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Keywords:

  • NK lymphocyte;
  • Activating coreceptor;
  • Ligand

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

NK-T-B antigen (NTBA) is a CD2 family member that functions as a coreceptor in human NK cell activation. Several receptor/ligand interactions occur between different members of this molecular family. In this study, in order to identify the natural ligand of NTBA, we produced a chimeric protein formed by the NTBA extracellular region fused with the Fc portion of human IgG1 (termed NTBA-Fc*). NTBA-Fc* specifically binds to NTBA cell transfectants but not to cells transfected with other CD2 family members including CD2, CD48, CD84, CD150, CD229, and CD244. Moreover, NTBA-Fc* also binds to NTBA+ but not to NTBA T cell lines. Enzyme-linked immunosorbent assays, plasmon resonance analysis, as well as NTBA-Fc*-mediated down-regulation of NTBA surface expression further confirmed the occurrence of NTBA/NTBA homophilic interaction. Functionally, in NK cells, NTBA-Fc* promoted a strong production of IFN-γ and TNF-α. Moreover, NTBA-transfected targets displayed increased susceptibility to NK-mediated killing as compared to untransfected cells and this effect was specifically inhibited by anti-NTBA mAb. Altogether our data indicate that NTBA is characterized by self recognition.

Abbreviations:
NTBA:

NK-T-B antigen

XLP:

X-linked lymphoproliferative disease

SH2D1A:

Src homology 2 domain-containingprotein 1A

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

NK-T-B antigen (NTBA) is a recently identified surface molecule expressed on NK, T, and B cells 1. In human NK cells, NTBA has been shown to act primarily as a coreceptor since it could trigger cytolytic activity only in cells expressing high surface densities of natural cytotoxicity receptors (NCR) 2. Molecular cloning revealed that NTBA is a memberof the Ig superfamily characterized by structural features that allowed its assignment to the CD2 family. This family also includes CD2 3 as well as CD48 4, CD58 5, CD84 6, CD150 (signaling lymphocyte activation molecule, SLAM) 7, CD229 (hLy9) 8, CD244 (2B4) 9, CS1 (19A, CD2-like receptor activating cytotoxic cells, CRACC) 1012, B lymphocyte activator macrophage-expressed (BLAME) 13, and CD84H1 14. These surface glycoproteins are characterized by similar structures consisting of a membrane distal IgV that lacks the typical disulfide bond, and a membrane proximal IgC2 domain, with the exception of CD229 that contains an IgV-IgC2 domains tandem repeat.

The genes encoding the CD2 family molecules are located on human chromosome 1 in two different gene complexes. In detail, CD244, CD150, CD48, CS1, CD229, CD84, BLAME, CD84H1, and NTBA map on the long arm at 1q21–24, whereas CD2 and CD58 are located on the short arm at 1p13. Structure similarities as well as gene localizations have therefore supported the hypothesis that the CD2 members arose from gene duplication events. Several studies have demonstrated that some members of the CD2 family are involved in adhesions between T lymphocytes and accessory cells and can generate intracellular activating signals 15. In addition, CD84, CD150, CD229, CD244, and NTBA contain in their cytoplasm tail tyrosine-based motifs (TxYxxV/I) capable of recruiting the Src homology 2 domain-containing protein 1A (SH2D1A), also termed SLAM-associated protein (SAP) 16. Mutations in the SH2D1A gene represent the genetic defect responsible for the X-linked lymphoproliferative disease (XLP), a severe inherited immune deficiency, characterized by the inability to control Epstein-Barr virus (EBV) infections 17, 18. Importantly, analysis of NK cells derived from XLP patients showed that the lack of SH2D1A molecule profoundly affects the function of these cells.

