Dec 13, 1999 - Michael Bennett 7 Vinay Kumar. Porunelloor A. Mathew. Molecular cloning of transmembrane and soluble forms of a novel rat natural killer cell ...
Immunogenetics (2000) 51 : 306–313
Q Springer-Verlag 2000
ORIGINAL PAPER
Pappanaicken R. Kumaresan 7 Susan E. Stepp Michael Bennett 7 Vinay Kumar Porunelloor A. Mathew
Molecular cloning of transmembrane and soluble forms of a novel rat natural killer cell receptor related to 2B4 Received: 1 July 1999 / Revised: 13 December 1999
Abstract Natural killer (NK)-cell recognition of target cells and cytolytic function are controlled by multiple receptor-ligand interactions. These receptors can transmit either positive or negative signals and belong to the lectin superfamily or immunoglobulin superfamily (IgSF). One member of the IgSF, 2B4, is expressed on the surface of all mouse and human NK cells and the subset of T cells that mediate NK-like killing. In both mouse and human, 2B4 is a transmembrane protein and is the counter-receptor for CD48. Northern blot analysis had indicated the existence of 2B4-related genes. Here we report the cloning of novel cDNAs (r2B4R) closely related to the rat 2B4. Unlike 2B4, rat NK cells express mRNA corresponding to both transmembrane (r2B4R-tm) and soluble (r2B4R-se) forms of r2B4R. r2B4R-tm contains an open reading frame encoding a polypeptide of 311 amino acid residues. The encoded protein has characteristics of type I transmembrane proteins with a 20-amino acid leader sequence, a 203-amino acid extracellular domain, a 23-amino acid transmembrane domain, and a 65-amino acid cytoplasmic domain. r2B4R-se encodes a protein of 205 amino acid residues without a putative transmembrane domain. Northern blot analysis and reverse transcriptasePCR analysis revealed that both transmembrane and soluble forms of r2B4R are expressed in interleukin-2activated NK cells. Key words Natural killer cells 7 Immunoglobulin superfamily 7 Rat 2B4R 7 2B4
P.R. Kumaresan 7 P.A. Mathew (Y) Department of Molecular Biology and Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA e-mail: pmathew6hsc.unt.edu Tel.: c1-817-7352120 Fax: c1-817-7352118 S.E. Stepp 7 M. Bennett 7 V. Kumar Immunology Graduate Program, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA
Introduction Natural killer (NK) cells are bone marrow-derived large lymphocytes that play a major role in immune surveillance against malignancy, and are part of the primary defense against bacterial and viral infections without prior sensitization (Trinchieri 1989). NK cells also mediate the acute rejection of major histocompatibility complex (MHC)-unmatched bone marrow transplants (George et al. 1997). NK-cell cytotoxicity and cytokine production have been proposed to be controlled by a balance of activating and inhibiting receptors of the NK cell (Colonna et al. 1999; Lanier et al. 1997). Resting NK cells express a number of surface molecules which, when stimulated, can activate their cytotoxic mechanism. These receptors include CD16 (FcgRIII), CD2, the NKR-P1 family, CD28, CD40, CD69, KARs, and 2B4 (Lanier 1998; Yokoyamma 1997). In addition, some members of the Ly49 and CD94/NKG2 family can also mediate activation signals to NK cells (Ryan and Seaman 1997). 2B4 is a novel member of the CD2 family of the immunoglobulin superfamily (IgSF) and is expressed on all mouse NK cells and a subset of T cells. It has been shown to transduce activation signals upon surface cross-linking with anti-2B4 monoclonal antibody (mAb) (Garni-Wagner et al. 1993; Mathew et al. 1993). 2B4 is also involved in the killing of skin tumor cells by mouse dendritic epidermal T cells (Schuhmachers et al. 1995a, 1995b). Mice have two isoforms of 2B4, which are produced by alternative splicing of a single 2B4 gene (Stepp et al. 1999). These two isoforms differ only in their cytoplasmic region and transduce opposing signals upon ligation with anti-2B4 mAb (Schatzle et al. 1999). Recently, an allelic variant of 2B4 has been reported that is expressed on NK cells from all mice strains tested (Kubota et al. 1999). In humans, only the activating form of 2B4 has been identified and characterized (Boles et al. 1999; Nakajima et al. 1999; Tangye et al. 1999). In both mouse and human, 2B4 is the
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counter-receptor for CD48 (Brown et al. 1998; Latchman et al. 1998). 2B4 hybridized with several transcripts in activated NK cells, indicating the presence of 2B4-related genes (Boles et al. 1999; Mathew et al. 1993). Here we report the molecular cloning of 2B4-related genes (r2B4R) expressed in rat NK cells. Unlike 2B4, r2B4R does not contain any immunoglobulin tyrosinebased inhibitory motif/ immunoglobulin tyrosine-based activation motif (ITIM/ITAM)-like sequence motifs in the cytoplasmic domain, suggesting that they may have a different role in NK cell function. We identified and characterized cDNAs corresponding to transmembrane and secreted forms of rat 2B4R.
