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The Journal of Immunology, 2003, 171: 3415-3425.
Copyright © 2003 by The American Association of Immunologists

KIR2DL4 Is an IL-2-Regulated NK Cell Receptor That Exhibits Limited Expression in Humans but Triggers Strong IFN-{gamma} Production1

Akiko Kikuchi-Maki, Sei-ichi Yusa, Tracey L. Catina and Kerry S. Campbell2

Fox Chase Cancer Center, Division of Basic Science, Institute for Cancer Research, Philadelphia, PA 19111


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Killer cell Ig-like receptor (KIR)2DL4 (2DL4, CD158d) was previously described as the only KIR expressed by every human NK cell. It is also structurally atypical among KIRs because it possesses a basic transmembrane residue, which is characteristic of many activating receptors, but also contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). We expressed epitope-tagged 2DL4 in an NK-like cell line to study receptor function. Three distinct 2DL4 cDNA clones were analyzed: one encoding the "conventional" 2DL4 with the cytoplasmic ITIM (2DL4.1) and two encoding different cytoplasmic truncated forms lacking the ITIM (2DL4.2 and 2DL4*). Surprisingly, one truncated receptor (2DL4.2), which is the product of a prevalent human 2DL4 allele, was not expressed on the cell surface, indicating that some individuals may lack functional 2DL4 protein expression. Conversely, both 2DL4.1 and 2DL4* were expressed on the cell surface and up-regulated by IL-2. Analysis of primary NK cells with anti-2DL4 mAb confirmed the lack of surface expression in a donor with the 2DL4.2 genotype. Donors with the 2DL4.1 genotype occasionally expressed receptor only on CD56high NK cells, although their expression was up-regulated by IL-2. Interestingly, Ab engagement of epitope-tagged 2DL4 triggered rapid and robust IFN-{gamma} production, but weak redirected cytotoxicity in an NK-like cell line, which was the opposite pattern to that observed upon engagement of another NK cell activating receptor, NKp44. Importantly, both 2DL4.1 and 2DL4* exhibited similar activation potential, indicating that the ITIM does not influence 2DL4.1 activating function. The unique activation properties of 2DL4 suggest linkage to a distinct signaling pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Natural killer cells play a vital role in the immune response to virus infection and cancer by providing linkage between innate and adaptive immunity. They can mediate direct lysis of susceptible target cells by releasing cytotoxic granules containing perforin and granzymes or by expressing ligands for apoptosis-inducing receptors on the target cell, such as Fas ligand (FasL)3 and TNF-related apoptosis-inducing ligand (1, 2, 3). In addition, NK cells secrete cytokines, such as IFN-{gamma} and TNF-{alpha}, during infection and inflammation (4). These cytokines are important for tumor and viral clearance, as well as lymphocyte recruitment and differentiation.

The physiological functions of NK cells are regulated by a balance of signals transmitted through activating receptors and inhibitory receptors (5, 6). A group of inhibitory receptors recognize MHC class I molecules on normal cells and dominantly suppress NK cell activation (7, 8). These MHC class I-binding inhibitory receptors contain at least one conserved immunoreceptor tyrosine-based inhibitory motif (ITIM) in their intracytoplasmic domain. In contrast, most NK cell-activating receptors are characterized by the presence of a basic amino acid in the transmembrane domain and the lack of an ITIM in the intracytoplasmic domain (9). The basic transmembrane residue associates noncovalently with acidic transmembrane residues in distinct signaling accessory proteins, named DAP10, DAP12, Fc{epsilon}RI{gamma}, and TCR{zeta}, which contain immunoreceptor tyrosine-based activation motifs or YxxM motifs in their cytoplasmic domains. KIRs constitute a family of polymorphic receptors with members that are activating or inhibitory (10). The activating forms possess a transmembrane lysine, exhibit short cytoplasmic domains that lack ITIMs, and are associated with DAP12, whereas inhibitory forms have long cytoplasmic domains with two ITIMs, lack a basic transmembrane residue, and lack DAP12 association.

KIR2DL4 (2DL4; CD158d) is the only member of the KIR family reportedly expressed in every NK cell clone analyzed from humans, as assessed by mRNA expression (11, 12), and has been identified in several lower primates (13, 14, 15, 16). This broad expression profile and evolutionary conservation strongly implies that the receptor serves a critical role in the biological function of NK cells. Because a 2DL4-specific mAb has only recently been described (17), however, the surface expression and function of 2DL4 protein has not been fully clarified. Interestingly, 2DL4 is structurally unique among KIRs, because it exhibits elements of both inhibitory and activating NK cell receptors: 1) the cytoplasmic domain possesses a single ITIM and 2) the transmembrane domain contains a basic arginine residue (18). In accordance with this, both inhibitory and activating functional properties have been described for 2DL4 (17, 19, 20). The cytoplasmic domain of 2DL4, containing the single ITIM, can exhibit strong Src homology 2-containing protein tyrosine phosphatase (SHP)-1-independent inhibitory function in isolation, when fused to the extracellular and transmembrane domains of KIR3DL1 (19) or when the 2DL4 transmembrane arginine residue is mutated (20). On the other hand, engagement of full-length 2DL4 has also been shown to trigger activating function with the unique capacity to induce IFN-{gamma} production, but not cytotoxicity in resting NK cells (17). This is distinct among most NK cell-activating receptors, which usually trigger both functional responses.

The extracellular domain of 2DL4 exhibits both D0 and D2 Ig-like domains, which classifies the receptor as a type II KIR, along with KIR2DL5 (21). 2DL4 does not recognize classical MHC class I molecules, but instead two groups have reported that it binds to the nonclassical HLA-G (22, 23, 24). HLA-G is normally expressed only on fetal-derived trophoblast cells that invade the maternal decidua and appear to create a barrier for maternal NK cell attack of the HLA-mismatched fetus (25, 26, 27). An attractive hypothesis has been proposed suggesting that the engagement of 2DL4 by HLA-G might stimulate IFN-{gamma} secretion but not cytotoxicity by uterine NK cells to support normal pregnancy and prevent fetal attack (24). In support of this hypothesis, NK cells are the predominant uterine lymphocyte population in pregnant women, and studies in mice by Croy and colleagues (28, 29) have shown that both NK cells and IFN-{gamma} are essential for promoting optimal placental vascularity and development.