Indeed, in the absence of SH2D1A molecule, the interaction of CD244 and NTBA with their ligands on EBV-infected cells has been shown to transduce inhibitory signals that render XLP-NK cells unable to kill EBV-infected targets 1921. While the ligand recognized by CD244, i.e. CD48, has been identified 22, 23, the molecule(s) recognized by NTBA is still unknown. Different heterophilic or homophilic receptor/ligand interactions have been reported to occur between other members of the CD2 family. In particular, human CD2 binds CD58 24, but it also binds with low affinity CD48 25, whereas CD150 26, CD84 27, and CS1 28 mediate homophilic interactions. In this study, using a soluble NTBA molecule we demonstrate that also NTBA recognizes itself. Importantly, we also show that NTBA/NTBA interaction induces both triggering of cytolytic activity and cytokine production in NK cells.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 Generation of NTBA-Fc chimeric soluble molecule

In attempt to identify the natural ligand of NTBA, we used a chimeric molecule containing the extracellular region of the protein fused with the Fc portion of human IgG1. To avoid possible Fc binding to Fcγ receptors (FcγR), three mutations were introduced in the hIgG1 hinge region (234 Leu [RIGHTWARDS ARROW] Ala, 235 Leu [RIGHTWARDS ARROW] Glu, and 237 Gly [RIGHTWARDS ARROW] Ala) 29. To verify whether the amino acidic substitutions indeed abrogated the FcγR binding, we first used non-mutated or mutated CD244 soluble proteins (termed CD244-Fc and CD244-Fc*, respectively). CD244 was chosen due to the fact that the CD244 ligand (CD48) is known already 22, 23. These soluble proteins were analyzed for their reactivity with the CD48FcγR+ MM6 cell line. As shown in Fig. 1A, CD244-Fc but not CD244-Fc* soluble molecules stained MM6. These results confirmed that the mutated soluble molecules do not bind to FcγR thus representing suitable reagents to be used when analyzing FcγR+ cells such as NK cell populations. Moreover, the amino acid substitutions introduced into the hinge region did not alter the ability of CD244-Fc* to recognize the specific ligand as demonstrated by its ability to stain LCL721.221 cells with an intensity comparable to that obtained using an anti-CD48 mAb (Fig. 1B).

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Figure 1.  Mutations in the hinge region of soluble molecules abrogate FcγR binding but do not alter the ligand recognition. (A) The CD48FcγR+ MM6 cell line was stained with CD244-Fc or with CD244-Fc* and analyzed by cytofluorimetry. PE-conjugated goat anti-human was used as second reagent. (B) The CD48+ LCL722.221 cell line was stained with anti-CD48 mAb followed by a PE-conjugated goat anti-mouse isotype-specific second reagent, or with CD244-Fc* followed by PE-conjugated goat anti-human second antibody. White profiles indicate staining with second reagent only.

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2.2 NTBA mediates homophilic interaction

Both heterophilic and homophilic interactions have been described between different members of the CD2 family 2228. We therefore explored the possibility that NTBA may recognize one or another member of this family. To this end, HEK-293T cells where transiently transfected with CD2, CD48, CD84, CD150, CD229, CD244, as well as NTBA. The surface expression of each molecule was controlled by cytofluorimetric analysis using specific mAb (Fig. 2). The various HEK-293T cell transfectants were then analyzed for their reactivity with the NTBA-Fc* chimeric molecule. NTBA-Fc* consistently stained NTBA transfectants while it did not display any reactivity with the other CD2 family members analyzed (Fig. 2), including CD58 that is constitutively expressed by this cell line.

Further, we tested representative T cell lines, including JA3 and HSB2 (that express high levels of NTBA) or PEER (displaying no surface reactivity with anti-NTBA specific mAb) (Fig. 3A). In agreement with the results obtained with cell transfectants, NTBA-Fc* molecules selectively stained NTBA+ T cell lines (Fig. 3A). Notably, cytofluorimetric analysis of other proteins belonging to the CD2 family, including CD2, CD48, CD58, CD84, CD150, CD229, and CD244, revealed that NTBA is the only one expressed by JA3 and HSB2 cell lines but absent on the PEER cell line. Altogether, these data strongly suggested that NTBA mediates homophilic interaction. It is of note that, in terms of mean fluorescence intensity, NTBA-Fc* staining was lower than that detected with CD244-Fc* in CD48 transfectants (see Fig. 2). This suggests that, as previously documented for the CD150/CD150 recognition 26, NTBA may mediate a low-affinity homophilic recognition.

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Figure 2.  NTBA-Fc* selectively binds to NTBA cell transfectants. HEK-293T cells transiently expressing the indicated molecules were stained with the corresponding specific mAb, NTBA-Fc*, and CD244-Fc* soluble molecules. PE-conjugated goat anti-mouse isotype-specific or PE-conjugated goat anti-human were used as second reagents. Cells were analyzed by cytofluorimetry. Dotted profles indicate staining with second reagent only.

