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Role of CD38 and Its Ligand in the Regulation of MHC-Nonrestricted Cytotoxic T Cells¹

Alessandra Cesano^*, Sophie Visonneau^*, Silvia Deaglio, Fabio Malavasi^, and Daniela Santoli^2,*

^* The Wistar Institute of Anatomy and Biology, Philadelphia, PA; Laboratory of Cell Biology, Department of Genetics, Biology and Medical Chemistry, University of Turin, Turin, Italy; and Institute of Biology and Genetics, University of Ancona, Ancona, Italy

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human CD38 is a type II transmembrane glycoprotein that regulates lymphocyte adhesion, proliferation, and cytokine production. The mAb Moon-1 recognizes a ligand for CD38 (CD38L) and specifically inhibits CD38-mediated cell adhesion. To analyze the role of CD38 and its ligand in MHC-nonrestricted T cell activation, we examined the effects of Moon-1 and the anti-CD38 mAb IB4 on the effector functions of the IL-2-dependent T cell line TALL-104 (CD3/TCR-ß⁺, CD8⁺, CD56⁺) and of LAK cells (90% CD3⁺). TALL-104 cells were almost 100% reactive with both mAbs, whereas the reactivity of LAK cells for IB4 and Moon-1 ranged from 10 to 60% among different donors. From 78 to 94% of the cytotoxic CD8⁺/CD56⁺ LAK subset was CD38L⁺. Like mAb OKT3 (anti-CD3), and at variance with IB4, Moon-1 drastically enhanced the cytotoxicity of TALL-104 and CD8⁺ LAK cells against a resistant tumor target. Granule exocytosis did not appear to play a role in Moon-1-induced cytotoxicity. Moreover, neither IB4 nor Moon-1 induced [Ca²⁺]_i mobilization in LAK and TALL-104 cells. Whereas stimulation of CD3 and CD38 resulted in a dramatic induction of cytokine (granulocyte-macrophage-CSF, IFN-, TNF-, and TNF-ß) release by both TALL-104 and LAK cells, ligation of CD38L was not followed by cytokine production in TALL-104 cells. Thus, cytotoxicity and cytokine release are independently regulated, at least in this system. These data demonstrate that CD38 and its ligand can regulate some T cell functions using signaling pathways distinct from those of CD3.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human CD38 Ag is a type II 45-kDa transmembrane glycoprotein, prevalently expressed by immature and activated T and B lymphocytes, plasma cells, monocytes, peripheral blood NK cells and, at a lower epitope density, other cells and tissues (1, 2, 3, 4). A unique characteristic of CD38 is its ability to act as coreceptor, inducing activation and proliferation signals in lymphocytes (1, 3). In particular, early studies indicated that human CD38 is physically associated with the TCR/CD3 complex, surface Ig/CD19, and CD16 on T, B, and NK cells, respectively, and can function as a channel for activation signals in these cells (5, 6, 7, 8). CD38 also reportedly mediates a selectin-like binding to endothelial cells, thus acting as an adhesion molecule (9). More recently, CD38 was shown to have ADP-ribosyl (ADPR)³ cyclase and hydrolase activities, leading to the conversion of NAD⁺ into cyclic ADPR and hydrolysis of this molecule to ADPR (10, 11, 12). This function has potential biologic implications, since cyclic ADPR, as the likely natural ligand of the ryanodine receptor, regulates cytoplasmic Ca²⁺ currents (13, 14, 15). However, the in vivo function of CD38 remains unclear, mainly due to lack of evidence concerning its ligand repertoire.

A ligand for human CD38 Ag (CD38L) was recently defined based on the ability of murine mAb Moon-1 (which reacts with a single-chain 120 kDa molecule expressed mainly by endothelial cells, monocytes, platelets, NK cells and, to a lesser extent, by T, B, and myeloid cells) to significantly inhibit CD38-mediated adhesion (15). Further studies showed that the human CD38L recognized by Moon-1 corresponds to the CD31 (PECAM-1) Ag, a well-characterized endothelial adhesion molecule (16).

Accumulating evidence indicates that CD38 triggering induces proliferation of human T and NK cells and expression of a discrete pattern of cytokines (5, 17). However, little is know about the functional effects of CD38L triggering, other than the reported increase in cytoplasmic calcium concentrations in T cell lines treated with mAb Moon-1 (18).

