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Ly-49P Activates NK-Mediated Lysis by Recognizing H-2D^{d 1}

Elizabeth T. Silver^*, Dong-Er Gong^*, Chew Shun Chang^*, Abdelaziz Amrani, Pere Santamaria and Kevin P. Kane^2,*

^* Department of Medical Microbiology and Immunology, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada; and Department of Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada

	Abstract

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Little is known regarding the ligand specificity of Ly-49 activating receptor subfamily members expressed by NK cells. A new Ly-49 activating receptor related to Ly-49A in its extracellular domain, designated Ly-49P, was recently cloned from 129 strain mice. We independently cloned an apparent allele of Ly-49P expressed by nonobese diabetic and nonobese diabetes-resistant mouse strain NK cells. We found it to be reactive with the A1 Ab thought to recognize a polymorphic epitope expressed only by the Ly-49A inhibitory receptor of the C57BL/6 strain. Rat RNK-16 cells transfected with Ly-49P mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49P can activate NK-mediated lysis. We determined that RNK-16 lysis of Con A blasts induced by Ly-49P was MHC dependent, resulting in efficient lysis of H-2D^d-bearing targets. We found that the D^d 1/2 domain is required for Ly-49P-mediated RNK-16 activation, as determined by exon shuffling and transfection. Thus, Ly-49P is the second activating Ly-49 receptor demonstrated to induce NK cytotoxicity by recognizing a class I MHC molecule.

	Introduction

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Ly-49 molecules are disulfide-bonded homodimeric type II transmembrane proteins belonging to the C-type lectin receptor superfamily (1). Ly-49 receptors are expressed on murine NK cells and certain subsets of murine T cells (2, 3, 4). Multiple Ly-49 receptors can be expressed simultaneously by an individual NK cell (5, 6). A number of Ly-49 family members recognize class I MHC molecules, including Ly-49A, -C, and -G, and serve to inhibit NK function (7, 8, 9). Inhibitory Ly-49 receptors, as with CD94/NKG2A and a variety of Ig domain-containing receptors, inhibit NK function by recruiting the SH2 domain-containing tyrosine phosphatase SHP-1³ to disrupt tyrosine kinase-dependent, membrane-proximal, signaling events (10). Following inhibitory Ly-49 receptor engagement, phosphorylation of tyrosine within an immunoreceptor tyrosine-based inhibitory motif (ITIM) present on the cytoplasmic tail of each receptor subunit facilitates binding and activation of the SHP-1 phosphatase (10). Ly-49 receptor expression on NK cells can be modulated by class I MHC expression. For example, expression of the H-2D^d ligand for Ly-49A in vivo can reduce the expression of Ly-49A on NK cells and correspondingly influence the class I density threshold for regulating NK lytic activity against H-2D^d-expressing targets (11). The purpose of inhibitory Ly-49 receptors appears to be to inhibit potentially autoaggressive NK function when self class I MHC expression on adjacent cells is in a normal density range. However, should the class I density drop significantly as a result of a disruption in class I expression, due to viral infection for example, Ly-49 inhibition is reduced or discontinued for lack of sufficient ligand, and NK lysis can ensue. This is the missing self hypothesis first proposed by Kärre and co-workers (12) and functions as a paradigm for NK inhibitory receptor function.

The Ly-49 family also includes Ly-49D and -H. These receptors lack ITIMs, do not recruit SHP-1, and are unable to function as inhibitory receptors. Instead, they associate with the disulfide-bonded immunoreceptor tyrosine-based activation motif-containing signaling adapter molecule DAP12 and can stimulate transmembrane signaling initiated by tyrosine phosphorylation events (13, 14). Association of Ly-49D or -H with DAP12 is through a charge interaction between a conserved arginine in the transmembrane segment of Ly-49D and -H and a glutamic acid in the transmembrane segment of DAP12 (13). Engagement of Ly-49D or Ly-49H on NK cells with FcR-mediated target cell-bound Abs induces NK-mediated target cell lysis by the mechanism of reverse Ab-dependent cell-mediated cytotoxicity (rADCC) (15, 16). Ly-49D has been shown to recognize the H-2D^d class I MHC molecule, and this event results in NK activation (17, 18, 19, 20). Thus, an activating Ly-49 receptor recognizes a class I MHC molecule. The Ly-49D⁺ NK subset of some strains of mice has also been shown to mediate MHC-specific and Ly-49D-dependent allogeneic bone marrow rejection (21). These findings potentially complicate interpretation of the missing self hypothesis, since they would not be readily predicted within the context of this model. Recently, the Ly-49D molecule was also shown to recognize undefined xenoantigens, resulting in NK lysis of certain xenogeneic target cells (22, 23). Although able to stimulate NK cells, the role of activating Ly-49 receptors in regulating NK cell function remains unclear.