Materials and methods Cell purification and cell culture RNK-16, a rat NK leukemia cell line, was cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (Hyclone laboratories, Logan, Utah), 2 mM glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids (complete media; Gibco BRL, Gaithersberg, Md.). Rat spleen adherent lymphokine-activated killer (ALAK) cells were prepared as previously described with slight modification (Reynolds et al. 1981). Briefly, Rat A-LAK cells were generated by culturing nylon wool-nonadherent spleen cells in T75 flasks (Corning, Corning, N.Y.) in RPMI-1640 complete medium prepared as for RNK16 cells with 55 mM b-mercaptoethanol (Gibco), 50 mg/ml gentamycin, and 1000 units/ml human recombinant interleukin-2 (rIL-2) for 3 days at 37 7C in 5% CO2. On the 3rd day of culture, the nonadherent cells were removed by gentle washing with warm complete RPMI media. The adherent cells were further cultured and fed every 2–3 days with LAK culture medium, maintaining IL-2 at a concentration of 1000 units/ mL. Cells were harvested at 4–5 days of culture. cDNA library screening and sequence analysis A cDNA library made from the RNK-16 cell line was a kind gift from H. Young, NIH, Frederick, Md. It was constructed in the lgt11 vector, at the EcoRI cloning site (Smyth et al. 1992). 1.5!10 6 plaques were lifted onto N c nylon membrane (Amersham, Piscatawy, N.J.) and membranes were prehybridized at 65 7C using Blotto as blocking buffer. The membranes were then hybridized with a 32P-dCTP-labeled full-length m2B4L cDNA probe as described by Feinberg and Vogelstein (1983) for 18 h. Membranes were washed four times for 5 min in 2!sodium chloride and sodium citrate solution (SSC), 0.1% sodium dodecylsulfate (SDS) at room temperature, twice for 30 min at 60 7C in 1!SSC, 0.1% SDS, and subjected to autoradiography for 18 h. After three successive screenings, positive clones were selected and l DNA was isolated from positive clones by the method of Lee and Clark (1997). The size of the insert was identified by EcoRI digestion and sequencing was performed with phage vector-specific primers (Automated Sequencing Service Facility, Molecular Oncology Center, UT Southwestern Medical Center, Dallas, Tex.). Northern blot analysis Twenty micrograms of total RNA from RNK-16 cells or A-LAK cells was run on 1.2% formaldehyde gels and transferred onto a nylon membrane by conventional methods (Sambrook et al.
1989). RNA blot hybridizations were performed using a 32PdCTP-labeled full-length r2B4R-tm cDNA. The probe was labeled using the Megaprime DNA Labeling Kit (Amersham) following the manufacturer’s instructions. Blots were prehybridized with Denhart’s hybridization buffer (5!SSC, 5!Denhart’s solution, 0.5% SDS, 50 mM sodium phosphate buffer pH 6.5, and 100 mg single-strand DNA/ml) for 4 h and a 32P-dCTP-labeled probe was added in the hybridization solution. After 18 h hybridization, the membranes were washed four times with 2!SSC at room temperature, then twice for 30 min at 65 7C in 1!SSC, 0.1% SDS, and subjected to autoradiography.