In the present study, we analyzed the role of CD38L, CD38, and CD3 in MHC-nonrestricted T cell activation by comparing the effects mediated by mAbs Moon-1, IB4, and OKT3 (directed against CD38L, CD38, and CD3 molecules, respectively) on the effector functions of an IL-2-dependent CD8⁺ cytotoxic T cell line (TALL-104) (19, 20, 21), of IL-2-activated PBMC (LAK cells) and of positively selected CD8⁺ or CD4⁺ LAK subsets.

	Materials and Methods

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Preparation of lymphokine-activated killer (LAK) cell subsets

PBMC from healthy donors were separated by centrifugation on AccuPrep lymphocyte gradients (Accurate Chemical, Westbury, NY), depleted of monocytes by double adherence to plastic for 1 h at 37°C and cultured for 5 days in Iscove’s modified Dulbecco’s medium (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FBS (Atlanta Biologics, Norcross, GA) (complete medium) and human rIL-2 (Chiron, Emeryville, CA) (200 U/ml). The resulting cells were designated unfractionated LAK cells. In some experiments, aliquots of these cells were labeled with either FITC-CD4 or FITC-CD8 mAbs (21) (Sigma Chemical Co., St. Louis, MO) and sorted using an Ortho Cytofluorograf cell sorter. The purity of the sorted populations was always 95%.

Cell lines

TALL-104 cells were maintained at 37°C in 10% CO₂ in complete medium supplemented with 100 U/ml rhIL-2. The human leukemic cell line K562 and the mouse leukemia cell line 70Z used as targets were also maintained in complete medium: 70Z cells require addition of ß-mercaptoethanol (10^-5 M) (Sigma Chemical Co.) for continuous growth (21). All cell lines were Mycoplasma free by repeated PCR testing, using an American Type Culture Collection (Rockville, MD) kit.

Immunofluorescence analysis

Single immunofluorescence analysis was performed using the following mAbs: OKT3 (CD3); OKT4 (CD4); OKT8 (CD8) (Ortho Pharmaceutical, Raritan, NJ); B67.1 (CD2); and B159.5.2 (CD56) (gifts from Dr. Bice Perussia, Jefferson Medical School, Philadelphia, PA); IB4 (CD38); and Moon-1 (CD38L/CD31), previously described in detail (18). Indirect immunofluorescence was performed as described (21) with FITC-conjugated goat anti-mouse Ab (Sigma Chemical Co.) as second anti-mouse Ig reagent. Double and triple immunofluorescence was performed using phycoerythrin (PE)-labeled anti-CD56 mAb, FITC-labeled anti-CD4 or anti-CD8 mAbs (Sigma Chemical Co.) and biotin-labeled Moon-1 plus R670-conjugated avidin (Sigma Chemical Co.). Cells (5000) were analyzed on a FACScan flow cytometer (Becton Dickinson, Lincoln Park, NJ) gated to exclude nonviable cells.

Proliferation assay

Unfractionated LAK cells, CD8⁺ or CD4⁺ LAK cells, and TALL-104 cells were seeded at 10⁵/well in 96-well microtiter plates (Becton Dickinson) in the presence of IL-2 (200 U/ml in the case of LAK cells and 100 U/ml in the case of TALL-104 cells), with or without mAbs OKT3, IB4, or Moon-1 at the indicated concentrations. [³H]TdR (2 Ci/mmol, Amersham, Arlington Heights, IL) was added 48 h later (1 µCi/well), and isotope incorporation was measured after an 18-h incubation using a beta counter (Packard Instruments, Downers Grove, IL). Data are expressed as mean counts per minute.

Apoptosis assays

TALL-104 cells grown in IL-2 (100 U/ml) (10⁶/ml) were incubated for 18 h in the presence or absence of increasing concentrations of mAb IB4 (1, 5, or 10 µg/ml), and apoptosis was measured by two different methods: 1) permeabilized cells were stained with propidium iodide (PI, 50 µg/ml, Sigma Chemical Co.), and the amount of DNA fragmentation was calculated by flow cytometry, as described (22); 2) terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) of DNA fragmentation sites in nuclei was performed using a commercial kit (Boehringer-Mannheim, Indianapolis, IN) according to the manufacturer’s instructions. Briefly, permeabilized cells were incubated for 1 h at 37°C with FITC-12-dUTP (0.3 nmol), dATP (3 nmol), 2 µl of CCl₂ (25 mM), TdT (25 U), and TdT buffer in a total reaction volume of 50 µl. The reaction was stopped by adding 2 µl of EDTA (0.5 M). After two washings, samples were analyzed using a FACScan (23).

