Early expression of triggering receptors and regulatory role of 2B4 in human natural killer cell precursors undergoing in vitro differentiation
Abstract
In this study we analyzed the progression of cell surface receptor expression during the in vitro-induced human natural killer (NK) cell maturation from CD34+ Lin− cell precursors. NKp46 and NKp30, two major triggering receptors that play a central role in natural cytotoxicity, were expressed before the HLA class I-specific inhibitory receptors. Moreover, their appearance at the cell surface correlated with the acquisition of cytolytic activity by developing NK cells. Although the early expression of triggering receptors may provide activating signals required for inducing further cell differentiation, it may also affect the self-tolerance of developing NK cells. Our data show that a fail-safe mechanism preventing killing of normal autologous cells may be provided by the 2B4 surface molecule, which, at early stages of NK cell differentiation, functions as an inhibitory rather than as an activating receptor.
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Natural killer (NK) cells are capable of discriminating between normal cells and cells that have lost MHC class I expression as a result of tumor transformation or viral infection. This NK cell capability is the outcome of the concerted function of MHC class I-specific receptors that inhibit cytotoxicity upon recognition of their MHC ligands and of triggering receptors that induce NK cells to kill as a result of their interaction with non-MHC ligands.
In humans, the inhibitory receptors include the Ig-like killer inhibitory receptors (KIR) specific for HLA-A, -B, or -C, the CD94/NKG2A heterodimer, specific for HLA-E and the ILT2/LIR1, characterized by a broad specificity for different HLA class I molecules (1–3). The receptors responsible for NK cell activation in the process of target cell lysis have remained elusive until recently. They represent a heterogeneous molecular family composed of NKp30 (4), NKp44 (5, 6), and NKp46 (7, 8), all confined to NK cells, which have been termed collectively “natural cytotoxicity receptors” (NCR) (9, 10). Another triggering receptor, the NKG2D, is expressed by both NK cells and cytolytic T lymphocytes (11, 12). The 2B4 surface molecule, mostly functioning as an activating coreceptor, is expressed by NK cells and by a subset of cytolytic T lymphocytes (13, 14), whereas the NKp80, another recently identified coreceptor, is essentially confined to NK cells (15).
The surface expression of various inhibitory receptors during the NK cell development from CD34+ cell precursors has recently been analyzed, but no such information is available for the various triggering receptors and coreceptors involved in natural cytotoxicity. Studies on the NK cell development have been based mostly on the analysis of progenitors derived from fetal liver, cord blood, thymus, or adult marrow. These progenitors, characterized by the CD34+ Lin− surface phenotype, differentiated into mature NK cells when cultured under appropriate conditions, including the use of different cytokines and stromal cells (16–23). For example, in the presence of IL-2 as the only added cytokine, the development of precursors toward NK cells depended on their direct contact with stromal ligands (24). On the other hand, the requirement for stroma could be bypassed when NK cell progenitors were cultured in the presence of a mixture of cytokines including IL-15, IL-7, Flt3-L (FL), and stem cell factor (SCF) (16–23). In the present study we addressed the question of the sequential surface expression and function of the various triggering or inhibitory receptors during human NK cell differentiation from CD34+ Lin− cell precursors.
Methods
Monoclonal Antibodies (mAbs).
The following mAbs were used in this study: JT3A (IgG2a anti-CD3), c127 (IgG1 anti-CD16), KD1 (IgG2a anti-CD16), c218 (IgG1 anti-CD56), FS280 (IgG2a anti-CD56), A6–220 (IgM anti-CD56), BAB281 (IgG1 anti-NKp46), KL247 (IgM anti-NKp46), AZ20 (IgG1 anti-NKp30), F252 (IgM anti-NKp30), Z231 (IgG1 anti-NKp44), KS38 (IgM anti-NKp44), ON72 and BAT221 (IgG1 anti-NKG2D), MA152 (IgG1 anti-NKp80), MAR206 (IgG1 anti-CD2), XA185 (IgG1 anti-CD94), Z270 (IgG1 anti-NKG2A), EB6 (IgG1 anti-p58.1), GL183 (IgG1 anti-p58.2), PAX180 (IgG1 anti-p50.3), FS172 (IgG2a anti-p50.3), Z27 (IgG1 anti-p70), Q66 (IgM anti-p140), F278 (IgG1 anti-LIR1), PP35 (IgG1 anti-2B4), S39 (IgG2a anti-2B4), MA344 (IgM anti-2B4), MA127 (IgG1 anti-NTB-A), and ON56 (IgG2b anti-NTB-A) were produced in our laboratory. J4–57 (IgG1 anti-CD48) and RMO52 (IgG2a anti-CD14) mAbs were purchased from Coulter. PE-HPCA2 (anti-CD34) and TU145 (IgM anti-CD48) mAbs were purchased from Becton Dickinson.