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2.3 Analysis of NTBA homophilic interaction by ELISA and plasmon resonance

In order to further support the results described above, we performed an ELISA assay. Plates, coated with serial dilution of NTBA-Fc* or CD244-Fc* (negative control) soluble molecules, were incubated with NTBA-Fc* labeled with biotin. As shown in Fig. 3B, also under these experimental conditions the occurrence of an NTBA/ NTBA homophilic interaction could be documented. In particular, a typical prozone preceded the maximal NTBA/NTBA binding corresponding to the concentration of approximately 30 ng/well of coated NTBA-Fc* molecule. In no instances significant NTBA binding to CD244 could be detected.

We therefore analyzed the NTBA/NTBA interaction using surface plasmon resonance. In detail, purified NTBA-Fc* and CD244-Fc* were covalently coupled to different flow cells of a CM5 sensorchip to obtain a final concentration of 1.5 ng/mm2 (1,500 RU). The efficiency of NTBA-Fc* binding was assessed using the anti-NTBA specific antibody (data not shown).

Next, soluble NTBA-Fc* was diluted to obtain serial concentrations ranging from 10 nM to 25,600 nM. The profiles obtained with the serial dilutions clearly indicate interaction between the two parts (Fig. 3C). Profiles represent the specific binding of soluble NTBA-Fc* to immobilized NTBA-Fc* after subtraction of the background signal corresponding to the interaction of soluble NTBA-Fc* to immobilized CD244-Fc*. When soluble CD244-Fc* was passed over both flow cells, no significant increase in the resonance signal was recorded.

Moreover, the utilization of BIAcore 3000 platform enabled us to perform a kinetic analysis of such homophilic interaction. Indeed, considering the bivalent structure of NTBA-Fc*, we obtained double values of kon and koff related to the interaction of the two binding domains. Using the bivalent analyte model provided by the BIAevaluation software, we saw that the first interaction event (kon1 = 4.52×103 M–1s–1) occurs around 100-fold faster than the second one (kon2 = 0.05×103 M–1s–1). Also the dissociation rates suggest two different events (koff1 = 0.09×103 s–1; koff2 = 2.31×104 s–1). The dissociation constant at the equilibrium KD was calculated as a ratio between the values of dissociation and association rates of the first binding event, resulting to be 19.7 μM.

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Figure 3.  NTBA-Fc* staining of NTBA+ T cell lines and detection of NTBA/NTBA interaction by ELISA and BIAcore 3000 analyses. (A) The indicated cell lines were stained with the anti-NTBA specific mAb or NTBA-Fc* followed by appropriated PE-conjugated second reagents. Dotted profiles represent cells incubated with second reagents only. (B) Plates coated with the indicated serial dilutions of NTBA-Fc* or CD244-Fc* soluble molecules were incubated with NTBA-Fc* labeled with biotin followed by Neutravidin HRP-conjugated second reagent. Data are representative of six independent experiments. (C) Sensogram profiles represent the specific binding of soluble NTBA-Fc* to immobilized NTBA-Fc* after substraction of the background signal corresponding to the interaction of soluble NTBA-Fc* to immobilized CD244-Fc*. Curves represent serial dilutions of NTBA-Fc*: 100 nM, 200 nM, 400 nM, 800 nM, 1,600 nM, 3,200 nM, 6,400 nM, 12,800 nM, and 25,600 nM. Kinetic values are reported.

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2.4 The homophilic NTBA/NTBA interaction induces down-regulation of NTBA surface expression

In order to asses whether the NTBA/NTBA interaction affected its own expression at the cell surface, the NK92 cell line was incubated overnight on plates coated with NTBA-Fc* or with CD244-Fc* as negative control. The surface expression of NTBA on cells cultured under one or the other condition was subsequently analyzed by cytofluorimetric analysis using specific mAb. As shown in Fig. 4, the NTBA fluorescence intensity significantly decreased in cells cultured on plates coated with NTBA-Fc* but not with CD244-Fc*. Under both conditions, we could never detect changes in the expression of other molecules such as NKG2D (Fig. 4), CD244, NKG2A, and CD56 (data not shown).