Cytotoxicity assay

Effector cells (TALL-104, unfractionated LAK, CD8⁺, and CD4⁺ LAK) were tested at four different concentrations in a 4-h ⁵¹Cr release assay against a fixed number (10⁴/well) of ⁵¹Cr-labeled murine leukemia 70Z cells. mAbs OKT3, IB4, Moon-1 and, in some experiments, Moon-1 F(ab)'₂ were added to the effector cells (final concentration, 1 µg/ml) immediately before adding the 70Z target cells. Specific ⁵¹Cr release was calculated from the mean of three replicates (19). In some experiments, effector cells were preincubated for 1 h with 1-µg/ml concentrations of each mAb, washed, and used in the 4-h ⁵¹Cr release assay.

N--Benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) esterase release assay

The ability of mAbs specific for CD38L, CD38, and CD3 to trigger the release of cytotoxic granules from TALL-104 and unfractionated LAK cells was evaluated. Effector cells (10⁶/well) were incubated for 4 h at 37°C in the presence or absence of mAb OKT3, IB4, and Moon-1 (final concentration, 1 µg/ml) either alone or in combination with K562 cells (10⁵/well). Cultures were then centrifuged at 800 rpm for 5 min, and cell-free supernatants were collected. Fresh medium (200 µl) was added to the pellets, and cell lysates were obtained by repeated freezing and thawing. BLT esterase activity was measured as described (24). Data are given as specific percent enzymatic activity released, based on the equation [(E - S)/(T - S)] x 100, where E is the number of enzymatic units in the supernatant of experimental wells, S is the number of enzymatic units in the supernatant of unstimulated wells, and T is the total number of BLT esterase units contained in the cells in each well. Total BLT esterase content in K562 cells was constantly <1% of that in TALL-104 and unfractionated LAK cells.

Cytokine production

Unfractionated LAK cells from three different donors and TALL-104 cells (10⁶/ml) were incubated at 37°C for 18 h in the presence or absence of mAb OKT3, IB4, or Moon-1 (final concentration, 1 µg/ml). Cell-free supernatants were then harvested, filtered, and tested for the presence of IFN-, TNF-, TNF-ß, and granulocyte-macrophage-CSF using commercially available ELISA kits specific for each cytokine (Endogen, Boston, MA) (25).

Calcium measurements

Intracellular calcium was measured using the calcium-sensitive fluorochrome Indo-1 (Molecular Probes, Eugene, OR). Unfractionated LAK and TALL-104 cells (5 x 10⁶/ml) were loaded with 3 to 5 mM indo-1 (final concentration) in HBSS/10 mM HEPES buffer, pH 7.4, containing 1 g/l D-glucose and 1 mM CaCl₂, for 45 min at 37°C. Cells were then placed in a thermostat-regulated (37°C) and magnetically stirred fluorometer (ELITE Cytometer) cuvette where [Ca²⁺]_i was measured as described (26). The mAbs tested (OKT3, IB4, Moon-1, and goat anti-mouse Ig) (Sigma Chemical Co.) were added to the buffer at a concentration of 10 µg/ml. Ionomycin (Sigma Chemical Co.) was added at a final concentration of 0.25 µg/ml.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD38L expression by cytotoxic T cells

The human leukemic T cell line TALL-104, established and characterized in this laboratory (19-21), was used in this study as a model of MHC-nonrestricted cytotoxic T cells. TALL-104 cells can kill a broad range of tumors across several species, while sparing cells from normal tissues (19). Tumor cell lysis by TALL-104 cells occurs by a perforin-mediated pathway and/or a FAS-dependent mechanism (19, 20, 21). Moreover, cytokines released by TALL-104 cells on contact with tumor targets (TNF-, TNF-ß, TGF-ß, IFN-) exert cytostatic effects on tumor cell growth (19, 20, 21).

