. mAb's were used alone (a) or simultaneously in different combinations (b). A total mAb concentration of 10 µg/ml was used for each experimental condition. In all experiments, CB19 mAb (anti-CD19) was used as the control mAb to measure spontaneous cell death, as defined in Fig. 7. Bottom panel: isometric contour plots show the scatter parameters of the same cells before DNA staining. Arrow indicates the population of dead cells. The results are derived from one of three independently performed experiments.

CD38-induced apoptosis and expression of Fas molecule (CD95)
Fas receptor is a cell surface molecule that mediates apoptosis (35) and can be up-regulated after T cell activation induced by mitogens or anti-CD3 mAb (42). In Jurkat cells, the increase in apoptosis observed after ligation of CD38 with the IB4 mAb was paralleled by increased expression of CD95 ( Fig. 10a, left panel). In these cells, CD95 expression increased further after simultaneous stimulation with the anti-CD38 mAb IB4 and the anti-CD28 mAb CD28.2 ( Fig. 10c, left panel). CD28 ligation alone failed to induce any detectable modification of CD95 expression ( Fig. 10b, left panel).

View larger version (27K): [in this window] [in a new window]   Figure 10. Up-regulation of the CD95 molecule parallels CD38-mediated apoptosis. Cell surface expression of CD95 (left), CD3 (middle), or class I (right) after ligation of the CD38 (a), CD28 (b), and CD38 + CD28 (c) molecules in Jurkat cells. Overlaid histograms represent surface expression profiles of Jurkat cells cultured in the presence of the above-mentioned mAb's (open histograms) for 20 h at 37°C vs. cells cultured with an irrelevant anti-CD19 (CB19) (Y axis: number of events; X axis: fluorescence intensity). The results are derived from one of three independently performed experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This paper reports on the results of an ongoing attempt to define the events implemented in different cell lines by ligation of the CD38 receptor by agonistic mAb. CD38 is a surface molecule endowed with multiple functions, the catalytic one being the most studied due to its peculiarity and complexity. The potential of transducing signals once engaged by agonistic mAb or the CD38L has recently become the focus of interest by several groups. Katada et al. (20) defined the signaling steps in human myeloid cells whereas Howard et al. (16) addressed attention to the B cells, due to the marked expression of CD38 in mouse by such cells. Zubiaur et al. (22) transferred the analysis to human T cells, reporting that CD38 ligation is followed by activation of the Raf-1/MAP kinase and CD3-/ZAP-70 signaling pathway. A question of general interest is whether CD38 has developed an independent signaling pathway or, alternatively, shares (partly or totally) the existing pathways, as the specific associations with surface receptorial molecules seem to hint (23). The question could also be of central relevance when one tries to elucidate the modalities by which this ectoenzyme operates in an environment (i.e., biological fluids) where the substrate (i.e., NAD⁺) is almost completely absent and whose final product (i.e., cADPR) is used intracytoplasmatically.

In trying to answer these questions, we expanded on some original observations on human CD38 as a signaling molecule by monitoring the levels of cytoplasmic Ca²⁺, which is taken as a sensitive indicator of biological relevance. The results indicate that CD38 plays a role in regulating cytosolic levels of Ca²⁺, even though the magnitude of the response is lower and less rapid than that elicited via TCR/CD3. The dynamics and modalities of the effects mediated via CD38 led us to conclude that the Ca²⁺ is mobilized from internal and external stores, likely through phosphorylation of PLC-1, as inferred from PMA treatments and confirmed by direct analysis of PLC-1.

The firm conclusion is that the first events marking the action of CD38 signaling are achieved by means of increased Ca²⁺ levels, as seen in the murine model (15). Only mAb's reactive with the epitope where the catalytic function is localized are able to deliver the signal (or signals) driving Ca²⁺ mobilization and other relevant functions (vide infra the apoptosis) (43). Other evidence to support this inference comes from recent studies where the role of the agonistic mAb is taken by the CD38L, used either as soluble molecule or as a molecule expressed by surface cells (44, 45).