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Figure 4.  NTBA-Fc* mediates NTBA modulation in NK cells. NK92 cell lines either untreated or incubated on NTBA-Fc*- or CD244-Fc*-coated plates were analyzed by cytofluorimetry for NTBA and NKG2D surface expression. Dotted profiles indicate staining with second reagent only.

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2.5 NTBA/NTBA homophilic interaction results in cytokine production in normal but not in XLP NK cells

We next analyzed whether NTBA-Fc* soluble molecules could induce cytokine production by NK cells. The experiments were performed using polyclonal NK cells derived from either healthy donors or XLP patients. XLP-NK cells were previously shown to express NTBA molecules with inhibitory rather than activating function 1. As shown in Fig. 5, NTBA-Fc* induced strong production of both IFN-γ and TNF-α cytokines in normal NK cells. Notably, this NTBA-Fc*-induced cytokine release was higher than that induced by anti-NTBA mAb and was comparable to that triggered by anti-CD16 mAb (used as positive control). On the other hand, in agreement with the altered function of NTBA, incubation of XLP-NK cells with either NTBA-Fc* or anti-NTBA mAb had no effect. mAb-mediated engagement of CD16 consistently resulted in high levels of both cytokines (Fig. 5), thus demonstrating that XLP-NK cells are potentially capable of producing these cytokines in response to appropriate activating stimuli. The specificity of NTBA-Fc* in inducing IFN-γ and TNF-α production was further confirmed by blocking experiments using anti-NTBA mAb. As shown in Fig. 5, in healthy NK cells anti-NTBA, but not anti-NKG2D mAb used as control, virtually abrogated the NTBA-Fc*-induced production of cytokines.

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Figure 5. NTBA/NTBA interaction induces both IFN-γ and TNF-α production in normal NK cells. Polyclonal NK cell populations from healthy or XLP donors were stimulated with plate-bound (pb.) NTBA-Fc* or the indicated mAb, either in the absence or in the presence of pretreatment (pt.) with NTBA- or NKG2D-specific mAb. IFN-γ (A) and TNF-α (B) productions were assessed by ELISA. Data are representative of five independent experiments.

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2.6 The NTBA/NTBA interaction induces the NK-mediated killing of target cells

In order to analyze the role of NTBA/NTBA interaction in the NK-mediated killing of target cells, the human NK92 cell line was assessed for its ability to kill monkey COS-7 cells either untransfected or transiently transfected with the NTBA construct. The NK92 cell line expresses NTBA, CD244, NKp30, NKp44, and NKG2D, but lacks the NKp46 receptor 1. As shown in Fig. 6A, NK92 cells displayed some cytolytic activity against untransfected COS-7 cells. This is likely to depend upon the human NKG2D recognition of conserved ligand(s) expressed on monkey cells 30, since it was sharply inhibited by mAb-mediated masking of NKG2D. On the contrary, in agreement with the fact that NTBA is absent on COS-7 cells, the mAb-mediated masking of NTBA had no inhibitory effect. Importantly, the NK92-mediated lysis of NTBA-transfected COS-7 cells was significantly higher than that of untransfected cells. Moreover, NK92-mediated killing of cell transfectants was inhibited by anti-NTBA mAb, and was virtually abrogated by the simultaneous mAb-mediated masking of NTBA and NKG2D (Fig. 6A).

Similar results were obtained using the human HEK-293T cell line either untransfected or transfected with the NTBA construct. In this case, the NK cell-mediated killing was triggered via NKG2D, since HEK-293T cells express several NKG2D ligands, including MHC class I-related chain A (MICA) and UL16-binding protein (ULBP)-1, ULBP-2, and ULBP-3 31. Accordingly, lysis of HEK-293T cells was inhibited by anti-NKG2D mAb. Also in this case the lysis of NTBA-transfected HEK-293T cells was higher than that of untransfected cells. Moreover, addition of anti-NTBA mAb inhibited the NK-mediated lysis of NTBA cell transfectants (Fig. 6B). Altogether, these data provide evidence that the homophilic recognition represents the molecular basis for NTBA-dependent function.