The TALL-104 cell line requires exogenous addition of rhIL-2 (100 U/ml) for continuous growth and expression of cytotoxic function. TALL-104 cells can also be expanded in vivo without administration of rhIL-2 by engraftment in SCID mice (27). Once recovered from the murine tissues, TALL-104 cells are devoid of cytotoxic activity but can be readapted to growth in culture in the presence of rhIL-2. In these conditions, TALL-104 cells progressively acquire cytotoxic function first against NK-sensitive tumor targets (within 2 wk) and later on (by the 4th week) against more resistant targets (27). Flow cytometric analysis of CD38 and CD38L expression by this T cell line revealed coexpression of CD38 and CD38L at high intensity on virtually all TALL-104 cells at any time during their propagation in IL-2 (Table I); by contrast, expression of CD56 increased over time in culture in IL-2 and CD4 progressively decreased. Similar analysis using healthy donors PBMC cultured for 5 days in the presence of rhIL-2 (200 U/ml) indicated high variability among donors in the reactivity of PBMC with IB4 (anti-CD38) and Moon-1 (anti-CD38L) (ranging from 10 to 62%) and a lower fluorescence intensity (Table II). Interestingly, these differences did not correlate with any individual variation in the levels of other T cell markers tested (i.e., CD3, CD2, CD8, CD4, or CD56) and probably reflect the existence of a genetic polymorphism for CD38 (12). As with TALL-104 cells, CD56 levels in LAK cells increased with time in culture with IL-2 (Table II).

View larger version (45K): [in this window] [in a new window]   FIGURE 1. mAb Moon-1 reactivity with PBMC. PBMC from two donors were stained for three-color immunofluorescence using FITC-conjugated anti-CD8 or anti-CD4 mAbs, PE-conjugated anti-CD56 mAb, and biotin-labeled Moon-1 plus R670-conjugated avidin. The top four panels for each donor show a two-color analysis of total PBMC. The bottom left panel for each donor shows the percentage of Moon-1+ cells in the double-positive CD8+/CD56+ (left). Data are representative from three experiments with PBMC from two different donors for assay.

Cytotoxicity assays, using TALL-104 cells or LAK cells (unfractionated or as CD4⁺ and CD8⁺ subsets) as effectors and the lysis-resistant murine leukemic cell line 70Z (28) as targets, showed that OKT3 induced cytotoxic activity in all four effector cell populations tested (p < 0.01 relative to controls), whereas Moon-1 was a potent inducer in TALL-104 cells, somewhat less potent in CD8⁺ LAK cells (p < 0.01 relative to controls for both populations), and ineffective in CD4⁺ or unfractionated LAK cells (Fig. 2). Levels of 70Z cell killing induced by Moon-1 in both TALL-104 cells and CD8⁺ LAK cells were comparable with those induced in the same cells by OKT3. By contrast, CD38 stimulation using the agonistic mAb IB4 had no effect on the killer activity of LAK cells (fractionated or not) and induced only marginal cytotoxicity in TALL-104 cells (p < 0.05) (Fig. 2). Similar results were obtained whether the effectors were pretreated with the mAbs and washed before addition of ⁵¹Cr-labeled cells (not shown), or the stimuli were present during the assay. Experiments using Moon-1 F(ab')₂ mAb to rule out potential interference by the Fc region also gave similar results (not shown).

View larger version (34K): [in this window] [in a new window]   FIGURE 2. MHC-nonrestricted killer activity induced by Moon-1. TALL-104 cells, unfractionated LAK cells, and CD8+ or CD4+ LAK cells were incubated for 4 h at 37°C with 51Cr-labeled murine 70Z cells in the presence or absence of Moon-1, IB4, or OKT3 mAbs (final concentration, 1 µg/ml). Four different E:T ratios were used. Data are representative of three experiments with similar results.