Reverse transcriptase-PCR Total RNA was isolated from RNK-16 cells and rat A-LAK cells by the RNAzol method following the manufacturer’s instructions (Tel-test, Friendswood, Tex.). First-strand cDNAs were prepared in a 20-ml reaction volume containing 1!first-strand buffer, 0.1 M dithiothreital, 0.5 mM dNTP, 5 mg of total RNA, 200 units modified SuperScript (II) reverse transcriptase and 250 ng of random primers (Gibco). PCR was performed with forward primer 1F and reverse primer 1R that amplify both r2B4R-tm and r2B4R-se forms. The oligonucleotide primers used in this study are given in Table 1. The PCR mixture contained 0.1 mm of each primer, 0.2 mM dNTPs, 2 mM MgCl2, 1!PCR buffer, I unit Taq DNA polymerase (Gibco), and 5 ml of cDNA reaction product in a reaction volume of 50 ml. PCR conditions were 94 7C for 4 min, 35 cycles of 94 7C for 1 min, 55 7C for 1 min, 72 7C for 1 min, followed by extension at 72 7C for 7 min. First-strand cDNA synthesis was confirmed by performing PCR using GAPDH primers. The amplified products were subcloned into the PCR2.1 vector (Invitrogen, San Diego, Calif.) and sequenced using vector-specific primers.
Southern blot analysis Genomic DNA was extracted from rat liver by conventional methods (Sambrook et al. 1989) and digested with EcoRI, BamHI, XbaI, and HindIII restriction endonucleases (Gibco BRL). The digested DNA was subjected to horizontal agarose gel electrophoresis and blotted onto nitrocellulose membranes (Amersham) by conventional methods (Sambrook et al. 1989). The membrane was prehybridized for 5 h at 65 7C in prehybridization buffer (1 mM ethylenediamine tetraacetic acid, 0.5 M sodium phosphate, pH 7.2 and 7% SDS). Full-length a 32P-dCTP-labeled r2B4R-tm cDNA probe was added to the same buffer and hybridization was continued for 18 h at 65 7C. The membrane was washed with a buffer containing 40 mM sodium phosphate, pH 7.2, 1% SDS at 65 7C for 1 h, and exposed for autoradiography.
Results Isolation and characterization of a cDNA encoding a soluble form of r2B4R We screened an RNK-16 cDNA library constructed in lgt11 vector using full-length mouse 2B4 long (m2B4L) cDNA as a probe and isolated five different cDNA clones. After EcoRI digestion of the positive clones, two different sizes of inserts (1.4 and 1.2 kb) were obtained. Three of the clones contained the 1.4-kb insert and two of the clones had the 1.2-kb insert. The inserts were systematically purified, cloned, and sequenced. The 1.4-kb inserts contained full-length clones, while
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Fig. 1 A The nucleotide sequence and predicted amino acid sequence of r2B4R-se (GenBank accession number AF156988). The signal peptide is underlined. Glycosylation sites in the extracellular domain are boxed. B Hydrophilicity plot of the r2B4R-se putative peptide sequence determined by the Kyte-Doolittle method
the smaller clones contained partial cDNAs. One of the 1.2-kb partial cDNA clones differed from the others. The nucleotide sequence of one of the full-length clones, R7A3, contained a single open reading frame of 615 nucleotides encoding a polypeptide chain of 205 amino acids. The sequence contained a 118-nucleotide 5b untranslated region and a 600-nucleotide 3b untranslated region (Fig. 1A). The predicted protein of 205 amino acids has a potential N-terminal hydrophobic re-
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gion of 20 amino acids, presumably representing a secretory signal. The predicted protein does not contain another hydrophobic region characteristic of transmembrane proteins (Fig. 1B). Moreover, the absence of a GPI-anchored signal sequence (SSG) at the C-terminal of the protein suggests that it is not a GPI-anchored membrane-bound protein (Medof et al. 1996). The predicted protein showed 89% similarity to rat 2B4 (P.R. Kumaresan and P.A. Mathew, unpublished data). However, the protein does not contain the signaling motif (TxYxxV/I) present in rat, mouse, and human 2B4. Therefore, we designate this form as the soluble form of rat 2B4R (r2B4R-se). The predicted pro-
tein contains three potential N-linked glycosylation sites (Asn-X-Ser/Thr), suggesting that it may be glycosylated.