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Figure 6. NTBA/NTBA interaction induces NK cell triggering. The NK92 cell line was analyzed for cytolytic activity against COS-7 or HEK-293T cells either untransfected or transfected with the NTBA construct. Cytolytic assays were performed either in the absence of mAb (white bars) or in the presence of mAb to the indicated molecules (black bars) used alone or in combination. The E/T ratio used was 15:1. Data are representative of four independent experiments. The SD of the means of the triplicates was <5%.

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

In this study we provide evidence that NTBA, a molecule recently identified by our group, is capable of mediating homophilic interactions. Moreover, the NTBA/NTBA interaction results in NK cell triggering leading to target cell killing and lymphokine release. It has been postulated that the various members belonging to the CD2 family arose by duplications of a common ancestral gene encoding a molecule that displayed homophilic adhesion 16. According to this concept, the known ligands of the CD2-related proteins are represented by one or another member of this molecular family. In particular, it has been shown that these molecular interactions are either heterophilic, such as CD2/CD58, CD2/CD48, and CD244/CD48, or homophilic, such as CD150/CD150, CD84/CD84, and CS1/CS1 2228.

Using different approaches, we could demonstrate that the NTBA coreceptor has the capability of self recognition. Indeed, we show that soluble NTBA-Fc* selectively binds to NTBA cell transfectants while it does not recognize other members of the CD2 family, including CD48, CD58, CD84, CD150, CD229, and CD244. At the present it cannot be ruled out that NTBA might bind to emerging members of the CD2 family or to CS1, BLAME, or CD84H1, which were not analyzed in this study. The NTBA homophilic interaction has also been confirmed by ELISA assays and plasmon resonance. In this context, the low affinity of the NTBA/NTBA interaction is evident observing the BIAcore profile data. Indeed, the rapid dissociation that follows the end of the injection indicates that, similar to other homophilic interactions occurring between other members of the CD2 family, also NTBA is characterized by weak homophilic binding. Moreover, NTBA-Fc* induced a significant modulation of NTBA expression at the NK cell surface. Importantly, the NTBA/NTBA interaction resulted in functional activation of NK cells resulting in the enhancement of NK-mediated cytolytic activity and in the induction of cytokine production including both IFN-γ and TNF-α. In addition, it is of note that stimulation of NTBA by specific mAb was less efficient in terms of cytokine production as compared to NTBA-Fc*. These data are reminiscent of those obtained by comparing the cytokine production by NK cells in response to anti-NKG2D mAb or NKG2D-specific ligands such as MICA. In this case, stimulation of NK cells with soluble MICA but not with the anti-NKG2D mAb resulted in cytokine release 32. It has been shown that mAb-mediated engagement of CD150 and CD244 also enhances INF-γ production in T cells and in the YT cell line, respectively 33, 34.

Interestingly, in XLP patients a defect in INF-γ production upon engagement of CD244, CD84, or CD150 has been described 16. Moreover, in NK cells derived from these patients both CD244 and NTBA have been shown transduce abnormal inhibitory signals resulting in sharp inhibition of cytotoxicity against EBV-infected cells 1, 1921. This may contribute to the pathogenesis and to the progression of the disease since B-EBV-infected cells are characterized by high levels of expression of CD244- and NTBA-specific ligands, i.e. CD48 and NTBA itself, respectively. Thus, the functional impairment of the cytolytic activity and cytokine release may dramatically concur to the failure of viral clearance in XLP patients.

Finally, in the early steps of NK cell maturation, characterized by the expression of NCR in the absence of HLA class I-specific inhibitory receptors, both CD244 and NTBA molecules display inhibitory function (due to a later expression of SH2D1A) that contributes to self tolerance towards surrounding hematopoietic cell precursors 35. In this regard, the identification of the NTBA ligand may contribute to better define the mechanisms of self tolerance at early stages of NK cell differentiation.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Preparation of chimeric constructs

The cDNA sequence encoding the extracellular domains (including the leader sequence) of CD244 was amplified from codon 1 to codon 221. The coding primer used contained a SalI restriction site, while a BamHI restriction site was included in the complementary primer. The PCR product was digested with the appropriate restriction enzymes, purified using Perfectprep Gel Cleanup kit (Eppendorf) from 0.8% agarose gel, and ligated to Sal1-BamHI-cut pRB1 expression vector in frame with the cDNA sequence coding for hIgG1.