Purification of Human Cord Blood CD34+ Progenitors and Culture Conditions.
Umbilical cord blood (UCB) samples from full-term newborns were collected at the Department of Gynaecology Istituto Giannina Gaslini (Genoa, Italy) upon informed consent of the mothers. UCB mononuclear cells were obtained by Ficoll–Hypaque density gradient centrifugation. CD34+ cells were separated from mononuclear cells by using the MACS system (Miltenyi Biotec, Auburn, CA). Cells obtained in this manner were routinely ≥98% pure. CD34+ Lin− cells were cultured in 24-well plates at a concentration of 1–3 × 104 in 1.5 ml of MyeloCult H5100 medium (StemCell Technologies, Vancouver) supplemented with 10% human AB serum (ICN), 5% FCS (Euroclone, Wetherby, U.K.), and the indicated cytokines. These progenitors were cultured in the presence of MS-5 (21), a murine stromal cell line that was grown to confluence in 24-well plates before use. Purified recombinant human IL-15, FL, SCF, and IL-7, purchased from PeproTech (London), were used at 20 ng/ml.
Phenotypic Analysis and Cytolytic Activity of NK Cells.
At regular intervals during culture the phenotype of growing cells was analyzed by using a FACScan one- or two-color fluorescence cytofluorimetric analysis (7, 8). Purified CD56+ NK progenitors were tested for cytolytic activity in a 4-h 51Cr-release assay against human (FcγR+ K562 cell line, EBV+ Raji Burkitt lymphoma, FO-1 melanoma), or murine (FcγR+ P815 mastocytoma cell line) targets (4, 5). In other experiments immature NK cells were tested under the same conditions against purified immature myelomonocytic cells that differentiate in vitro from CD34+ precursors together with NK cells. The concentrations of the various mAbs added were 0.5 μg/ml for redirected killing or 10 μg/ml for masking experiments. The effector-to-target cell (E/T) ratios are indicated in the figure legends.
Reverse Transcription–PCR Analysis.
Total RNA was extracted from NK immature precursors or mature NK population by using RNAClean (TIB-MOLBIOL, Genoa, Italy), and oligo(dT)-primed cDNA was prepared by the standard technique. The SH2D1A cDNA (632 bp) was amplified with the following primers: 5′ CAG CGG CAT CTC CCT TG (SH2D1A-3 ORF frw) and 5′ TTT CAA AGC TCC TCA CTA TG (SH2D1A-4 ORF rev). Amplification was performed for 30 cycles: 30 s at 94°C, 30 s at 55°C, 30 s at 72°C. The set of primers SHP1-up (from nucleotide 155 to 173): 5′-TTC GGA TCC AGA ACT CAG G and SHP1-down (from nucleotide 736 to 755): 5′-TGC AAA CTC TCA AAC TCC TC were used to amplify the 601 bp of SHP-1 cDNA (30 cycles PCR: 30 s at 95°C, 30 s at 58°C, and 30 s at 72°C). Both PCR reactions were performed with Taq-Gold (Perkin–Elmer/Applied Biosystems) after preactivation of 15 min at 95°C. The PCR products were resolved into a 0.8% agarose gel.
Results
Early Expression of Triggering NK Receptors by Immature NK Cells Differentiating from CD34+ Lin− Cell Precursors.