Fig. 2 A The nucleotide sequence and predicted amino acid sequence of r2B4R-tm (GenBank accession no. AF156989). The signal peptide is underlined. Glycosylation sites in the extracellular domain are boxed. The transmembrane domain is double underlined. The lysine amino acid in the transmembrane domain is indicated by an arrowhead. B Hydrophilicity plot of the r2B4R-tm putative peptide sequence determined by the Kyte-Doolittle method
310 Table 1 PCR primer sequences and their relative position to r2B4R-tm and r2B4Rse cDNA (from the first base of the respective clones)
Primer
1F 1R 2F 2R 3F 3R
Oligonucleotide sequences
5b-CTCATCAGGGCCAAGATTGTG-3b 5b-TCCACACACAGAAGCAAATGATGG-3b 5b-TAGACATCAGAGCACCTGGAG-3b 5b-CTTCTGGGCTCCACTTATCA-3b 5b-TATGGCACCCACTGGGAGAACC-3b 5b-AAGGGACACTCTGACAGCTCTGC-3b
Isolation and characterization of a cDNA encoding a transmembrane form of r2B4R One of the partial cDNA clones obtained differed in nucleotide sequence from the other clones. Comparison with the full-length clone and translation of the partial coding region revealed that this cDNA clone corresponded to a transmembrane form of r2B4R. It was identical with the r2B4R-se, except that it conFig. 3 Comparison of predicted amino acid sequence of rat 2B4R with mouse and human 2B4. Mouse and human 2B4 residues matching those of r2B4R-tm and r2B4R-se are shaded. Predicted transmembrane domains are boxed. Compiled with the Genetics Computer Group Wisconsin software package
Relative nucleotide position r2B4R-tm
r2B4R-se
165–185 800–777 1–21 1351–1331 586–609 714–691
105–125 730–707 61–81 1283–1263 P P
tained a 131-bp addition in the open reading frame that resulted in a hydrophobic transmembrane region. To obtain the full-length cDNA corresponding to this transmembrane form, we designed two distal primers (2F and 2R) based on the r2B4R-se sequence and reverse transcriptase-PCR was performed using total RNA from RNK-16 (Table 1). Two fragments of F1.3 kb and F1.2 kb were amplified and subcloned as described in Materials and methods. The sequence
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analysis of four clones (R3a, R3b, R4a, and R5a) from two different PCR reactions showed that the 1.3-kb fragment corresponded to a transmembrane form (r2B4R-tm) and the 1.2-kb fragment corresponded to the soluble form (r2B4R-se) of rat 2B4R. The nucleotide sequence of the 1.3-kb rat 2B4R cDNA clone comprised 1351 nucleotides, encoding a polypeptide chain of 311 amino acid residues (Fig. 2A). The predicted protein has an N-terminal hydrophobic region of 20 amino acids, presumably representing a secretory signal sequence. The predicted mature protein has an extracellular domain with 203 amino acid residues, a 23-amino acid hydrophobic membrane-spanning domain, and a 65-amino acid cytoplasmic domain. The presence of a hydrophobic region suggests that this rat 2B4R form is a transmembrane form (Fig. 2B). Therefore, we designate this cDNA as r2B4R-tm. The predicted protein has six potential N-linked glycosylation sites (Asn-X-Ser/Thr), in the extracellular domain, suggesting that the protein may be glycosylated. It has one putative protein kinase C phosphorylation site in the cytoplasmic domain. Comparison of r2B4R-tm and r2B4R-se forms at the nucleotide level showed that there is a 131-nucleotide deletion in the cDNA corresponding to the C2 domain of the r2B4R-se form. Except for this deletion, the two forms showed 100% identity at the nucleotide level, indicating that these isoforms