Mutagenesis was performed using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). Briefly, CD244-Fc/ pRB1 was amplified, using high-fidelity Pfu DNA polymerase, in the presence of a pair of oligonucleotide primers complementary to each other. The primers were designed in order to contain sequences coding for the three amino acids that we wanted to mutagenize (muta-up: 5′-CCCGAGGCACCTGAAGCAGAGGGGGCACCGTCAGTCTTCC, and muta-down: 5′-GGAAGACTGACGGTGCCCCCTCTGCTTCAGGTGCCTCGGG). The parental DNA template was digested by DpnI restriction endonuclease, specific for methylated and hemimethylated DNA sequences. The mutated vector was then transformed in bacteria. Sequencing checked the correct DNA sequence of the plasmid. This mutagenized plasmid, termed CD244-Fc*/pRB1, was used to subclone the extracellular regions of NTBA (from codon 1 to codon 346) to obtain the NTBA-Fc*/pRB1 construct. All the DNA plasmids were sequenced using d-Rhodamine Terminator Cycle Sequencing kit and a 3100 ABI automatic sequencer (Perkin Elmer-Applied Biosystems, Foster City, CA).

4.2 Production of soluble receptors

Plasmids encoding CD244-Fc, CD244-Fc*, or NTBA-Fc*/pRB1 were transiently transfected into HEK-293T using the non-liposomal FuGene-6 reagent (Roche, Monza, Italy) following the manufacturer's instruction. Transfected cells were cultured using Dulbecco's modified Eagle's medium supplemented with 10% ultra-low IgG FCS (Gibco BRL). The supernatants were collected on days 4 and 8 after transfection. Soluble chimeric proteins were purified by affinity on Protein A-Sepharose column (Amersham Biosciences). Protein concentration was determined using Bio-Rad protein assay. SDS-PAGE and silver staining established the purity of the soluble receptors.

4.3 HEK-293T transfection

The cDNA coding for the proteins belonging to the CD2 family were amplified starting from RNA extracted from different cells or cell lines. The primer sets used in this study are listed in Table 1; amplifications were performed for 30 cycles (30 s at 95°C, 30 s at the annealing temperature, and 30 s at 72°C) utilizing TAQ-GOLD (Perkin Elmer-Applied Biosystems) after a pre-activation for 10 min at 95°C. The obtained PCR products were cloned into pcDNA3.1/V5-His-TOPO expression vector (Invitrogen, Carlsbad, CA) and sequenced.

HEK-293T cells were transfected with CD2-, CD48-, CD84-, CD150-, CD229-, CD244-, or NTBA-pcDNA 3.1 plasmids using non-liposomal FuGene-6 reagent (Roche) following the manufacturer's instruction. The transfections were checked with mAb specific for the different CD2 family members and isotype-matched PE-conjugated goat anti-mouse second reagent. In particular the following mAb have been used: MAR206 (anti-CD2, IgG1), CO202 (anti-CD48, IgM), 152.1D5 (anti-CD84, IgG1; Ancell Corporation), IPO-3 (anti-CD150, IgG1; Kamiya Biomedical Company), pp35 (anti-CD244, IgG1), MA127 (anti-NTBA, IgG1) and Hly-9.1.25 (anti-hLy9, IgG1; kindly provided by P. Engel, Corporacio Sanitaria Clinic, Barcelona, Spain). To perform staining with soluble receptors, about 200,000 cells were incubated with 5 μg CD244-Fc* or with 7 μg NTBA-Fc* fusion protein for 30 min at 4°C; the Fc portion of human IgG chimeric molecules was detected using PE-conjugated goat anti-human antibodies (Southern Biotechnology Associates, Inc.). Cell acquisition was performed on FACScan flow cytometer (Becton Dickinson) and data analyzed using Cell Quest software (Becton Dickinson).

Table 1. Primer sets used in this study
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4.4 ELISA assay

Ninety-six-well plates (M. Medica, Milano, Italy) were coated overnight at 4°C with serial dilutions of NTBA-Fc* or 2B4-Fc* soluble receptors, washed (PBS pH 7.5, 0.05% Tween-20) and saturated with PBS containing 5% BSA overnight at 4°C. After three washes, 50 μl of NTBA-Fc* (4 μg/ml, PBS pH 7.5) labeled with EZ-LINK SULFO-NHS-LC-LC-BIOTIN (Pierce, Rockford, IL) was added to each well. Plates were washed three times and revealed using 1/2,000 dilution of Neutravidin horseradish peroxidase (HRP)-conjugated second reagent (Pierce). Optical density was read at 405 nm using the TECAN SUNRISE diagnostic photometer (TECAN, Salzburg, Austria).