Highly purified CD34+ Lin− cell populations isolated from UCB did not express NK cell markers, including CD56, NKp46, and NKp30 (Fig. 1a). These populations containing hemopoietic cell precursors were cultured with IL-15, IL-7, FL, and SCF in the presence of MS-5, a murine stromal cell line shown to support and to amplify the human NK cell differentiation from CD34+ cell progenitors (21). Under these culture conditions CD34+ Lin− cells (104 cells per well) underwent proliferation and progressively lost the expression of CD34 surface antigen (not shown). After 20 days of culture ≈4–5 × 105 cells per well were recovered. A fraction of these cells were CD56+ (Fig. 1b), whereas most of the remaining cells were differentiating toward the myelomonocytic cell lineage as indicated by the expression of CD33 and CD14 (not shown). The CD56+ cell subset did not express other NK cell markers, including NKp46 (Fig. 1b), NKp30, NKG2D, CD16, CD94, NKp80 or KIR, T (CD3) and B cell (CD19) markers, and myeloid cell markers (CD14 or CD33) (not shown). Although, at this time, the surface density of CD56 molecules was low, a progressive increment of fluorescence intensity occurred at later stages of differentiation. Thus, after 30 days of culture, a fraction of cells were CD56bright (Fig. 1b). The acquisition of the CD56bright phenotype preceded the expression of NKp46 (Fig. 1b) and NKp30 (not shown). Thus, at day 45 of culture, most CD56bright cells coexpressed these activating receptors (Fig. 1c). At this stage, both receptors were expressed at low surface densities, but their fluorescence intensities progressively increased with time. Indeed, cells with a NKp30/NKp46bright phenotype could be detected after 65 days of culture (Fig. 1 b and d). Remarkably, at day 45, CD94/NKG2A and other HLA class I-specific inhibitory receptors were still undetectable (Fig. 1c). Indeed, the expression of CD94/NKG2A occurred at later stages and >60% of cells expressed this inhibitory receptor at day 65, whereas KIR+ cells represented a minor subset (2–8%) (Fig. 1d). At this stage a larger cell fraction expressed ILT2/LIR1 (15–25%) (not shown), CD16 (30–40%), and NKp80 (30–40%) (Fig. 1d). The expression of NKG2D was slightly increased as compared with day 45. Similar results were obtained in seven independent experiments from different UCB.
Figure 1
Acquisition of Natural Cytotoxicity During NK Cell Development.
Next, we assessed the cytolytic activity of NK cells undergoing differentiation in parallel with the expression of triggering receptors. In this context, previous data showed that mature NK cells express variable surface densities of NCR and that this expression correlated with the magnitude of the natural cytotoxicity (25). Thus, NCRbright NK cells were characterized by high cytolytic activity, whereas the NCRdull ones were poorly cytolytic against most NK-susceptible target cells (25). As illustrated above, the NK cell progenitors undergoing differentiation in culture displayed slowly progressing increases in the surface densities of NKp30 and NKp46. This observation allowed us to analyze the cytolytic activity of NK cells at discrete stages of maturation. Immature NK cells (CD56+) were tested against the K562 target cell line (FcγR+ CD48−). K562 cells are characterized by a high susceptibility to lysis by mature NK cells, and they are also suitable for redirected killing analysis because of the surface expression of FcγR. As shown in Fig. 2a, NK cell progenitors acquired cytolytic activity in a stage-related fashion. Thus, after 30 days, cells characterized by the CD56+, NKp30−, NKp46−, CD94/NKG2A− surface phenotype (see Fig. 1b) did not lyse K562 cells. According to their NCR− phenotype, no cytotoxicity could be triggered in redirected killing in the presence of (IgG1) anti-NKp46 (Fig. 2a) or anti-NKp30 (not shown) mAbs. These data indicate that immature NK cells that do not express NCR are not cytolytic against classical NK susceptible target cells. At day 45 the surface expression of low amounts of NCR (NCRdull) coincided with the appearance of low levels of cytotoxicity against K562 (Fig. 2a). At day 65, the higher surface density of NKp46 and NKp30 receptors coincided with a sharp increase in the magnitude of both spontaneous and anti-NCR mAb-induced cytolytic activity that reached levels comparable with mature NK cells (Fig. 2a).
Figure 2
To assess directly whether the cytolytic activity acquired by developing NK cells was indeed dependent on NCR expression and function, the cytolytic assay was also performed in the presence of anti-NCR mAbs of IgM isotype, to avoid cross-linking by the Fcγ receptors expressed on K562 target cells. The presence of these mAbs allows disruption of the interaction between the NCR and their ligands. Indeed, the combined masking of NKp46 and NKp30 by specific mAbs resulted in a sharp inhibition of lysis of K562 mediated by immature (day 45) NK cells (Fig. 2b). At this stage, the contribution of NKG2D to the lysis of K562 was negligible because of the low expression of this molecule on immature NK cells. In mature NK cell populations derived from peripheral blood (Fig. 2c) and in immature NK cells at day 65 (not shown) both NCR and NKG2D contributed to the NK-mediated recognition and lysis of K562 cells.
The cytolytic activity of immature NK cells was tested also against additional NK-susceptible target cells (e.g., the HLA class I-negative human melanoma FO-1) (4). Again, the cytotoxicity against these targets paralleled the expression of NKp30 and NKp46 and was inhibited by specific mAbs (not shown).