Serine esterase release into the supernatant is a convenient indicator of degranulation by CTL and LAK cells (24). Analysis of granule exocytosis induced by OKT3, IB4, and Moon-1 mAbs in TALL-104 and unfractionated LAK cells revealed significant (p < 0.01) and reproducible secretion of BLT esterase by TALL-104 cells on triggering with either OKT3 or IB4 mAbs, both alone and in combination with K562 cells (Fig. 3). By contrast, Moon-1 did not affect degranulation of this cell population regardless of whether it was used alone or with K562 cells (Fig. 3). Serine esterase release by different populations of LAK cells (stimulated with OKT3, IB4, and Moon-1 mAbs alone or in combination) was, on the contrary, low and extremely variable from donor to donor, thus rendering its interpretation difficult.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Percentage of specific BLT esterase activity released by TALL-104 cells (cultured in IL-2 for 3 or 9 wk) and unfractionated LAK cells (from two donors) after 4 h of stimulation with soluble Moon-1, IB4, or OKT3 mAbs (final concentration, 1 µg/ml) either alone or in conjunction with the tumor cell line K562 (E:T ratio, 10:1). Data are representative of three experiments (with a total of six LAK donors) with similar results.

Comparison of mAbs Moon-1, IB4, and OKT3 for the ability to induce cytokine release in TALL-104 and LAK cells (Fig. 4) indicated high levels of cytokine production by both effectors after triggering of CD3 and CD38 (p < 0.0001), consistent with previous studies (5, 17, 20). Interestingly, Moon-1 induced significant cytokine release in unfractionated LAK cells but not in TALL-104 cells (p < 0.0001).

View larger version (52K): [in this window] [in a new window]   FIGURE 4. Cytokine release in TALL-104 and unfractionated LAK cells after stimulation with soluble Moon-1, IB4, and OKT3 mAbs. Cells were incubated for 18 h at 37°C in the presence or absence of each mAb at a final concentration of 1 µg/ml. Cytokine levels were measured in cell-free supernatants by ELISA. Data are representative of three experiments with different LAK donors. GM-CSF, granulocyte-macrophage-CSF.

Moon-1 had no effect on the IL-2-dependent proliferation of TALL-104 cells and marginally increased that of LAK cells (p < 0.05), while OKT3 up-regulated proliferation in both populations (p < 0.001) (Fig. 5). Interestingly, IB4 stimulated the proliferative response of LAK cells (both unfractionated and CD8⁺ or CD4⁺ subsets) but inhibited that of TALL-104 cells in a dose-dependent manner (p < 0.001) (Fig. 5). PI staining studies to determine whether the decrease in [³H]TdR uptake in TALL-104 cells after CD38 triggering reflected cell cycle arrest or cell death showed that 25% of TALL-104 cells incubated with increasing doses of IB4 were apoptotic at the highest dose of mAb used (10 µg/ml) (Fig. 6). FITC/TUNEL assay confirmed the apoptotic death of TALL-104 cells treated with mAb IB4; this test proved to be twice as sensitive in detecting apoptotic cells in the cultures (50%).

View larger version (21K): [in this window] [in a new window]   FIGURE 5. Effect of Moon-1, IB4, and OKT3 mAbs on proliferative activity of TALL-104 cells, unfractionated LAK cells, CD4+ or CD8+ LAK cell subsets (cultured in rhIL-2, 200 U/ml), and freshly isolated PBMC from two donors. Cells were seeded at 105/well in microtiter plates in the presence or absence of each mAb at the indicated concentrations or at 1 µg/ml. [3H]TdR was added 48 h later, and isotope incorporation measured after 18 h. Data are representative of three experiments with different donors.

View larger version (32K): [in this window] [in a new window]   FIGURE 6. Induction of apoptotic (APOP) cell death by mAb IB4 in TALL-104 cells. Cells grown in IL-2 (100 U/ml) (106/ml) were incubated overnight in the presence or absence of mAb IB4 at the indicated concentrations. Apoptosis was measured by PI staining (left panels) and the FITC/TUNEL assay (right panels) as described in Materials and Methods. In the FITC/TUNEL assays, K indicates the percentage of apoptotic cells. Grn, green; Int, intensity.

Analysis of the effects of CD38L, CD38, and CD3 stimulation on intracellular Ca²⁺ levels revealed no induction of intracellular Ca²⁺ mobilization in TALL-104 and LAK cells by Moon-1 or IB4 (Fig. 7). However, when Moon-1, but not IB4, bound to the TALL-104 cell surface was cross-linked with goat anti-mouse Ig, a small but reproducible increase in [Ca²⁺]_i was observed (Fig. 7). Under the same test conditions, direct binding of the CD3 molecule to both TALL-104 and LAK cells was constantly followed by a sustained increase in [Ca²⁺]_i levels; a further [Ca²⁺]_i rise was triggered by cross-linking with goat anti-mouse Ig (Fig. 7).