4.5 Modulation of surface molecules

Flat-bottom ELISA plates (96 wells/plate) were coated overnight at 4°C with 50 μg/ml (100 μl) of NTBA-Fc* or CD244-Fc*. NK92 cells (105) were added to coated or uncoated plates and incubated for 18 h at 37°C. Cells were washed twice and used to determine the surface phenotype.

4.6 Cytokine production

IFN-γ and TNF-α production from polyclonal NK cells was measured in supernatants using ELISA (BIOSOURCE Int., Inc., Camarillo, CA). NK cells were incubated in 96-well U-bottom tissue culture plates (5×105 cells/ml). Cells were cultured in RPMI 1640 medium supplemented with 2 mM glutamine, 50 μg/ml penicillin, 50 μg/ml streptomycin, and 10% heat-inactivated FCS (Invitrogen, Life Technologies) in the presence of 1 ng/ml of rIL-12, purchased from Peprotech Inc. (London, GB). Purified NTBA-Fc* or purified mAb (anti-NTBA, MA127, IgG1; anti-NKG2D, BAT221, IgG1; anti-CD16, c127, IgG1) were used as plate-bound proteins at concentrations of 50 μg/ml (NTBA-Fc*) or 10 μg/ml (mAb). In some conditions, NK cells were first incubated with anti-NTBA (MA127, IgG1 mAb) or with anti-NKG2D (BAT221, IgG1 mAb) (10 μg/ml) for 30 min at room temperature 32.

4.7 Cytolytic activity

Enriched NK cells were isolated by incubating PBL with anti-CD3 (JT3A), anti-CD4 (HP2.6), and anti-HLA-DR (D1.12) mAb (30 min at 4°C) followed by goat anti-mouse-coated Dynabeads (Dynal, Oslo, Norway; (30 min at 4°C) and immunomagnetic depletion 1. CD3CD4DR cells were cultured on irradiated feeder cells in the presence of 100 U/ml rIL-2 (Proleukin; Chiron Corp., Emeryville, CA) and 1.5 ng/ml PHA (Gibco Ltd., Paisley, Scotland) in order to obtain polyclonal NK cell populations. NK cells were tested for cytolytic activity against untransfected or NTBA-transfected monkey COS-7 or human HEK-293T cells in a 4-h 51Cr-release assay as previously described 1. The concentration of the various mAb added for masking experiments was 10 μg/ml. The E/T ratios are indicated in the legend (Fig. 6).

4.8 BIAcore 3000 analysis

The BIAcore system instrument (Pharmacia Biosensor AB, Uppsala, Sweden) and its use have been described previously 36. In the BIAcore system, the binding of analytes to immobilized ligands is measured in arbitrary units (RU) and there is a linear relationship between the mass of protein bound to the immobilized ligand and the RU observed. Purified NTBA-Fc* at 500 μg/ml in 50 mM sodium acetate buffer at pH 4.5 was covalently immobilized on the dextran matrix of a CM5 chip by manual immobilization. The running buffer used in these experiments (20 mM Hepes buffer pH 7.4, containing 150 mM NaCl, 5 mM NaCl, 5 mM EDTA and 0.05% Tween-20) was also used to dialyze the protein and to dilute the samples for the injection. Each diluted sample was passed over both immobilized NTBA-Fc* and CD244-Fc* channels at a flow rate of 30 μl/min, setting 3 min of contact and a dissociation time of 180 s. Purified specific anti-NTBA mAb (MA127) was injected over the surface at a concentration of 0.5 μg/ml. Regenerations between injections were performed with 10 mM HCl.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

This work was supported by grants awarded by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), Istituto Superiore di Sanità (I.S.S.), Ministero della Sanità, Ministero dell'Università e della Ricerca Scientifica e Tecnologica (M.I.U.R.), and Consiglio Nazionale delle Ricerche, Progetto Finalizzato Biotecnologie. Also the financial support of Fondazione Compagnia di San Paolo, Torino, Italy, is gratefully acknowledged.

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