Altogether, these results indicate that the acquisition of cytolytic activity by developing NK cells parallels the surface expression of NKp30 and NKp46. Maximal cytotoxicity against suitable targets was detectable at late stages of maturation when NK cells had expressed both NCRs and NKG2D and their cytolytic activity was mediated by all these triggering receptors, as occurs in mature NK cells.
2B4 Displays Inhibitory Rather Than Activating Function in Immature NK Cells.
The expression of triggering receptors (NCR) before the HLA-specific inhibitory receptors poses the question of why immature NK cells do not kill normal surrounding cells. This could be explained by the presence of a still undefined self-reactive inhibitory receptor. Another distinguished possibility would be the involvement of surface receptors such as 2B4 mediating signals of opposite sign depending on their association with different intracytoplasmic polypeptides (27). This possibility was taken into consideration because, in preliminary experiments, the mAb-mediated engagement of 2B4 in a redirected killing assay against P815 (FcγR+) murine target cells resulted in inhibition of cytolysis mediated by immature NK cells. Notably, in mature NK cells, human 2B4 is involved in triggering the NK-mediated natural cytotoxicity (13, 14) and this effect depends on its association with a small cytoplasmic molecule termed Src homology 2 (SH2) domain-containing protein [SH2D1A; or signaling lymphocyte activation molecule (SLAM)-associated protein (SAP); refs. 26 and 27]. This molecule apparently competes with SHP phosphatases for binding to the cytoplasmic tail of 2B4 (26). Remarkably, in patients with X-linked lymphoproliferative disease (XLP), lacking SH2D1A molecules because of critical mutations in the SH2D1A-encoding gene, the cross-linking of 2B4 leads to inhibition rather than activation of cytotoxicity (27).
To evaluate the possibility that 2B4 may play a role during the NK cell maturation, we analyzed its expression and function at different stages of development. Cytofluorimetric analysis revealed that 2B4 was surface expressed already in freshly isolated CD34+ cell progenitors (Fig. 3a), and its expression remained unchanged during the whole NK cell differentiation process. The 2B4 ligand, represented by CD48 (28), was expressed at low density in fresh CD34+ cells but, upon culture, was up-regulated in all differentiating cells, including both immature NK cells and bystander myelomonocytic cell precursors (Fig. 3a). Immature NK cell populations cultured for 45 days were assessed for cytolytic activity in a redirected killing assay against P815 target cells that are relatively resistant to spontaneous NK-mediated killing (Fig. 3b) (10). Although mAb-mediated cross-linking of NKp46 induced triggering of cytolytic activity, cross-linking of 2B4 failed to induce cytotoxicity but rather exerted an inhibitory effect. This inhibitory effect could be demonstrated better in experiments in which mAb-mediated cross-linking of NKp46 and 2B4 was induced simultaneously. These data indicate that, in immature NK cells (similar to NK cells from XLP patients), the engagement of 2B4 can lead to down-regulation of the NCR-mediated triggering. This was further confirmed in experiments in which immature NK cells were analyzed for cytotoxicity against CD48− (FO-1) or CD48+ (EBV+ Raji Burkitt lymphoma) target cells (Fig. 3c). Different from CD48− target cells, CD48+ cells were resistant to lysis by immature NK cells whereas they were highly susceptible to lysis by mature NK cells (Fig. 3c). mAb-mediated blocking of the 2B4/CD48 interactions could restore, at least in part, the cytolytic activity of immature NK cells against CD48+ targets (Fig. 3c). This effect could be further incremented by the simultaneous mAb-mediated blocking of NTB-A, a surface molecule that has recently been shown to bind SH2D1A in normal NK cells (29). Because even under these conditions the reconstitution of cytotoxicity mediated by immature NK cells was not complete, it is possible to speculate that additional receptors delivering inhibitory signals may be implied in the recognition of ligands expressed by Raji cells. These receptors might even be represented by still-undefined members of the SH2D1A-binding family.
Figure 3
The inhibitory effect mediated by 2B4 at day 45 was not detected further at day 65 of culture (Fig. 3b), but, as expected, in mature NK cells the mAb-mediated cross-linking of 2B4 resulted in triggering of the cytolytic activity (not shown). A similar 2B4-mediated phenomenon in NK cells from XLP patients reflected the lack of SH2D1A molecules (27). To verify whether immature NK cells also were characterized by a defect of SH2D1A, cells cultured for 45 days (surface phenotype: NKp46+, NKp30+, NKG2A−, LIR1−, KIR−) were analyzed for the expression of SH2D1A transcripts. Reverse transcription–PCR analysis failed to reveal SH2D1A transcript in these immature NK cells, whereas SHP-1 transcript was detected in all samples analyzed (Fig. 4a). Although not shown, expression of SH2D1A could be detected at later stages of NK differentiation (i.e., day 65).