In this study, we utilized retroviral transduction to express N-terminal FLAG-tagged versions of 2DL4 in the NK-92 cell line. This allowed us to analyze the surface expression of 2DL4, as well as its function. To determine the role of the ITIM in the full-length receptor, three 2DL4 clones were analyzed: one that contains the cytoplasmic ITIM in frame (2DL4.1), and two others that contain cytoplasmic frame shifts resulting in truncated cytoplasmic domains that lack the ITIM (2DL4.2 and 2DL4*). We report the lack of surface expression of the product of a widely prevalent truncated receptor genotype (2DL4.2). In addition, the surface expression of 2DL4 on NK-92 cells and primary NK cells is shown to be up-regulated by IL-2. We further provide evidence that 2DL4 is indeed an activating receptor that strongly stimulates IFN-{gamma} production and the up-regulation of cell surface activation markers. Interestingly, direct comparison between 2DL4 and NKp44 revealed significant differences in their activating capacities, with 2DL4 stimulating stronger IFN-{gamma} production, but weaker redirected cytotoxicity. Importantly, the same activation potential was observed for 2DL4 isoforms that possess or lack the cytoplasmic ITIM, indicating that the ITIM does not influence activating function. Finally, engagement of 2DL4 can up-regulate FasL to trigger apoptosis of appropriate target cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and culture

The IL-2-dependent NK-like cell line, NK-92 (a gift from Dr. C. Lutz, University of Iowa, Iowa City, IA), was maintained as previously described (19). NK-92 cells were passed with fresh IL-2-containing medium every 4 days. The murine mastocytoma, P815, was cultured as previously described (19). Jurkat T cells were cultured in RPMI 1640 medium (Life Technologies, Rockville, MD) containing 10% FBS (HyClone Laboratories, Logan, UT), 2 mM L-glutamine, 100 µg/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES (pH 7.4), 1 mM sodium pyruvate (all supplements from Life Technologies), and 50 µM 2-ME (Fisher, Pittsburgh, PA). The amphotropic retroviral packaging line, Phoenix-Ampho, was provided by Dr. G. Nolan (Stanford University, Stanford, CA). Phoenix-Ampho cells were cultured in DMEM medium (containing 10% FBS, 2 mM L-glutamine, 100 µg/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-ME, and 10 mM HEPES) and passed before reaching confluence. NK3.3 cells were kindly provided by Dr. J. Kornbluth (St. Louis University School of Medicine, St. Louis, MO) and grown as previously described (19). For analysis of primary NK cells, blood samples were obtained from volunteer adult donors that were recruited by written informed consent under procedures approved by our Institutional Review Board. PBMC were isolated with Ficoll-Hypaque (Amersham Biosciences, Uppsala, Sweden) and cultured in RPMI 1640 medium containing 5% autologous serum or human AB serum (ICN Biomedicals, Aurora, OH), nonessential amino acids, 2 mM L-glutamine, 100 µg/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 50 µM 2-ME, and 500 U/ml recombinant human IL-2 (Roche, Basel, Switzerland; generously provided by the Biological Resources Branch, National Cancer Institute, Frederick, MD). Primary cells were split with fresh IL-2 every 3 days, beginning on day 1 after initiation. All cell culture was performed at 37°C in humidified 7% CO2 atmosphere.

Antibodies

The anti-FLAG mAb, M2, was purchased from Sigma-Aldrich (St. Louis, MO). PE-conjugated anti-CD69 mAb, biotin-conjugated anti-human FasL mAb (NOK1), and PE-conjugated anti-CD25 mAb were purchased from BD PharMingen (San Diego, CA). For intracellular staining, permeabilized cells were incubated with PE-conjugated anti-human IFN-{gamma} mAb (B27) or an isotype control, PE-conjugated mouse IgG1 (Caltag Laboratories, Burlingame, CA). PE-conjugated goat anti-mouse {kappa} (Southern Biotechnology Associates, Birmingham, AL) and PE-conjugated streptavidin (BD PharMingen) were used as secondary reagents. Protein G-purified anti-CD56 mAb, B159.5.2, and anti-NKp44 mAb, 3.43.13 (both mouse IgG1 isotypes) were prepared from the hybridomas, which were kindly provided by Dr. B. Perussia (Thomas Jefferson University, Philadelphia, PA) and Dr. M. Colonna (Washington University, St. Louis, MO), respectively. Anti-KIR2DL4 mAb (64, mouse IgM isotype) (17) was kindly provided by Dr. E. Long (National Institutes of Health, Rockville, MD). For the detection of 2DL4 on primary PBMC, cells were sequentially stained with anti-2DL4 (1 µl ascites/50 µl), biotinylated rat anti-mouse IgM (b-7-6, produced in our laboratory), and PerCP-conjugated streptavidin (BD PharMingen). For identification of the NK cell population (CD3-CD56+), PBMC were subsequently stained with FITC-conjugated anti-CD3 mAb (Immunotech, Marseilles, France) and PE-conjugated anti-CD56 mAb (Immunotech). All stained cells were analyzed on a FACScan (BD Biosciences, Mountain View, CA).

cDNA constructs

Total mRNA was extracted from NK3.3 cells with RNAzol reagent (Tel-Test, Friendswood, TX). The following gene specific primer sequences for KIR2DL4 were used to generate full-length cDNA from immediately after the leader by RT-PCR (C.therm. polymerase for RT-PCR; Boehringer Mannheim, Mannheim, Germany): forward primer containing EcoRI site 5'-TAGCTGAATTCACACGTGGGTGGTCAGGA-3' and reverse primer containing XhoI site 5'-TACGACTCGAGTGCTCTAAGATGGAGACTCAC-3'. RT-PCR products were subsequently amplified by PCR using the Pfx polymerase (Invitrogen). Three PCR products, subcloned from pBluescript, were named 2DL4.1 (corresponding to GenBank accession AF034772 containing a string of 11A at the beginning of the region encoding the cytoplasmic domain), 2DL4.2 (corresponding to GenBank accession BC028137 containing 10A) and 2DL4* (not found in GenBank, but 12A at the same location). The cDNAs were subcloned into the pFLAG-CMV3 vector (Sigma-Aldrich) to introduce a leader and N-terminal FLAG epitope tag (DYKDDDK) before the start of the mature polypeptide receptor (see Fig. 1A). NKp44 cDNA was cloned with the FLAG epitope sequence inserted between the endogenous leader and the mature polypeptide (see Fig. 1, A and B) as will be detailed elsewhere.4 All four constructs containing leader and FLAG tag sequence were ligated into the bicistronic retroviral expression vector, pBMN-IRES-EGFP (generously provided by Dr. G. Nolan, Stanford University), to produce recombinant retrovirus for generation of NK cell lines with stably integrated cDNA. Cells transduced with this retroviral system coordinately express the introduced cDNA and enhanced green fluorescent protein (EGFP). Integrity of all constructs was confirmed by sequencing in the Fox Chase Cancer Center Automated DNA Sequencing Facility (Perkin-Elmer Applied Biosystems, Shelton, CT).



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  		FIGURE 1. Protein structure and expression of FLAG-tagged 2DL4.1, 2DL4.2, 2DL4* and NKp44. A, Schematic representations of the protein products from the receptor cDNA constructs that were generated. D0 and D2 are highly conserved Ig-like domains in the KIR family, and transmembrane (TM) and cytoplasmic (Cyt) domains are shown. Hatched portions of cytoplasmic domains represent alternative cytoplasmic sequences that result from frame shifts. Each construct carried FLAG-epitope tags (at the N terminus) for detection and stimulation. B, Amino acid sequence of the amino terminal, transmembrane, and cytoplasmic domains of receptor constructs. Dashes represent identity and dots represent either intervening sequence or sequence that continues for 74 or 22 amino acids for 2DL4.1 and NKp44, respectively. An asterisk signifies a termination codon. Underlined sequence represents the position of the ITIM. The sequences of 2DL4.1, 2DL4.2, and NKp44 are representative of the GenBank accession numbers AF034772, BC028137, and AJ225109, respectively. The cDNA sequence of 2DL4.1 has a string of 11 adenines (11A) at the point of the cytoplasmic frame shift, whereas 2DL4.2 and 2DL4* have 10A and 12A, respectively. C, The surface expression of 2DL4.2 was not detected on NK-92 cells. The cDNAs encoding FLAG-tagged receptors were transduced into NK-92 cells with recombinant retrovirus. Parent cells and EGFP+ transduced cells were stained with anti-FLAG mAb in combination with PE-conjugated goat anti-mouse {kappa} secondary reagent and analyzed by flow cytometry. Results are representative of numerous separate retroviral transduction experiments. D, 2DL4.2 protein was expressed in NK-92 cells. NK-92 cells and receptor-transduced NK-92 cells were lysed with 1% n-dodecyl {beta}-D-maltoside and sequential immunoprecipitates were prepared with anti-CD56 and then anti-FLAG mAbs. Each lane represents immunoprecipitation from a lysate of 40 million cells. Samples were separated on 10% SDS-PAGE gels under reducing condition, and immunoblot analysis was performed with anti-FLAG mAb.