View larger version (49K): [in this window] [in a new window]   FIGURE 7. Effects of mAbs Moon-1, IB4, and OKT3 on intracellular Ca2+ levels of TALL-104 and unfractionated LAK cells. Cells (5 x 106/ml) were loaded with Indo-1 and stimulated directly in the fluorimeter cuvette with each mAb at a final concentration of 10 µg/ml. Ca2+ levels were measured using an ELITE Cytometer as described in Materials and Methods. Goat anti-mouse Ig and ionomycin were added at final concentrations of 10 and 0.25 µg/ml, respectively. Arrows indicate the time when stimuli were added to the cells. Results are representative of three experiments performed under the same conditions with different LAK donors.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

On the basis of the association of CD38 with B and TCRs (31) and the role of this molecule in inducing cytokine synthesis and release by NK and T cells (5, 17), we addressed the potential role of CD38 signaling and ligation in immune-mediated effector function after lymphocyte activation and the biologic significance of CD38/CD38L interaction as compared with CD3 signaling on T cells. Our analysis focused on MHC-nonrestricted cytotoxic T cells activated by short (LAK) or long (TALL-104) term growth in rhIL-2. Immunofluorescence analysis revealed that the IL-2-dependent leukemic T cell line TALL-104 (a clonal CD3/TCRß⁺ CD8⁺ population) expresses high concentrations of CD38 and its ligand, regardless of the state of functional maturation of these cells along the cytotoxic pathway. In fact, both molecules were coexpressed by TALL-104 cells even during early stages of propagation in IL-2, when they have not yet acquired a fully cytotoxic CD8/CD56 phenotype (19, 27). By contrast, LAK cells expanded from the peripheral blood of healthy donors displayed different levels of CD38 and CD38L on their surface; three-color immunofluorescence analysis revealed a high expression of CD38L (74 to 94%) on the CD8⁺/CD56⁺ LAK subset, suggesting a role for this molecule in the functional activation of T cells with the cytotoxic phenotype.

Our cytotoxicity assays used as target the FcR-negative murine leukemia cell line 70Z, which is not recognized by Moon-1, IB4, or OKT3 (not shown) and is highly resistant to necrotic cell death induced by MHC-nonrestricted effectors (32). IB4 triggering did not affect the MHC-nonrestricted killing ability of TALL-104 and LAK cells against this target, whereas striking stimulatory effects were induced by mAbs Moon-1 and OKT3 on both TALL-104 cells and the CD8⁺ fraction of LAK cells. Since, as previously described (33), TALL-104 cells do not express any FcR molecule (CD16, CD32, or CD64) either at the protein level (as judged by immunofluorescence analysis) or at the mRNA level, it is unlikely that their cytolytic effect against 70Z was mediated through a reverse Ab-dependent cellular cytotoxicity mechanism. Moreover, the fact that the F(ab')₂ of Moon-1 mediated the same effect excludes that cytotoxicity was dependent from FcR involvement. The mechanism of killing induced by Moon-1 in this particular experimental system is under investigation in this laboratory. Preliminary data suggest that Moon-1 mAb induces Fas ligand on TALL-104 cells, at least at the mRNA level; however, it is unlikely that activation of Fas/Fas ligand pathway plays a role in 70Z cell killing because these cells are Fas negative. In addition, despite the fact that TALL-104 cells form conjugates with 70Z cells (32), the possibility that Moon-1 acts at the postbinding level by triggering the release of cytotoxic mediators in TALL-104 cells is unlikely in this system because of the inability of this mAb to induce cytokine release and degranulation. Another interesting observation in our study was the lack of Moon-1 activity in the unfractionated LAK populations and its presence in the CD8⁺ T cell subsets. These results were highly reproducible and likely reflect an enrichment process: in fact, although the number of CD8⁺ cells in unfractionated LAK cells is high, the percentage of noncytotoxic CD8^- CD31⁺ cells is also significantly high and justifies by itself a considerable sequestration of the CD31 mAb when these cells are present in the effector population. In contrast with previous data with OKT3 (19), we did not detect granule exocytosis after activation of the effectors through CD38L binding. Instead, our data suggest that stimulation via the CD3 or CD38L pathways, while leading to the same effects, involves different mechanisms that are selectively activated by one stimulus but not the other. We previously showed that binding of CD38L to CD38⁺/Moon-1⁺ Jurkat cells was followed, after a brief time lag, by a sustained increase in [Ca²⁺]_i, with a pattern that differed from that triggered via CD3 and CD38 on the same cells (18). In the present study, neither IB4 nor Moon-1 had any effect on intracellular Ca²⁺ mobilization of TALL-104 and LAK cells, although an increase in [Ca²⁺]_i was induced by CD38L ligation in TALL-104 cells after cross-linking of Moon-1 with goat anti-mouse Ig.