The Ly-49 family may be substantially larger than the commonly known members, Ly-49A through Ly-49I. Complex Southern blot hybridizations obtained with Ly-49-specific probes using genomic DNA from inbred mouse strains suggest that additional Ly-49 family members may remain to be identified and characterized (24). Recently, genes or gene segments of five potential additional Ly-49s, tentatively designated J, K, L, M, and N, have been identified in the C57BL/6 (B6) genome (25). In addition, two Ly-49 cDNAs, one encoding an inhibitory receptor designated Ly-49O and another encoding an apparent activating receptor designated Ly-49P, were recently cloned from the 129 mouse strain (26). The Ly-49P receptor is closely related to Ly-49A in its carbohydrate recognition domain (CRD), associates with DAP12, and triggers transmembrane signaling (26). These results suggest that Ly-49P may serve as an activating receptor. In this report we describe the independent cloning of Ly-49P from nonobese diabetic (NOD) and nonobese diabetes-resistant (NOR) NK cells and provide evidence that it can activate NK cell cytolytic activity by recognizing H-2D^d. Before this report, Ly-49D was the only activating member of the Ly-49 family shown to recognize a class I MHC molecule. Thus, Ly-49P joins Ly-49D in this capability. Understanding the diversity, ligand specificity, activation potential, and expression of activating Ly-49 members may provide insight toward understanding their function(s).

	Materials and Methods

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Animals

Five- to 8-wk-old female BALB/c (H-2^d), C57BL/6 (H-2^b), NOD (H-2K^d,D^b), NOR (H-2K^d,D^b), B10.BR (H-2^k), B10.D2 (H-2^d), B10.S (H-2^s), BALB.B10 (H-2^b), and BALB.K (H-2^k) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Additional 5- to 8-wk-old NOR and NOD mice were obtained from the University of Alberta mouse breeding facility (Edmonton, Canada).

Hybridomas and Abs

Hybridomas producing the following Abs: A1(IgG2a), anti-Ly-49A (27); MK-D6(IgG2a), anti-IA^d (28); 11-4.1(IgG2a), anti-H-2K^k (29); 14-4-4S(IgG2a) anti-IE^k (30); BBM.1(IgG2b), anti-human ß₂-microglobulin (31); B27 M1(IgG2a), anti-HLA-B7,27 (32); Y3(IgG2b) anti-K^b (33); 34-2-12S(IgG2a), anti-H-2D^d (3 domain) (34, 35); 34-5-8S(IgG2a), anti-H-2D^d (1/2 domain) (34, 35); and 2.4G2(rat IgG2b) anti-mouse Fc- receptor (36); were obtained from American Type Culture Collection (Manassas, VA), except A1, which was obtained from Dr. James Allison (University of California, Berkeley, CA). Abs were prepared by ammonium sulfate precipitation and PBS dialysis of tissue culture supernatants obtained from hybridomas grown in protein-free hybridoma medium. Purified G28 (IgG2a) anti-rat CD8 (37), F23.1(IgG2a) anti-mouse Vß 8.1 and 8.2 TCRs (38, 39); DX5 (rat IgM, PE-coupled), which recognizes NK cells (3); 145-2C11 (hamster IgG, PerCP-coupled), which recognizes the CD3 chain (40); and isotype control Abs, G155-178 (mouse IgG2a, fluorescein-coupled), anti-TNP, A19-3 (hamster IgG) anti-TNP, R4-22 (rat IgM, PE-coupled) of unknown specificity, and A23-1 (rat IgG2c) of unknown specificity were purchased from PharMingen (San Diego, CA). The Ly-49A-reactive YE1/48 (rat IgG2c) Ab (24), was provided by Dr. Fumio Takei (Terry Fox Laboratories, Vancouver, Canada). Tissue culture supernatants were also obtained from hybridomas grown in RPMI 1640, 8% heat-inactivated FCS, 20 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin for use in cell surface staining. FITC-coupled rat anti-mouse IgG, goat anti-rat IgG, and mouse anti-rat IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