are derived from a common gene. The predicted proteins showed 91% similarity. The difference between the r2B4R-tm and the r2B4R-se forms at the protein level is due to the 131nucleotide deletion in r2B4R-se which results in a frameshift in the translational reading frame, which introduces a stop codon in r2B4-se before the predicted hydrophobic transmembrane domain. Comparison of rat 2B4R Comparison of r2B4R-tm and r2B4R-se with rat 2B4 (P.R. Kumaresan and P.A. Mathew, unpublished data) showed 94% and 89% similarity, respectively. Both r2B4R-tm and r2B4-se also showed 77% similarity to mouse 2B4 (m2B4L as well as m2B4S) and 58% similarity to human 2B4. Alignment of the protein sequences of r2B4R-tm and r2B4R-se forms with mouse and human 2B4 forms is shown in Fig. 3. The protein sequence was analyzed with a pileup program in the GCG package (Genetics Computer Group, Madison, Wisc.). The comparison showed that the C2 domain is highly conserved in rat 2B4R and mouse and human 2B4 forms. Unlike mouse and human 2B4, the 2B4Rtm contains a charged lysine residue in its transmembrane domain, suggesting possible association with other signaling molecules. A search was made for other IgSF-related proteins that may have homology with 2B4R molecules using BLAST search (http:/ /www.ncbi.nlm.nih.gov/cgi-bin/BLAST). It revealed that both r2B4R-tm and r2B4R-se forms show high
similarity with CD48 (46% each), and signaling lymphocyte activation molecule (SLAM) (45% and 49%, respectively).
Expression of mRNA corresponding to soluble and transmembrane forms of r2B4R Northern blot analysis of total RNA from RNK-16 cells and A-LAK cells yielded three major bands of approximately 5, 2.8, and 2 kb in size when using full-length r2B4R-tm as a probe. These three mRNA species could represent closely related transcripts or differentially spliced products of the same gene. Some of the hybridizing bands may be rat 2B4 because of the high homology (80% identity) of r2B4R-tm with r2B4 at the nucleotide level (P.R. Kumaresan and P.A. Mathew, unpublished data). Total RNA isolated from rat nonadherent LAK cells hybridized weakly with the above probe suggesting that r2B4R may also be expressed in a subpopulation of T cells (Fig. 4). The expression patterns of r2B4R-tm and r2B4R-se were analyzed by reverse transcriptase-PCR of total RNA isolated from rat A-LAK cells. PCR was performed with primers that could amplify both r2B4R-tm and r2B4R-se forms. Two DNA fragments of 0.7 kb and 0.56 kb, corresponding to r2B4R-tm and r2B4R-se were amplified from IL-2-activated rat NK cells (Fig. 5). Sequence analysis of the fragments further confirmed that the 0.7-kb and 0.56-kb fragments corresponded to r2B4R-tm and r2B4R-se, respectively.
Fig. 4 Northern blot analysis. Twenty-five micrograms of total RNA from YAC, RNK-16, A-LAK, and nonadherent LAK (NA-LAK) cells was electrophoresed in a 1.2% formaldehyde denaturing gel, blotted, and hybridized with 32 P-labeled full-length r2B4Rtm probe. Hybridization of the membrane with a b-actin gene control probe is shown at the bottom. Molecular-weight markers are shown to the left
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size is about 27 kb (Stepp et al. 1999). The pattern obtained by Southern blot, using a 128-bp probe specific for r2B4R-tm (nucleotides 586–714), identified the same restriction fragments that hybridized to fulllength r2B4R-tm and r2B4R-se probes, suggesting that both forms are derived from the same gene (data not shown).