Consistent with previous studies (5, 17, 20), both OKT3 and IB4 were strong inducers of cytokine release in TALL-104 and LAK cells; however, CD38L binding with Moon-1 triggered this function only in unfractionated LAK cells. These data indicate that induction of cytokine release and cytotoxicity in MHC-nonrestricted effectors can be independently regulated. Moreover, because TALL-104 cells lack FcR on their surface while LAK cells are a polyclonal population that expresses FcR molecules, our data suggest that differential cross-linking of the mAbs may be responsible for the differences in responses seen in the LAK vs TALL-104 populations.

On the basis of the known ability of mAbs OKT3 and IB4 to stimulate proliferation in normal lymphocytes (5), we also analyzed the effects of CD38L binding on the proliferative activity of TALL-104 and LAK cells. Our results confirmed the up-regulatory activity of OKT3 and showed that Moon-1 failed to induce cell proliferation in either effector. Interestingly, IB4 had stimulatory effects on LAK (CD4⁺ and CD8⁺) cells but inhibited TALL-104 cell proliferation, due in part to the induction of apoptotic cell death in 50% of these cells. In this respect, using a multiprobe RNase protection assay, we have recently observed an up-regulation of mRNA for the FLICE gene (a novel FADD-homologous ICE/CED-3-like protease which is recruited by the CD95 death-inducing signaling complex) (34) in TALL-104 cells stimulated overnight with IB4 mAb (unpublished observations).

Together, our results provide the first demonstration for a modulatory role of CD38 and CD38L on the IL-2-dependent growth and MHC-nonrestricted cytotoxic function of CD8⁺ cells. The observation that CD38L ligation selectively activates cytotoxicity in both TALL-104 and LAK cells but fails to induce cytokine production, granule release, and proliferation in the TALL-104 cell model indicates the utilization of different signaling pathways by CD3 and CD38L. Given the important role of these molecules in lymphocyte/HEC adhesion, the high level expression of CD3 and CD38L on effector T cells suggests their biologic significance in adoptive therapy of cancer. In this context, both LAK and TALL-104 cells have been shown to migrate to the tumor site, infiltrate the tumor vasculature and exert potent antitumor effects in experimental animal models. While the potentiating effects of OKT3 on the efficacy of adoptively transferred LAK cells are being tested in clinical trials, further preclinical studies will define whether CD38L binding in vivo favors T cell penetration and killing of resistant tumors through direct or indirect (cytokine-mediated) mechanisms.

	Acknowledgments

 
We thank Jeffrey Faust for assistance at the cell sorter and the Editorial Department of The Wistar Institute for preparing the manuscript.

	Footnotes

 
¹ This work was supported by American Cancer Society Grant DH-107C (D.S.), Associazione Italiana per la Ricerca sul Cancro (Milan, Italy), Telethon (Rome, Italy), and AIDS and TB special projects (Istituto Superiore di Sanità, Rome, Italy) (F.M.).

² Address correspondence and reprint requests to Dr. Daniela Santoli, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address:

³ Abbreviations used in this paper: ADPR, ADP-ribosyl; LAK, lymphokine-activated killer; PE, phycoerythrin; TdT, terminal deoxynucleotidyl transferase; BLT, N--benzyloxycarbonyl-L-lysine thiobenzyl ester; HEC, human endothelial cell; CD38L, ligand for human CD38 antigen; PI, propidium iodide; TUNEL, TdT-mediated dUTP-biotin nick end-labeling; rh, recombinant human.

Received for publication July 2, 1997. Accepted for publication October 14, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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