Rat tumor cell lines

RNK-16, a spontaneous F344 rat strain NK cell leukemia cell line (41) was provided by Dr. Mary Nakamura (University of California, San Francisco, CA). The RNK-16 cells were maintained in RPMI 1640 supplemented with 10% FCS, L-glutamine, penicillin, streptomycin, and 5 x 10^-5 M 2-ME (RNK medium). YB2/0 is a rat myeloma (42) we obtained from American Type Culture Collection, and it was maintained in RPMI 1640 supplemented with L-glutamine, penicillin, streptomycin, and 1 mM sodium pyruvate (YB2/0 medium).

COS-7 cells

COS-7 SV40-transformed African green monkey kidney cells were provided by Dr. John Elliott (University of Alberta, Edmonton, Canada).

PCR cloning of Ly-49 transcripts

IL-2-activated NK cells were prepared from spleen cells harvested from 6- to 10-wk-old female mice as described by Smith and co-workers (2). Total cellular RNA was isolated from NOD and NOR IL-2-activated NK cells with TRIzol reagent (Life Technologies, Burlington, Canada), and reverse transcribed using SuperScript II (Life Technologies) with an oligo(dT) primer. Ly-49 transcripts were amplified with Advantage cDNA Polymerase (Clontech, Palo Alto, CA) using the sense cloning primer 5'-CCCAAGATGAGTGAGCAGGAGG-3' and the antisense cloning primer 5'-GAGAGTCAATGAGGGAATTTATCC-3'. The PCR products were purified using QIAquick spin columns (Qiagen, Santa Clarita, CA) and directly ligated into the TA cloning vector p123T (Mo Bi Tec, Gottingen, Germany). Individual cDNA clones were divided into two groups: ITIM-encoding clones, which could be PCR-amplified using the antisense cloning primer with the sense primer 5'-GGAGACTCAAGGGCCCAGAG-3', and ITIM-lacking clones, which could be PCR-amplified by using the antisense cloning primer with the sense primer 5'-CTRRAAAAGCT GGCCTCAGAGTY-3' (where R = A/G and Y = C/T).

To verify the nucleotide sequences of the ends of the open reading frames and to obtain sequence of the noncoding gene flanking regions for cloning, we performed 5'- and 3'-rapid amplification of cDNA ends (5'- and 3'-RACE) for NOR Ly-49A and Ly-49P. Poly(A)⁺ RNA was isolated from total RNA obtained from IL-2-activated NK cells using Oligotex spin columns (Qiagen) and used to synthesize a cDNA library with the Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA). The following gene-specific primers were used: for Ly-49P 5'-RACE, 5'-CTTGAGGAGACGCTGAAGCCTAG-3'; and for Ly-49P 3'-RACE, 5'-GCCAGC TTTTCTAGGCTTCAGC-3'. Ly-49A required nested primers: for 5'-RACE, 5'-TGGTGAGATTTCATAAATCTGCAGGAT-3' followed by 5'-TGGCTACAGAAGGTGTTCATTCCAC-3'; for 3'-RACE, 5'-TCCTGCAGATTTATGAAATCTCACCAT-3' followed by 5'-ATGAACTTCCAGTGGAATGAACACCT-3'. With the sequence data thus acquired, we designed the following cloning primers to obtain both NOD and NOR full length cDNA clones by RT-PCR: for Ly-49A, 5'-TTCCTCCACCAGAACCACTTCTTG-3' (sense) and 5'-AGAAGATCTGTCCAGTCTCAACA GGG-3' (antisense); for Ly-49P, 5'-TTTAAAAGAGAACATACTCTACATCCTC-3' (sense) and the same antisense primer as Ly-49A. Ly-49D was cloned by RT-PCR from NOD and NOR first strand cDNA using the same primers and conditions as were used to previously clone Ly-49D from C57BL/6 cDNA (43). Sequencing reactions were performed using a dideoxy terminator method and analyzed on an ABI 373A automated sequencer (Applied Biosystems, Foster City, CA).