 
Retroviral transduction of NK-92 cells

Phoenix-Ampho cells were transfected with the pBMN vector containing one of the cDNA constructs using Lipofectamine Plus reagent (Life Technologies) as previously described (19). Supernatants (1 ml) of these transfectants grown for 2 days in serum-free Opti-MEM medium (Life Technologies) were collected, mixed with Lipofectamine Plus reagent, and added to 1–2 x 106 NK-92 cells in a 24-well plate (3526, Corning, Corning NY), which was then centrifuged at 2000x g for 45 min at room temperature. After 5–7 days, EGFP-positive cells were sorted on a FACSVantage SE cell sorter (BD Biosciences) in the Fox Chase Cancer Center Cell Sorting Facility.

KIR2DL4 genotyping

Primers complementary to the 5' end of exon 6 of the KIR2DL4 gene (sense; 5'-CCA GAC ACC TGC ATG CTG-3') and the middle of intron 6 (antisense; 5'-TCC CTG TTC ACT GTT CTG TGT-3') were designed to amplify a 600-bp genomic product. Genomic DNA was prepared from IL-2 cultured PBMC from each donor with phenol/chloroform and ethanol precipitation. PCR mixtures contained 20 pmol of each primer, 40 nM MgCl2, 4 nmol dNTP mix, 0.5 U AmpliTaq, 10x PCR buffer (all from Invitrogen) and 50–100 ng genomic DNA in a final reaction volume of 20 µl. The following thermal cycler conditions were used: 1 cycle of 3 min at 94°C, 35 cycles of 30 s at 94°C/30 s at 55°C/30 s at 68°C, and 1 cycle of 3 min at 68°C. PCR products were purified directly with the Wizard PCR Preps DNA Purification System (Promega, Madison, WI) or from an agarose gel using a Gel Extraction kit (Qiagen, Valencia, CA). Products were directly sequenced in both directions using the sense primer (same as previously described, from exon 6) or a nested antisense primer (5'-TTG GGC CAG AGA CTT TCC TG-3') with the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA) and analyzed on an ABI PRISM Model 377 DNA Sequencer in the Fox Chase Cancer Center DNA Sequencing Facility. Because polymerase slippage is common across long sequence repeats, especially poly(A) (30, 31), these events always accounted for minor peaks in our sequencing reactions after the poly(A) tract. Therefore, sequencing reactions were performed in both sense and antisense directions from at least three separate PCR to assure that the patterns were consistent and representative of the corresponding genomic DNA sequence.

Immunoprecipitation and immunoblotting

Parent NK-92 cells and 2DL4.1-, 2DL4.2-, and 2DL4*-transduced NK-92 cells (40 million/sample) were washed three times in HBSS (Life Technologies), and lysed for 30 min on ice in 1 ml/sample of lysis buffer, containing 1% n-dodecyl {beta}-D-maltoside (ULTROL Grade; Calbiochem, La Jolla, CA), 150 mM NaCl (Fisher), 10 mM Tris-HCl (pH 7.5) (Fisher), 2 mM Na3VO4 (from 100x stock boiled 5 min before addition), 0.4 mM EDTA (Fisher), 10 mM sodium fluoride (Sigma-Aldrich), 1 mM Pefabloc (Roche, Indianapolis, IN), and 1 µg/ml each of leupeptin, aprotinin, and soybean trypsin inhibitor (Sigma-Aldrich). Lysates were cleared of nuclear/cytoskeletal components by centrifugation at 20,800 x g for 15 min at 4°C. Lysates were sequentially immunoprecipitated for 90 min at 4°C with anti-CD56 mAb (B159.5.2) and anti-FLAG mAb (2 µg/sample precoupled to 30 µl protein G-agarose). All immunoprecipitates were washed five times with ice-cold 0.2% n-dodecyl {beta}-D-maltoside buffer (same components as lysis buffer) and resuspended in Laemmli reducing sample buffer (10% DTT). Immunoprecipitated samples were boiled for 3 min before separation on discontinuous 10% SDS-PAGE. Proteins were electrophoretically transferred to Immuno-Blot polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) and blocked with 5% skim milk (ACME, Salt Lake City, UT) in TBST buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20). Blocked membranes were probed initially with mouse anti-FLAG mAb (2 µg/ml) and secondarily with HRP-coupled donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Immunoblotted proteins were visualized by chemiluminescence using the ECL detection reagents (Amersham, Arlington Heights, IL).

Cell stimulation with plate-bound Ab

Purified mAb (10 µg/ml in 0.1 M sodium carbonate/bicarbonate buffer, pH 9.5) was added at 50 µl/well to flat-bottom 96-well plates (PRO-BIND; BD Biosciences) or 500 µl/well to 24-well plates (3526, Corning). The plates were sealed with parafilm and incubated for 3 h at 37°C. Before use, the plates were washed twice with culture medium. 1–3 x 105 cells/well were added to 96 flat-well plates or 5 x 105 cells/well to 24-well plates, and plates were incubated at 37°C in a 7% CO2 incubator.

Redirected cytotoxicity assay

NK-92 cells were tested for redirected cytotoxicity against the Fc{gamma}RII/III+ P815 murine mastocytoma cell line in a 3–4 h 51Cr release assay in 200 µl medium/well. The P815 target cells (2 x 106 cells) were labeled with 100 µCi 51Cr (5 mCi/ml, stock product 2030B; NEN, Boston, MA) for 60–90 min and incubated with effector cells in V-bottom 96-well plates (Costar, Cambridge, MA). Spontaneous release and maximal release of 51Cr were determined by incubating in medium alone or 1% Triton X-100, respectively. The percentage of specific lysis was determined as follows: [(mean cpm experimental release - mean cpm spontaneous release)/(mean cpm maximal release - mean cpm spontaneous release)] x 100. To engage specific receptors in the redirected assay, mAb (1 µg/ml) were mixed with P815 cells for 5 min before effector cell addition.

Intracellular staining for IFN-{gamma}

NK-92 cells (5 x 105/well: 24-well plate, 2 x 105/well: 96-flat plate) were cultured with plate-bound anti-FLAG mAb or phorbol ester (PMA, 8 ng/ml) + Ca2+ ionophore (A23187, 0.1 µg/ml) for 3 or 6 h (entire period or last 4 h in the presence of brefeldin A (10 µg/ml; Sigma-Aldrich), respectively). After the incubation, the cells were fixed with 4% paraformaldehyde (Fisher), permeabilized with 0.1% saponin-containing PBS, stained with PE-conjugated anti-IFN-{gamma} mAb or IgG1 control mAb, and analyzed on a FACScan.