Figure 4
Inhibitory 2B4 Molecules Allow Immature NK Cells to Spare Bystander Cell Precursors.
In an attempt to verify whether the inhibitory function of 2B4 could play any physiologic role in self-tolerance during NK cell maturation, we analyzed the cytolytic activity mediated by NK precursors at day 45 against autologous purified immature myelomonocytic cells. As mentioned above, these cells differentiate together with NK cell precursors in cultures derived from CD34+ Lin− cells and are characterized by high surface expression of CD48, i.e., the 2B4-specific ligand (see Fig. 3a). As shown in Fig. 4b, immature NK cells (day 45), although lacking HLA class I-specific inhibitory receptors, were poorly cytolytic against purified (autologous) myelomonocytic cells. Their cytolytic activity could be significantly rescued (>50%) by mAb-mediated blocking of 2B4. Controls included polyclonal NK cell populations derived from an XLP patient or from a normal individual. Also, in the case of the XLP patient, the NK-mediated lysis of myelomonocytic precursors was significantly increased on mAb-mediated blocking of 2B4 molecules (Fig. 4b). In contrast normal (allogeneic) mature NK cells were highly cytolytic, and lysis was not modified under the same experimental conditions. Although not shown, the NK-mediated lysis of myelomonocytic precursors was NCR dependent as indicated by mAb-mediated blocking experiments. These data suggest that immature NK cells may spare surrounding precursors cells by a mechanism involving 2B4 as in XLP patients. However, immature NK cells displayed some degree of cytolytic activity against myelomonocytic precursors even in the absence of mAb-mediated blocking of 2B4 (Fig. 4b). This observation suggests that immature NK cells are not completely turned off by the inhibitory signals generated by 2B4, as also demonstrated by the ability of NK cells to undergo proliferation and maturation in a microenvironment containing CD48+ cells. It is conceivable that the incomplete down-regulation of NK cell function may allow the delivery of signals needed for NK precursors to progress toward a mature phenotype. These data provide an interesting explanation of how the early expression of 2B4, accompanied by a late expression of SH2D1A, may lead to the negative control of natural cytotoxicity during NK cell development.
Discussion
Taken together, our data provide additional insight into the process of human NK cell maturation. A remarkable finding was the early expression of triggering receptors mediating natural cytotoxicity. Because this event preceded the expression of HLA class I-specific inhibitory receptors, it posed a serious problem about to how to avoid the NK cell-mediated attack to surrounding cells at the site of NK cell maturation. Importantly, we show that the 2B4 receptor, expressed early during NK cell maturation, may provide this necessary fail-safe device.
Human CD34+ cell progenitors, cultured in vitro in the presence of suitable cytokines, differentiated into NK cells, most of which expressed the CD94/NKG2A inhibitory receptor (19, 30, 31). Almost no KIR expression could be detected in these cultures, unless suitable stromal cells providing undefined signals were used to support the NK cell differentiation (22, 23). Similar results were obtained in mice, in which the expression of Ly49 inhibitory receptors required both stroma and cytokines (32, 33). Thus, according to these reports, the NK cell self-tolerance at a relatively early stage of human or murine NK cell maturation would be ensured by the expression of CD94/NKG2A that precedes that of human KIR or murine Ly49. Our present study, allowing a better dissection of the early events occurring during the human NK cell differentiation, indicated that the current models of self-tolerance during NK cell development should be reconsidered. Although the education processes that finally give rise to a (self-tolerant) NK cell repertoire are still poorly defined, they may require signals provided by both triggering and inhibitory receptors that are sequentially expressed at the cell surface. Of the different models that have been proposed to explain the integration between triggering and inhibitory receptors during the NK cell development (reviewed in ref. 34), one is compatible with our present data. This model that is based, at least in part, on recent studies in mice, suggests that the first receptors to be expressed during NK cell maturation are (still undefined) triggering receptors. Their engagement with self-ligands, however, would not result in induction of cytotoxicity but would be limited to the delivery of signals responsible for the subsequent expression of MHC-specific inhibitory receptors (CD94/NKG2A and, subsequently, KIR or Ly49). In line with this model, we show that NKp30 and NKp46, two major