Transfections

COS-7. COS-7 cells were grown in Opti-MEM I (Life Technologies, Grand Island, NY) containing 5% heat-inactivated FCS (Medicorp, Montreal, Canada). DNA containing the coding regions of Ly-49A and Ly-49P cDNA were inserted into the XbaI-EcoRI sites of the mammalian expression vector pCI-neo (Promega, Madison, WI). Sequence encoding the mature form of the mouse DAP12 protein was inserted into the pFLAG-CMV-1 expression vector (Sigma-Aldrich, Oakville, Canada) to create an epitope-tagged chimeric protein. Vectors were then transfected into COS cells using Lipofectamine (Life Technologies).

YB2/0 cells. A cDNA encoding H-2D^d cloned from S49.1 T lymphoma cells was inserted into the expression vector pCDNA3 and electroporated into YB2/0 using 180 mV and 960 µF. A chimeric class I molecule, referred to as K^b/D^d, was produced by ligating the 1/2 domain portion of H-2K^b to the 3, transmembrane, and cytoplasmic portion of H-2D^d at the common FspI site. This chimeric cDNA was inserted into the expression vector pCI-neo and electroporated into YB2/0 as described above. Transfected cells were immediately cloned in 96-well microtiter plates in YB2/0 growth medium supplemented with 1 mg/ml of G418 for drug selection (Life Technologies).

RNK cells. The coding region of Ly-49P was inserted into the XhoI-XbaI sites of the bicistronic expression vector BSREN, which was provided by A. Shaw, Washington University (St. Louis, MO), and transfected into RNK-16 cells using the protocol described by Nakamura et al. (10). Four million cells were transfected with 20 µg of linearized plasmid by electroporation at 180 mV and 960 µF. Transfected cells were cloned in 96-well microtiter plates in RNK medium supplemented with 1 mg/ml G418 for drug selection.

Preparation of nylon wool-nonadherent spleen cells and generation of IL-2-activated LAK

Single-cell suspensions of spleen cells from 5- to 7-wk-old C57BL/6 and NOD mice were prepared with tissue grinders. After osmotic lysis of RBC, the cell suspensions were passaged over nylon wool columns. The nylon wool-nonadherent (NWNA) spleen cells were either analyzed immediately by FACS or cultured for 6 days at a concentration of 2 x 10⁶ cells/ml in RPMI 1640/10% FCS supplemented with sodium pyruvate, nonessential amino acids, 5 x 10^-5 M 2-ME, and 1000 U/ml human rIL-2 expressed in and isolated from Escherichia coli, before FACS analysis.

Flow cytometry

Approximately 48 h after transfection, COS-7 cells were incubated for 30 min with A1 or isotype control mAb MK-D6. The cells were washed with PBS, incubated with FITC-labeled secondary Ab, washed again with PBS, and then fixed with 2% formaldehyde for analysis on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). YB2/0 cells as well as the H-2D^d and H-2K^b/D^d transfectants of YB2/0 were incubated with either rat IgG (Sigma) or normal rat serum before staining with the mAbs 34-2-12S or 14-4-4S, followed by FITC-conjugated rat anti-mouse IgG. YB2/0 transfectants expressing the chimeric class I molecule H-2K^b/D^d were also analyzed by FACScan with the Y3 H-2K^b-specific mAb and the BBM.1 isotype control after blocking with normal rat serum, followed by FITC-conjugated secondary rat anti-mouse IgG and formaldehyde fixation. RNK-16 transfectants were incubated with normal rat serum for 30 min before addition of the mouse IgG2a mAbs A1 and B27 M1 followed by FITC-conjugated rat anti-mouse IgG, then fixed and analyzed by FACScan. Cells were gated for forward and side scatter.

Three-color analysis of NWNA cells

Freshly isolated NWNA spleen cells or 6-day IL-2-activated NWNA spleen cells were preincubated with normal mouse serum to block nonspecific Ab binding to Fc receptors before Ab incubation. Abs used for analysis were PerCP-coupled 2C11 (anti-mouse CD3), PE-coupled DX5 (anti-mouse pan-NK cell), and either fluorescein-coupled A1 or YE1/48 with a fluorescein-coupled mouse anti-rat second Ab. Isotype control Abs used were A19-3-PerCP, R4-22-PE, G155-178-fluorescein, and A23–1. Cells were gated for forward and side scatter.