ELISA for IFN-{gamma}

NK-92 cells (2 x 105 cells/well: 96 flat-well plate) were stimulated with plate-bound mAbs for 20 h, and IFN-{gamma} release was measured in the supernatant by ELISA kit according to the manufacturer’s instructions (BD PharMingen).

Fas-mediated DNA fragmentation assay

Target cell DNA fragmentation was measured using a modification of the JAM assay (32, 33). NK-92 cells were pelleted onto plate-bound mAb and incubated for 4 h. Jurkat T cells were separately labeled for 4 h with [3H]thymidine (1.0 mCi/ml; NEN) at a final concentration of 5 µCi/ml (5 x 105 cells/ml). Just before the assay, Jurkat cells were gently pelleted and washed once with prewarmed medium. [3H]Thymidine labeled Jurkat T cells (1 x 105 cells/well) were then pelleted with stimulated effector cells for 2 h in the presence or absence of EGTA/MgCl2 (added to medium at 5 mM and 10 mM final concentrations, respectively) and DNA was harvested onto glass fiber filters (Packard Instrument, Meriden, CT). In some experiments, anti-CD56 (2 µg/ml) or anti-FasL (1:10 dilution of biotin-conjugated) mAbs were added to effector cells 30 min before addition of target cells. 3H retained on the filter (intact DNA) was assayed by scintillation counting (Top Count NXT; Packard Instruments) and percentage of specific DNA fragmentation was calculated using the following formula: percentage of DNA degradation = [(the total cpm in the Jurkat T cells alone - the number of cpm after incubation with NK-92 cells)/the total cpm in the Jurkat T cells alone] x 100. Statistical analysis by the Student t test was performed using the Excel X program for Mac (Microsoft, Redmond, WA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of KIR2DL4 in the NK-92 cell line

To study 2DL4 in the absence of specific mAbs, we engineered N-terminal FLAG epitope-tagged forms of the receptor. We used RT-PCR to clone three versions of 2DL4 cDNAs from RNA derived from the human NK-like cell line NK3.3. We named the cDNAs 2DL4.1, 2DL4.2, and 2DL4* in the following study. These cDNAs encode proteins that differ substantially within the intracytoplasmic regions following lysine-246 (K246) (Fig. 1, A and B), due to frame shifts derived from variations in the number of a consecutive string of adenines in the cDNA, ranging from 10A to 12A. Several groups have previously reported cDNAs containing 10A or 11A sequences (34, 35, 36), and these are derived from distinct 2DL4 alleles that differ in the extreme 3' end of exon 6 (9A or 10A from exon 6 and an additional adenine from exon 7), for which multiple genomic sequences have been reported (9A exon 6, GenBank accession AC011501 and AF0031211; 10A exon 6, AL133414 and AF110035). The "conventional" allele of the 2DL4 cDNA, which we have named 2DL4.1 (11A, GenBank accession AF034772), encodes a protein with a cytoplasmic domain containing 115 amino acids, including a single ITIM. Another cDNA, which represents the widely reported alternative allele (10A, GenBank accession BC028137), encodes a short cytoplasmic domain containing only 11 amino acids and was named 2DL4.2. The profound intracytoplasmic difference and lack of the ITIM sequence suggest that the allele corresponding to 2DL4.2 exhibits alternative function. We also cloned a third cDNA named 2DL4* that contains a 12A sequence to encode 27 intracytoplasmic amino acids. Genomic or cDNA sequences corresponding to the 2DL4* cDNA have not previously been reported in GenBank. We cannot rule out that 2DL4* may have resulted from polymerase slippage during PCR, but it offered the capability to test a receptor with an alternative cytoplasmic domain, lacking the ITIM. In addition, we engineered a FLAG-tagged version of the NKp44 receptor (Fig. 1, A and B), which is an activating receptor that associates with DAP12 (37), and therefore, serves as a representative immunoreceptor tyrosine-based activation motif-coupled receptor for functional comparison with 2DL4.1.

We expressed the FLAG-tagged versions of 2DL4.1, 2DL4.2, 2DL4*, and NKp44 in the IL-2-dependent NK-like cell line, NK-92, using retroviral transduction with a bicistronic recombinant retrovirus that coordinately expresses both the cloned cDNA and EGFP, as previously described (19). EGFP-positive transduced cells were sorted and stained for receptor surface expression with anti-FLAG mAb. As shown in Fig. 1C, 2DL4.1, 2DL4*, and NKp44 were expressed on the surface of NK-92 cells in parallel with EGFP expression. Surprisingly, however, 2DL4.2 was not detected on the cell surface (Fig. 1C), despite the presence of significant intracellular protein (Fig. 1D). This result suggests that the highly prevalent human allele corresponding to 2DL4.2 (34, 35) encodes a protein that is retained inside NK cells and is incapable of reaching the cell surface. Therefore, we used NK-92 cells transduced with either 2DL4.1, 2DL4*, or NKp44 for comparative functional analysis of these three receptors.

Regulation of KIR2DL4 surface expression on transduced NK-92 cells by IL-2

In contrast to our previous studies with the inhibitory receptor, KIR3DL1 (19), we noted that the surface expression of both 2DL4.1 and 2DL4* varied on transduced NK-92 cells. Therefore, we used anti-FLAG mAb staining to assess the surface expression levels of both forms of the receptors at various time intervals (day 1, 2, 3, and 4) after passing the cells into fresh IL-2 containing medium. This analysis revealed that the expression of both 2DL4.1 and 2DL4* on transduced NK-92 cells was dependent upon IL-2 stimulation, with the highest expression being detected on days 1 and 2 following the IL-2 stimulation (activated cells) and progressive decline on days 3 and 4 (resting cells, see Fig. 2). Restimulation with fresh IL-2-containing medium on day 4 always restored the expression of 2DL4.1 and 2DL4* to the maximal levels, but never resulted in the up-regulation of 2DL4.2 surface expression (data not shown). The expression of 2DL4.1 and 2DL4* on transduced cells was completely lost when cultured with a low dose of IL-2 in the medium or if cultures were extended for 5 days (data not shown). In contrast, the expression of FLAG-NKp44 was nearly unchanged in response to fresh IL-2 in the medium (Fig. 2). Similarly, CD56 expression levels were virtually unchanged on 2DL4.1 and 2DL4* transduced NK-92 cells during the same time course (data not shown). Therefore, IL-2 dependent 2DL4.1 and 2DL4* expression in NK-92 cells seems to be a unique property of these receptors. Furthermore, although driven from the same long-terminal repeat promoter of the retroviral vector, the expression level of EGFP in the transduced cells remained unchanged for the interval (data not shown), indicating that the IL-2 dependent surface expression of 2DL4.1 and 2DL4* on NK-92 cells is regulated post transcriptionally. In addition, the same expression pattern was observed for 2DL4.1 and 2DL4*, indicating that the regulation of expression is independent of the majority of the cytoplasmic domain and may alternatively be regulated through an associated accessory protein.



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  		FIGURE 2. IL-2 dependent expression of 2DL4.1 and 2DL4* on NK-92 cells. Receptor-transduced NK-92 cells were stained with anti-FLAG mAb (thin histogram) or control mAb (thick histogram) in combination with PE-conjugated goat anti-mouse {kappa} secondary reagent on various days of culture. On day 0, cells were passed into fresh IL-2-containing medium. The histograms represent staining profiles performed on the same day from independent cultures with staggered initiation days. MFI values of anti-FLAG staining and control secondary alone staining (in parentheses) are shown atop each panel. The results are representative of three time course experiments.