triggering receptors involved in natural cytotoxicity in humans, are expressed before the CD94/NKG2A inhibitory receptor. However, immature NKp30+ NKp46+ CD94/NKG2A− NK cells displayed cytolytic activity in response to stimuli acting on triggering receptors. Thus, because NKp30 and NKp46 receptors were functional, one may ask how, in such immature (but cytolytic) NK cells, they can deliver signals resulting in cell differentiation without affecting self-tolerance. A possible explanation may be provided by studies on MHC class I knockout mice and on human HLA class I-deficient individuals (35–38). In these cases, the engagement (and the function) of MHC-specific inhibitory receptors (consistently present in NK cells from MHC-deficient subjects) is impaired because of the low levels of expression of MHC ligands. The resulting NK cells, however, do not kill autologous cells, thus implying the existence of a still-undefined fail-safe mechanism. Two models have been proposed to explain the self-tolerance of NK cells derived from class I-deficient subjects. According to the first model, lack of autoreactivity may be consequent to the down-regulation of stimulatory receptors involved in NK cell triggering. The second model proposes a role for a still-undefined inhibitory receptor for non-MHC ligands. Recent studies from our laboratory support the second hypothesis because both the expression and the function of the major triggering NK receptors (NKp30, NKp46, and NKG2D) was apparently normal in HLA class I-deficient individuals (TAP2 −/−) (39). A similar scenario could be envisaged for developing NK cells analyzed in the present study. Indeed, these cells, although expressing triggering receptors, still lack MHC class I-specific inhibitory receptors. Thus, the inhibitory receptor or receptors capable of extinguishing their cytolytic activity should be acquired very early during NK cell differentiation and its expression should be independent on signaling by stimulatory receptors. We provide evidence suggesting that 2B4 represents a suitable candidate to fulfill this role, rendering self-tolerant potentially autoreactive immature NK cells. Our finding that, in immature NK cells, 2B4 displays inhibitory function allowed understanding of how it is possible to block potentially harmful cells. The actual key is represented by the late expression of the SH2D1A molecule. Thus, in immature NK cells, 2B4 would replace class I-specific inhibitory receptors until the stage at which they are surface expressed. The expression of 2B4 precedes that of all of the presently known triggering NK receptors. Thus, 2B4 is expressed on CD34+ precursors, and its expression is maintained through the whole differentiation process toward mature NK cells. Because it is expressed in early hematopoietic precursors, it is possible that 2B4 may play a more general regulatory role in the process of hematopoiesis, possibly by blocking the induction of unwanted function(s) during the early steps of cell differentiation.
Abbreviations
- NK cells
- natural killer cells
- NCR
- natural cytotoxicity receptors
- UCB
- umbilical cord blood
- FL
- fetal liver tyrosine kinases 3 (Flt3)-ligand
- SCF
- stem cell factor
- KIR
- killer Ig-like receptors
- XLP
- X-linked lymphoproliferative disease
- SH2D1A
- Src homology 2 (SH2) domain-containing protein 1A
- SHP-1
- SH2-containing phosphatase 1
- E/T
- effector-to-target cell ratio
- PE
- phycoerythrin
Acknowledgments
We thank Ms. Cinzia Miriello and Ms. Tiziana Baffi for secretarial assistance. This work was supported by grants awarded by Associazione Italiana per la Ricerca sul Cancro, Istituto Superiore di Sanità, Ministero della Sanità, and Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Consiglio Nazionale delle Ricerche, Progetto Finalizzato Biotecnologie. Also the financial support of Telethon-Italy (Grant E.0892) is gratefully acknowledged. E.M. is recipient of a fellowship awarded by Fondazione Italiana per la Ricerca sul Cancro.
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Copyright © 2002, The National Academy of Sciences.
Submission history
Received: November 29, 2001
Accepted: February 4, 2002
Published online: March 26, 2002
Published in issue: April 2, 2002
Acknowledgments
We thank Ms. Cinzia Miriello and Ms. Tiziana Baffi for secretarial assistance. This work was supported by grants awarded by Associazione Italiana per la Ricerca sul Cancro, Istituto Superiore di Sanità, Ministero della Sanità, and Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Consiglio Nazionale delle Ricerche, Progetto Finalizzato Biotecnologie. Also the financial support of Telethon-Italy (Grant E.0892) is gratefully acknowledged. E.M. is recipient of a fellowship awarded by Fondazione Italiana per la Ricerca sul Cancro.
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