Discussion
Fig. 5 Reverse transcriptase (RT)-PCR analysis. The expression of r2B4R-tm and r2B4R-se forms in RNK-16 and A-LAK cells from F344 and Wistar rats. cDNA clones corresponding to r2B4R-tm and r2B4R-se were used as positive controls and the size of the amplified products is shown to the right. First-strand cDNA was synthesized from 5 mg of total RNA using superscript II, a modified RT. PCR was performed using 2 ml of cDNA mixture, under standard conditions for 35 cycles. The RT-PCR products were run on a 1.6% agarose gel and stained with ethidium bromide. PCR amplification of GAPDH from the same cDNA is shown at the bottom
Genomic DNA analysis Southern blot analysis of EcoRI, BamHI, HindIII, and XbaI restriction digests of rat genomic DNA showed hybridization of several bands with full-length r2B4Rtm probe (Fig. 6). The sizes of the different restriction fragments identified in Fig. 6 account for a gene of approximately 30 kb in rat. Characterization of a mouse 2B4 genomic clone indicated that the mouse 2B4 gene Fig. 6 Southern blot analysis. Twenty micrograms of ratgenomic DNA was digested with EcoRI, BamHI, HindIII, and XbaI restriction enzymes and electrophoresed in a 0.8% agarose gel, blotted, and probed with 32P-labeled fulllength r2B4R-tm cDNA. Molecular-weight markers are shown to the left
NK cells express several receptors that recognize MHC class I molecules on target cells. We have previously identified a non-MHC-recognizing receptor, 2B4, expressed on all NK cells and a subset of T cells. Northern blot analysis identified several transcripts in activated NK cells that hybridized to 2B4, suggesting the presence of 2B4-related genes. 2B4 belongs to the CD2 family of the IgSF. Most of the CD2 family members function as cell adhesion and stimulatory molecules, doing so as both membrane-bound and soluble forms. Soluble forms may be created either by breaking the membrane structure at some stage, as seen in CD48 (Smith et al. 1997) and CD58 (Hoffmann et al. 1996) molecules, or by deletion of domains during alternative splicing in mRNA processing, thus producing several isoforms, as with the molecule SLAM (CD150w) (Cocks et al. 1995). SLAM is an IgSF molecule expressed as a soluble, secreted, and a transmembrane form in activated T cells (Aversa et al. 1997) and in B cells (Punnonen et al. 1997). Here we identified a novel member of the CD2 family which is closely related to 2B4. Like other members of the CD2 family, based on the predicted protein structure, rat 2B4R also exists in both soluble and transmembrane forms. r2B4R-tm has a short cytoplasmic tail and does not contain any ITIM or ITAM signaling motifs. In mouse, m2B4L and m2B4S do not contain any consensus ITIM or ITAM motifs, but have sequences that show some similarity to these motifs (Mathew et al. 1993; Stepp et al. 1999). Ligation of m2B4S with anti-2B4 mAb stimulates NK-cell cytotoxicity, whereas the m2B4L form inhibits NK-cell cytotoxicity, indicating the presence of a divergent signaling mechanism (Schatzle et al. 1999). Certain isoforms of the mouse Ly-49 NK-cell receptors lack an ITIM sequence in their cytoplasmic domain and they associate noncovalently with small transmembrane proteins containing ITAM and transduce positive signals (Smith et al. 1998). The presence of a positively charged lysine residue in the transmembrane domain of r2B4R-tm and the absence of signaling motifs in the cytoplasmic domain indicate that it may also associate with other molecules for signal transduction. DAP12, a disulfide-bonded homodimer containing an ITAM in its cytoplasmic domain, was recently shown to associate noncovalently with membrane glycoproteins of the killer cell-inhibitory receptor (KIR) family in NK cells (Lanier et al. 1998a). Cross-linking of KIR-DAP12 complexes results in cellular activation, as demon-
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strated by tyrosine phosphorylation of cellular proteins and up-regulation of early activation antigens (Lanier et al. 1998b). At present, the function of r2B4R is unknown. Soluble molecules of the CD2 family have been reported to be involved in costimulatory functions. The soluble form of SLAM, a member of the CD2 family, activates B-cell function by enhancing the production of IgM, IgA, and IgG (Punnonen et al. 1997). The expression of both r2B4R-tm and r2B4R-se in rat NK cells indicates that they may be involved in similar types of functions as other CD2 family members. mAbs are being generated to study the specific role of r2B4R-tm and r2B4Rse in NK-cell biology. Acknowledgements We thank Drs. Bill Seaman and Erene C. Niemi (University of California, San Francisco, Calif.) for providing RNK-16 cells, and Dr. Howard A. Young (NIH, Frederick, Md.) for providing the RNK-16 cDNA library. The authors thank Drs. S. Chuang and K. Boles for valuable discussions. This work was supported by National Institutes of Health Grant AI 38938.
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