 
Up-regulation of KIR2DL4 by IL-2 on primary NK cells from only some donors

Next we tested whether surface expression of 2DL4 is also up-regulated by IL-2 on primary NK cells. PBMCs were isolated from six separate donors, cultured in IL-2, and stained every three days with anti-CD3 mAb, anti-CD56 mAb, and a recently described anti-2DL4-specific mAb that was kindly provided by Dr. Eric Long (17). When freshly isolated PBMCs were analyzed, they exhibited the typical pattern of a major CD3-CD56+ NK cell fraction and a minor CD3-CD56high fraction (Fig. 3). Interestingly, only two of three donors (donors 3A and 3D) clearly expressed 2DL4 on freshly isolated NK cells, and expression was predominantly restricted to the CD56high fraction (Fig. 3). After culture in IL-2 for 12 days, CD56 levels progressively increased in the entire NK cell population and three distinct 2DL4 staining profiles were observed within the NK cell fraction: 1) the two donors that expressed 2DL4 on the freshly isolated CD56high fraction (donors 3A and 3D) expressed increased levels of 2DL4 on the entire NK cell fraction, 2) two donors that either initially lacked or marginally expressed 2DL4 (donors 3B and 2G, respectively) gradually expressed substantial levels of the receptor within three to six days of culture in IL-2, and 3) two donors (donors 3C and 3E) lacked the receptor on freshly isolated cells and only marginally expressed the receptor on some of the NK cell fraction after 12 days of culture in IL-2. Interestingly, subsequent analyses of donor 3D at 3 and 5 weeks later did not show 2DL4 expression on his freshly isolated CD56high NK cells, although expression was induced by culture in IL-2, indicating that expression may be influenced by certain environmental factors, possibly minor infections. We conclude from these results that 2DL4 is variably expressed on NK cells from different individuals, it is only found on the CD56high NK cell fraction when expressed, cell surface levels of the receptor can be up-regulated by IL-2, and some individuals only have marginal capacity to express the receptor on NK cells after 12 days of culture in IL-2. These primary NK cell results correlate directly with our observations in NK-92 cells transduced with 2DL4.1 or 2DL4.2.



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  		FIGURE 3. Analysis of KIR2DL4 expression on resting and IL-2 cultured primary NK cells. PBMCs were isolated from six healthy human donors (3A, 3B, 3C, 3D, 3E, and 2G) and cultured for 12 days with IL-2 as described in Materials and Methods. Cells were simultaneously stained with PE-conjugated anti-CD56, FITC-conjugated anti-CD3, and PerCP-conjugated anti-2DL4 mAb (64). Histograms of 2DL4 staining are shown for lymphocytes gated for the CD3-CD56+ fraction and the CD3-CD56high fraction (freshly isolated cells on day 0) or the entire CD3-CD56+ fraction (days 3–12). CD3/CD56 staining profiles of the gated populations from a representative culture are shown in the top panels for each day of analysis. Gray lined histograms represent secondary alone staining (biotinylated anti-IgM + PerCP-Streptavidin) and dark line histograms indicate anti-2DL4 staining (with the same secondary reagents). Cells were split and supplemented with fresh IL-2 on days 0, 1, 4, 7, and 10. The different 2DL4 expression patterns did not correlate with specific age, gender, or race of the donors.

 
Correlations between KIR2DL4 genotype and receptor surface expression

We next tested whether the lack of 2DL4 surface expression on primary NK cells from donors 3C and 3E corresponds to a KIR2DL4.2 homozygous genotype and the expression of 2DL4 in the other donors corresponds to a KIR2DL4.1 genotype. For this analysis, genomic DNA was prepared from each donor’s NK cells and used as a template for PCR to synthesize a ~600 bp sequence spanning from exon 6 through intron 6 that encompasses the poly(A) tract at the 3' end of exon 6. These PCR products were sequenced in both directions and the sense sequencing reactions are shown in Fig. 4. Because one adenine in the transcribed mRNA is derived from the beginning of exon 7, a 10A genomic sequence in this region of exon 6 is characteristic of 2DL4.1 and a 9A genomic sequence corresponds to 2DL4.2. A distinct frame disruption in the sequence after 9A is characteristic of heterozygosity. Importantly, all donors that expressed 2DL4 on their NK cells were either homozygous for 2DL4.1 (10A, donors 3A, 3B, and 3D) or heterozygous for 2DL4.1 and 2DL4.2 (9A/10A, donor 2G). Further, the only donor that was homozygous for 2DL4.2 (9A, donor 3E) was incapable of expressing the receptor on NK cells. These results were consistent with our cDNA expression studies in NK-92 cells. Surprisingly, however, the other donor that did not express surface receptor was homozygous for 2DL4.1 (10A, donor 3C). We confirmed this phenotype and genotype in IL-2-stimulated NK cells prepared from a separate blood sample that was derived from donor 3C. Taken together, our results indicate that 2DL4.2 homozygous humans are incapable of expressing 2DL4, although the presence of at least one allele of 2DL4.1 generally produces a receptor that can reach the NK cell surface. The results from donor 3C, however, clearly demonstrate that alternative mechanisms must be responsible for a lack of 2DL4 surface expression in some individuals.



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  		FIGURE 4. Genotype analysis of KIR2DL4 from donors analyzed in Fig. 3. The genomic sequences at the 3' end of exon 6 were amplified from PBMC of six donors (3A, 3B, 3C, 3D, 3E, and 2G) by PCR and sequenced to determine 2DL4 genotypes. Sequences with 10A and no subsequent frame displacement are characteristic of a homozygous 2DL4.1 genotype (3A, 3B, 3C, and 3D), 9A sequences with no subsequent displacement indicates a 2DL4.2 homozygous genotype (3E), and 9A sequences followed by frame displacement indicates a heterozygous genotype (2G). The sequence results in this region were also confirmed using an antisense sequencing primer and sequencing from at least three genomic PCR that were generated from the PBMC of each donor. The 2DL4 expression profile for these donors from the data in Fig. 3 is indicated on the right.

 
KIR2DL4 exhibits unique activating function compared with NKp44

We next tested the biological impacts of 2DL4 engagement on receptor-transduced NK-92 cells. Our initial functional experiments were designed to test whether the transmembrane region of 2DL4, which contains an arginine, can link the receptor to activation signaling in NK-92 cells. To test the transmembrane region independently of the cytoplasmic ITIM sequence, we first compared functional capacities of the frame-shifted FLAG-2DL4*, which lacks the ITIM, with the FLAG-NKp44 in distinct transduced NK-92 cell populations. These cell lines allowed us to manipulate the receptors under identical conditions with the common triggering reagent, anti-FLAG mAb. For consistency and to avoid nonspecific activation effects in our functional studies, we always tested the transduced NK-92 cells on day 3 after IL-2, which we refer to as "resting" NK cells.

To directly compare the stimulation capacities of 2DL4* and NKp44, we engaged the receptors on resting NK-92 cells with anti-FLAG mAb to test for Ab redirected cytotoxicity toward the Fc{gamma}R+ P815 target cells (Fig. 5A). Interestingly, NKp44 engagement stimulated strong target cell lysis, whereas only weak cell lysis was detected upon engagement of 2DL4* on NK-92 cells, despite similar levels of receptor expression as assessed by anti-FLAG staining (Fig. 5A). Furthermore, when triggered with an anti-NKp44 mAb, the same levels of cytotoxicity were observed for both receptor-transduced cell populations (data not shown). These observations demonstrate a significant difference in the cytolytic capacities triggered by NKp44 and 2DL4.