A second conclusion that could reasonably be inferred from the results confirms the leading hypothesis of this work, that CD38 achieves its main effects by inducing a down-modulation of the TCR/CD3 complex, which would render CD38 a unique coreceptor. The results also indicate that CD38 signaling drives the expression of a panel of molecules [e.g., CD5 and CD28 (data not shown) and CD69] reported to be up-modulated by TCR/CD3 engagement. The effects observed after CD38 ligation are strictly linked to the expression of an efficient TCR/CD3 complex, its absence (either induced by modulation or by using clones with molecular defects in genes controlling chains of the complex) being correlated with failures both in Ca²⁺ mobilization and in the induction of expression of activation molecules. Knowing the importance of the signals ruled by TCR/CD3 on apoptosis, we assessed the role of CD38 in inducing cell death by evaluating morphological parameters and DNA staining. As expected, CD38 signaling was followed in Jurkat cells by death by apoptosis: this event was specifically inhibited by CsA and paralleled by up-regulation of the CD95 (Fas) molecule. Furthermore, the fraction of dead cells increases when CD38 signaling operates simultaneously with CD28.

In conclusion, the data led us to depict a scenario in T cells whereby CD38 leads a signaling pathway that has several steps in common with that ruled by the TCR/CD3 complex, even if the exact molecular mechanisms underlying such interactions are missing. An appealing explanation is that CD38 may operate through a chain of events contributing to modulation of the TCR/CD3 complex. Taking into account the short cytoplasmic tail of CD38, which is inadequate for signaling, one could hypothesize that the effects are mediated through lateral interactions between some of the CD3 chains, either in the intra- or extracytoplasmatic or in the transmembrane domains. It will be fascinating to validate such a model by assessing the associated molecules to be found on B and myeloid cells. Against such a model, where CD38 is simply parasitizing the main pathway of TCR/CD3, are several lines of evidence that include diverse effects on Ca²⁺, different signaling potentials on cells otherwise insensitive to such signals [e.g., cord blood lymphocytes (46) or lamina propria lymphocytes (31)], and the induction of cytokines. Indeed, Ausiello et al. (47) tested mRNA for a wide panel of cytokines, comparing the signals via CD38 and CD3 in PBL and purified T lymphocytes. The results confirm differences between the messages obtained, which differ in quality (interleukin-6, interferon-, and GM-CSF are the more prominent ones induced by CD38) and stabilization of messages (as is the case for interleukin-6), a striking feature shared with CD28. Now the study will be expanded in the direction of evaluating the interactions with CD2 in normal and disease conditions: it will also be interesting to highlight the effects on CD38 signaling in in vivo conditions where CD3 is inefficient (e.g., umbilical cord blood cells) and in diseases such as immunodeficiency (either genetically or acquired) and autoimmunity.

	ACKNOWLEDGMENTS

 
Thanks are given to Drs. A. Alcover and B. Alarcón for providing mutant Jurkat clones, and to Dr. B. Malissen and Mr. B. Johnson for supplying anti-CD28 and anti-CD38 mAb, respectively. This work was supported by grants from AIRC (Milan, Italy), TELETHON (Rome, Italy), AIDS, and TB Projects (ISS, Rome, Italy), Interministerial Commission of Science and Technology (SAF96–0117, CICYT, Spain), and an Italy-Spain Bilateral Project (CNR, Rome, Italy). CRT and CARIVERONA Foundations contributed to the development of this work. M.M. is Fellow of the Postgraduate School of Clinical Pathology, University of Torino Medical School, Torino, Italy. M.Z. is supported by a Contract of Reincorporation from the Ministry of Education and Culture, Spain. Thanks are extended to E. Ferrero, M.D., and S. Deaglio for reviewing the manuscript.

	FOOTNOTES

 
¹ Correspondence: F. Malavasi, M.D., Lab of Cell Biology, Via Santena 19, 10126 TORINO, Italy. E-mail: malavasi{at}molinette.unito.it

² Abbreviations: cADPR, cyclic ADP-ribose; Ca²⁺, calcium; PLC-, phospholipase C-; CsA, cyclosporin A; mAb, monoclonal antibody; TCR, T cell receptor; PVDF, polyvinylidene difluoride; Ig, immunoglobulin; FCS, fetal calf serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; pTyr, phosphotyrosine; PMA, phorbol 12-myristate 13-acetate.

Received for publication October 28, 1997. Accepted for publication January 9, 1998.

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