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  		FIGURE 5. Differential activation capacity induced by the engagement of 2DL4* vs NKp44. A, Weak redirected cytotoxicity was induced by engagement of 2DL4*, but strong cytotoxicity was induced by NKp44 engagement. Resting transduced cells (day 3 after IL-2) were stained with anti-FLAG mAb (M2; dark lined histograms in left panels) and assayed for Ab-redirected cytotoxicity against 51Cr–labeled Fc{gamma}R+ P815 target cells in the absence or presence of anti-CD56 mAb (B159, used as a negative control) or anti-FLAG mAb (M2) for 4 h (right panels). The results are representative of four experiments. B, IFN-{gamma} production was routinely more robust through engagement of 2DL4* than through NKp44. Transduced cells were stimulated with or without plate-bound anti-FLAG mAb for 3 h or 6 h. After cell fixation and permeabilization, intracellular staining was performed with PE-anti-IFN-{gamma} mAb (black histogram) or PE-mouse IgG1 (gray histogram; negative control). C, Late term IFN-{gamma} production is more potent through 2DL4* despite lower cell surface receptor expression when compared with NKp44. FLAG-2DL4* and FLAG-NKp44 transduced NK-92 cells were stained with anti-FLAG mAb (black histogram) in combination with PE-conjugated goat anti-mouse {kappa} secondary reagent or the secondary alone (gray histogram) on day 3 after IL-2. The same cells were stimulated with or without plate-bound anti-FLAG mAb or phorbol ester and calcium ionophore (PMA + A23187), culture supernatants were harvested after 20 h, and supernatants were analyzed by ELISA for IFN-{gamma} concentration. The results are representative of three experiments.

 
Next we assessed IFN-{gamma} production by resting NK-92 cells after cross-linking the receptors with plate-bound anti-FLAG mAb for 3 h to 20 h by intracellular staining (3 h and 6 h) and ELISA (20 h). Strong intracellular staining of IFN-{gamma} was detected at early time points in 2DL4* transduced NK-92 cells after anti-FLAG mAb engagement, whereas significantly lower levels were detected after the same treatment in the NKp44 transduced NK-92 cells (Fig. 5, B and C). In fact, significantly increased levels of IFN-{gamma} staining were detectable in 2DL4*-transduced NK-92 cells after only 3 h of stimulation. Consistently, about twice as much IFN-{gamma} secretion was detected by ELISA at later time points (20 h) in supernatants from NK cells stimulated through 2DL4 than through NKp44 (Fig. 5C). Interestingly, engagement of 2DL4* stimulated significantly more potent IFN-{gamma} production despite about 10-fold lower levels of receptor surface expression when compared with that of NKp44, as assessed by anti-FLAG mAb staining (Fig. 5C). In summary, our data showed rapid and robust IFN-{gamma} secretion in response to engagement of 2DL4* on NK-92 cells, which significantly exceeded that stimulated by the same mAb through NKp44 over a wide time course, despite significantly higher surface expression of NKp44.

We further tested whether stimulation through 2DL4* or NKp44 induces expression of several cell surface activation markers in cell NK-92 cells. Cross-linking of 2DL4* and NKp44 on resting transduced cells with plate-bound anti-FLAG mAb for 18 h resulted in the up-regulation of the cell surface activation markers CD69 (an activating receptor), CD25 (the IL-2R{alpha} chain), and FasL (data not shown). The degrees of up-regulation were similar for both receptors.

The intracytoplasmic ITIM sequence does not influence activating function of KIR2DL4

We next compared the activating capacities of 2DL4.1 and 2DL4* to determine the impact of the ITIM on activating function. When engaged with plate-bound anti-FLAG mAb, both receptors stimulated the up-regulation of all three activation markers, CD25, CD69, and FasL (data not shown). Importantly, both forms of 2DL4 (with or without the ITIM) increased activation markers to similar degrees, indicating that the ITIM does not influence activation through the full-length "conventional" receptor. In addition, there was no difference in IFN-{gamma} production stimulated through 2DL4.1 or 2DL4* when assayed by intracellular staining (3 or 6 h; Fig. 6A) or ELISA (12, 15, or 18 h; Fig. 6B), again indicating that the inhibitory ITIM domain does not influence receptor activating function. Finally, neither form of 2DL4 exhibited substantial redirected cytotoxicity toward P815 target cells, particularly when compared with stimulating with anti-NKp44 mAb in the same NK-92 cell populations (Fig. 6C). Therefore, in several biological assays, we did not detect any substantial negative (or positive) influence on 2DL4.1 activating function that could be attributed to the cytoplasmic ITIM. These results indicate that signals from 2DL4.1 appear to be derived from an associated accessory protein and are not influenced by the ITIM or the majority of the receptor intracytoplasmic domain.



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  		FIGURE 6. Similar activation capacities were observed for 2DL4.1 and 2DL4*. A, Comparable strong and rapid IFN-{gamma} production through 2DL4.1 or 2DL4* engagement on resting NK-92 cells. Transduced NK-92 cells (day 3 after IL-2) were stimulated with or without plate-bound anti-FLAG mAb for 3 or 6 h. After cell fixation and permeabilization, intracellular staining was performed with PE-anti-IFN-{gamma} mAb (thick line) or PE-mouse IgG1 (thin line, negative control). Results are representative of more than three experiments. B, Nearly identical induction of IFN-{gamma} release by engagement of 2DL4.1 or 2DL4* in transduced NK-92 cells at several later time points. Transduced NK-92 cells (day 3 after IL-2) were engaged or not with plate-bound anti-FLAG mAb for 12, 15, or 18 h. Supernatants were harvested and assayed by ELISA for IFN-{gamma} concentration. Error bars represent SD from the mean of three samples. Results are representative of more than three experiments. C, Comparatively weak redirected cytotoxicity was induced by Ab engagement of 2DL4.1 or 2DL4* on day 3 after IL-2 stimulation of NK-92 cells. Transduced cells (day 3 after IL-2) were assayed for mAb-redirected cytotoxicity against 51Cr–labeled Fc{gamma}R+ P815 target cells in the absence or presence of anti-CD56 mAb (B159, negative control), anti-NKp44 mAb (3.43.13), or anti-FLAG mAb (M2) for 4 h. Results are representative of three experiments.

 
KIR2DL4 engagement can stimulate NK cells to trigger Fas-mediated DNA fragmentation in Jurkat cells

Because we observed the up-regulation of FasL in response to engaging 2DL4 on NK-92 cells, we tested whether stimulation of NK-92 cells through 2DL4.1 could promote Fas-mediated DNA fragmentation of Jurkat cell targets, which express Fas (32). In these experiments, 2DL4.1-transduced NK-92 cells were stimulated for 4 h with plate-bound anti-FLAG mAb or anti-NKp44 mAb to induce the expression of FasL and then incubated with [3H]thymidine-labeled Jurkat T cells for 2 h. DNA from the cultures was harvested to measure specific DNA fragmentation. In this assay, target cell DNA fragmentation was induced after stimulation through either receptor (Fig. 7A), even in the presence of EGTA/MgCl2 to deplete calcium, which indicates that the elevated FasL promotes DNA fragmentation in Jurkat cells by a granule-independent mechanism. To confirm a role for FasL in this process, we assessed the effects of blocking FasL with specific mAb on the target cell DNA fragmentation. In these experiments, Jurkat DNA fragmentation induced by the stimulation of the NK cells through 2DL4.1 was inhibited partially by anti-FasL mAb, but not by anti-CD56 mAb (isotype control) (Fig. 7B). The results indicate that the FasL induced by 2DL4.1 engagement on NK-92 cells is functional and has the potential to promote DNA fragmentation in Fas+ target cells.



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  		FIGURE 7. Cross-linking 2DL4.1 on NK-92 cells enhances DNA fragmentation of Fas+ Jurkat target cells in a FasL-dependent manner. A, 2DL4.1 and NKp44 transduced NK-92 cells were preincubated for 4 h with or without plate-bound anti-FLAG mAb. [3H]Thymidine-labeled Fas+ Jurkat cells were subsequently added for 2 h in the presence or absence of 2 mM EGTA/2.5 mM MgCl2 (final concentration in medium) (E:T ratio = 1:1). [3H]Thymidine incorporation was then quantitated in harvested cells to determine percentage specific DNA fragmentation as in Materials and Methods. Student’s t test was performed and bracketed bars with *normal medium or with **EGTA/MgCl2 represent p < 0.05 compared with the corresponding unstimulated condition. B, 2DL4.1 transduced NK-92 cells were preincubated for 4 h with or without plate-bound anti-FLAG mAb and then pretreated with anti-FasL mAb (NOK1) or anti-CD56 mAb (as a negative control) for 30 min before the addition of [3H]thymidine-labeled Jurkat cells for 2 h (E:T ratio = 2:1). [3H]Thymidine incorporation was quantitated in harvested cells, and percentage-specific DNA fragmentation was calculated as described in Materials and Methods. Anti-FasL mAb and anti-CD56 mAb were present throughout the 2 h incubation. Student’s t test was performed and bracketed bars marked with asterisk represent p < 0.05 compared with the anti-FasL mAb treated, stimulated condition.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we utilized retroviral transduction to express and comparatively test the functions of FLAG epitope-tagged versions of the NK cell receptors 2DL4 or NKp44 in the human NK-like cell line, NK-92. The lack of Fc{gamma}RIII (CD16) on NK-92 cells eliminated concerns of Fc-mediated activation events from the stimulating IgG mAbs, which allowed us to directly compare functions of the two receptors by engaging each with a common anti-FLAG mAb. Differential capacities of 2DL4* and NKp44 to trigger cytotoxicity and produce IFN-{gamma} were observed under identical stimulation conditions in resting NK-92 cells. 2DL4* exhibited low redirected cytotoxicity and robust IFN-{gamma} production (Fig. 5), which is consistent with a previous report (17), whereas NKp44 exhibited strong redirected cytotoxicity and modest IFN-{gamma} production. Interestingly, we observed that 2DL4* stimulated more potent IFN-{gamma} production throughout a broad time course, despite significantly lower surface expression, as compared with NKp44. These observations strongly suggest that the two receptors transduce distinct downstream signals, which are likely triggered through different associated accessory proteins. Although NKp44 associates with DAP12, the accessory protein associated with 2DL4 has not yet been identified.

Our results and those of Long and colleagues (20) demonstrate that 2DL4 is an activating receptor, despite the inhibitory cytoplasmic motif. In fact, our comparisons between two forms of 2DL4 with radically different cytoplasmic domains (2DL4.1 with the ITIM and 2DL4* lacking the ITIM) showed no major differences in activating capacity, suggesting that the ITIM does not suppress activating functions (Fig. 6). This was unexpected because strong inhibitory function was previously demonstrated by the 2DL4.1 ITIM in isolation using modified 2DL4 receptors, either by replacing the cytoplasmic domain of KIR3DL1 with that of 2DL4.1 or by mutating the transmembrane arginine of 2DL4.1 (19, 20)]. The inhibitory capacity of the 2DL4.1 ITIM appears to be mediated through recruitment of the protein tyrosine phosphatase, SHP-2 (19). Whereas SHP-2 has been shown to contribute to negative signaling responses through some receptors (38, 39), recruitment of the phosphatase is more often involved in promoting positive signals through activating receptors (40, 41). Thus, it is important to consider the possibility that SHP-2 might contribute positive influences to 2DL4.1 function that are not evident in the functional assays used in this study. Further work is underway in our lab to address this possibility.

Our studies also demonstrated the lack of surface expression of a truncated form of 2DL4 (named 2DL4.2 in our studies) encoded by an allele that appears to be highly prevalent within the human population. Witt and colleagues (34) previously examined the frequency of the two 2DL4 alleles corresponding to 2DL4.1 and 2DL4.2 in two studies of Australian donors. Surprisingly, they found that both alleles were equally distributed in the donor populations, and 32.6% (n = 46) or 27.1% (n = 48) of individuals were homozygous for the 2DL4.2 genotype (35). Our results showed a lower representation of the 2DL4.2 allele, but our donor pool was limited. Although the identification of the two 2DL4 genotypes originally indicated differential function of receptors containing or lacking the intracytoplasmic ITIM, it is now apparent that the truncated form may not be expressed on the NK cell surface at all. Our studies of primary human cells support this possibility with only marginal expression of 2DL4 on NK cells from a donor (3E) exhibiting a homozygous 2DL4.2 phenotype, even after long-term culture in IL-2. Further, Witt et al. (35) showed that the predominant mRNA identified from NK cells of individuals homozygous for the 2DL4.2 genotype lacked the TM exon entirely, indicating further pressures against surface expression in those individuals. To standardize nomenclature for these allelic variations in this region of 2DL4, we propose adoption of the following: 1) KIR2DL4.1 (CD158d.1) for the ITIM-containing receptor (11A cDNA and 10A genomic exon 6) and 2) KIR2DL4.2 (CD158d.2) for the truncated receptor that is retained inside NK cells (10A cDNA and 9A genomic exon 6).

Our results complicate the attractive hypothesis that 2DL4 engagement with HLA-G plays an essential role in placental development by promoting IFN-{gamma} release, because maintenance of this nonexpressed genotype does not seem to be selected against in humans. Clearly, the potential relevance of 2DL4 in human pregnancy is more complicated than currently understood. Importantly, despite diminished placental development (28, 29), IFN-{gamma}-deficient and NK cell-deficient mice can still reproduce, and homozygosity of the 2DL4.2 allele in women does not correlate with the incidence of pre-eclampsia in later stages of pregnancy (35). Further, a recent report described a woman entirely lacking a 2DL4 gene who has had multiple normal pregnancies (42). It should also be noted that although two groups have published evidence that HLA-G is a ligand for 2DL4 (22, 23, 24), two additional reports have claimed that HLA-G is not the ligand (43, 44). These results indicate that alternative ligands may exist for 2DL4 and the receptor is not essential for normal pregnancy. It must be considered, however, that a beneficial role for 2DL4 in pregnancy is still possible under certain conditions. Therefore, the physiological impacts of the 2DL4.2 genotype on early stages of pregnancy should still be examined. However, it should also be considered that alternative 2DL4 ligands may mediate separate biological functions. For this reason, further population studies are also required to test whether different 2DL4 genotypes contribute to resistance to specific diseases, especially those that are more prevalent beyond childbearing ages, such as cancer.

The frame shift resulting from the missing adenine in the 2DL4.2 cDNA encodes only three subsequent amino acids before early termination (Fig. 1B). Interestingly, these three amino acids are methionine-leucine-leucine, thereby introducing a dileucine motif, known to bind the AP-1 clathrin adaptor, which mediates intracellular sorting of proteins from the trans-Golgi network to endosomes and lysosomes (45, 46, 47). Additionally, the only transmembrane 2DL4-like sequence identified in orangutan also ends prematurely with the methionine-leucine-leucine sequence (14), suggesting that this intracellular retention motif may serve some conserved biological purpose. Further analysis is necessary to confirm whether this motif contributes to the mechanism of intracellular retention of 2DL4.2. We cannot presently explain why one of our human blood donor (3C) exhibits a 2DL4.1-like genotype, yet does not express the receptor on the surface of NK cells, even after IL-2 culture. The result suggests that additional mechanisms may also prevent surface expression of 2DL4.1 in some individuals.

We also found that 2DL4 was only expressed on freshly isolated CD56high NK cells from some individuals, and expression of the receptor was up-regulated by IL-2 stimulation in NK-92 and primary NK cells (Figs. 2 and 3). Taken together, our results indicate that despite the reports that all NK cells express 2DL4 mRNA (11, 12), the receptor appears to only reach the surface of activated NK cells. This result supports a previous report that used a polyclonal Ab preparation to show 2DL4 expression only on decidual and placental NK cells from pregnant mothers, which also express high levels of CD56 (23). Our results with retrovirus-driven expression also indicate that IL-2-mediated up-regulation of 2DL4 is independent of most of the cytoplasmic domain and is apparently due to either posttranscriptional regulation or regulation by an associated protein. We cannot rule out the possibility that an alternative accessory protein (not found in NK-92 cells or only weakly found in IL-2 stimulated NK cells) may allow surface expression of 2DL4.2 in certain NK cell populations, especially because marginal 2DL4 expression was noted on NK cells from previously negative donors after 12 days of culture with IL-2 (Fig. 3). By analogy, recent reports (48, 49) have demonstrated that alternative cytoplasmic domains in the NKG2D receptor mediate selective association with different transmembrane accessory proteins in NK cells at different activation states. Additional experiments will be required to understand the regulation of 2DL4 surface expression, the roles of accessory protein association in surface expression, and their functional significance.

We also showed that 2DL4.1 engagement induced the up-regulation of FasL, which was functional, because it promoted DNA fragmentation in Jurkat cells in a Ca2+-independent manner that was partially blocked by anti-FasL mAb (Fig. 7). In the same way, previous studies have shown that upon stimulation with cytokines (50), appropriate target cells (3), or Fc{gamma}RIIIA ligands (51), functional FasL is up-regulated on NK cells and can induce Fas-mediated target cell death. Because redirected assays in this and the previous report (17) consisted of short-term 51Cr release assays, the importance of FasL-mediated killing triggered by 2DL4 may have been underestimated. Interestingly, a recent study of IL-12 plus IL-2 therapy suggested a unique beneficial interrelationship between IFN-{gamma} and the Fas/FasL pathways in mediating apoptosis, inhibition of tumor neovasculation, and overall tumor regression (52). Therefore, in a similar manner, local IFN-{gamma} production, such as that triggered through 2DL4, could sensitize tumor cells or virus infected cells to subsequent Fas-mediated bystander lysis by NK cells. In addition, our studies showed that anti-FasL mAb only partially blocked DNA fragmentation in Jurkat cells, suggesting the involvement of other apoptosis-inducing elements, such as TNF-related apoptosis-inducing ligand (2) or other TNF family members (53, 54, 55). It should also be noted that 2DL4-induced expression of CD25 (IL-2R{alpha} chain) and CD69 (an activation receptor) (56, 57) indicates that, in addition to promoting strong IFN-{gamma} production and Fas-mediated target cell cytotoxicity, engagement of the receptor may prime NK cells to respond subsequently to IL-2 and target cells in vivo.

Functionally, our data show that 2DL4.1 can stimulate rapid and potent IFN-{gamma} production by resting NK cells that is not influenced by its cytoplasmic ITIM motif. Potent IFN-{gamma} production through 2DL4.1 may play at least two important functional roles in vivo: support of pregnancy and/or contribution to a cytokine-specific innate immune response. Interestingly, uterine NK cells express high levels of CD56, which is characteristic of NK-92 cells and the CD56high NK cell subset that exclusively expressed 2DL4 in our studies of primary cells. Importantly, the CD56high CD16low/- cells are an important minor subset of the NK cell pool because they produce abundant immunoregulatory cytokines, such as IFN-{gamma}, TNF-{alpha}, and IL-10, are less effective mediators of Ab-dependent cellular and natural cytotoxicity, and can escape the vasculature to access lymph nodes and presumably reach sites of primary tumors and viral infections (58, 59, 60, 61, 62). IFN-{gamma} has been shown to provide effective antiviral and antitumor innate immune functions (63, 64). Therefore, although questions of ligands and surface expression remain, appropriate engagement of 2DL4.1 can selectively stimulate NK cells to elicit a strong IFN-{gamma} response, which has the potential to contribute significant biological benefits, but only in individuals capable of expressing this receptor.

Note added in proof.

Goodridge et al. (65) recently published similar evidence for differential expression and IL-2-mediated upregulation of KIR2DL4 that is consistent with our results.


   Acknowledgments
 
We thank Dr. Eric Long, Dr. Marco Colonna, Dr. Bice Perussia, Dr. Garry Nolan, Dr. Jackie Kornbluth, Dr. Charles Lutz, and the Biological Resources Branch of the National Cancer Institute for reagents; Dr. Luis Sigal (Fox Chase Cancer Center) for scintillation counting; and Dr. Mel Bosma and Dr. Dietmar Kappes for helpful comments on the manuscript. We also thank the Fox Chase Cancer Center DNA Synthesis, DNA Sequencing, Flow Cytometry, and Cell Culture Facilities for reagents and technical support.


   Footnotes
 
1 This work was supported by Grant CA83859 (to K.S.C.) from the National Institutes of Health and a grant from the Pennsylvania Tobacco Health Research Fund. The research was also supported in part by National Institutes of Health Centers of Research Excellence Grant CA06927 and an appropriation from the Commonwealth of Pennsylvania. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute. Back

2 Address correspondence and reprint requests to Dr. Kerry S. Campbell, Fox Chase Cancer Center, Institute for Cancer Research, 7701 Burholme Avenue, Philadelphia, PA 19111. E-mail address: KS_Campbell{at}fccc.edu Back

3 Abbreviations used in this paper: FasL, Fas ligand; ITIM, immunoreceptor tyrosine-based inhibitory motif; KIR, killer cell Ig-like receptor; A, adenine; EGFP, enhanced green fluorescent protein; SHP, Src homology 2-containing phosphatase. Back

4 K. S. Campbell, S. Yusa, and T. L. Catina. NKp44 triggers NK cell activation through DAP12 association that is not influenced by a putative cytoplasmic inhibitory sequence. Submitted for publication. Back

Received for publication April 7, 2003. Accepted for publication July 30, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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