Generation of Con A-activated blast target cells

Con A-activated T cell blasts were prepared from spleen cells of various mouse strains. Fifteen million spleen cells were cultured at 5.0 x 10⁶ cells/ml in RPMI 1640 with 10% heat-inactivated FCS, 2-ME, and 3.0 µg/ml Con A for 48 h. Blast cells were recovered either after three washes in RPMI 1640 medium or following Ficoll-Hypaque separation.

Cytotoxicity assays

Target cells were labeled at 37°C with 100–150 µCi of Na⁵¹CrO₄ (⁵¹Cr; Mandel/NEN Life Science Products, Guelph, Canada), for 1 h if tumor cells or for 1.5 h if Con A blast cells. Following extensive washes, 1 x 10^{4 51}Cr-labeled target cells were incubated for 4–5 h at 37°C in V-bottom microtiter plates with RNK-16 cells or RNK-16 cells transfected with Ly-49P at various E:T cell ratios in triplicate. After the incubation, plates were centrifuged for 5 min, and 100 µl of supernatant was removed and counted in a gamma counter. The percent specific lysis was determined as [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100. To perform the rADCC experiments, untransfected RNK-16 cells and the 1B9 Ly-49P transfectant of RNK-16 were preincubated for 15 min with 5 µg/ml of the A1, G28, or F23.1 Abs or with medium alone before addition of FcR-expressing YB2/0 target cells and subsequent 4-h cytotoxic assay as described above. For Ab inhibition experiments, Abs were incubated with soluble PA (2 µg/10 µg of mAb; Sigma, St. Louis, MO) for 30 min before addition to effector or target cells. Effector or target cells were preincubated with the mAb and PA for 15 min before the cytotoxicity assay. The mAb were present throughout the cytotoxicity assay at a final concentration of 5 µg/10⁶ effector cells or 5 µg/ml when the mAb was directed against a target cell Ag. All cytotoxicity assays were repeated a minimum of three times.

	Results

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Predicted amino acid sequences of Ly-49A, Ly-49D, and Ly-49P of different mouse strains

The Ly-49 family includes both activating and inhibitory receptors (1). Collectively, Ly-49 molecules may contribute to a balance of positive and negative signals that control murine NK activities as well as those of certain subsets of T cells (3, 7, 8, 9, 18). Most studies of Ly-49 gene family members have used the B6 strain as a prototype. It is clear from recent studies, however, that the diversity and expression of Ly-49 members can differ among strains (44). Furthermore, complex hybridization patterns obtained in Southern blot analyses of genomic DNA from multiple inbred mouse strains using Ly-49A and Ly-49C cDNA probes suggest that additional Ly-49 members may exist (24). We chose to examine Ly-49 receptor expression and function in a mouse strain other than B6, one in which immune dysregulation can occur, resulting in spontaneous disease. The NOD mouse spontaneously develops autoimmune disease at an early age (45), while the NOR mouse does not, although it shares many, but not all, NOD genetic loci (46). We designed oligonucleotide primers to clone cDNAs encoding putative activating and inhibitory Ly-49 family members by RT-PCR and used RNA obtained from IL-2-activated NK cells of NOD and NOR mouse strain origin as sources of transcripts. In addition to cDNAs encoding well-characterized Ly-49 family members, we cloned a noninhibitory Ly-49 family member that possessed high amino acid identity in its CRD with the inhibitory Ly-49A molecule. Sequence identities with a newly described activating Ly-49 present in the 129 mouse strain, designated Ly-49P by Makrigiannis et al. (26), indicate that we have cloned the NOD strain allele of Ly-49P. The NOR strain expresses Ly-49P identical in sequence to Ly-49P^NOD.

We also cloned a single cDNA sequence from both NOD and NOR NK cells encoding a novel allele of Ly-49A, since it has a very high identity of 99.1 and 99.6% on the nucleotide level, and 98.9 and 99.2% on the amino acid level, with B6 and BALB/c Ly-49A alleles, respectively (data not shown; Fig. 1A). The NOD (and NOR) Ly-49A allele is closer in sequence to both B6 and BALB/c alleles than they are to each other, with 99.0% nucleotide (not shown) and 98.1% amino acid identities (Fig. 1A). The predicted Ly-49P^NOD amino acid sequence lacks an ITIM sequence and thus is not an inhibitory receptor, yet a comparison of the Ly-49P^NOD sequence with Ly-49A^B6 indicates an amino acid identity of 83.3% for the entire molecule and 92.9% for the CRD (Fig. 1A). Similarly, a comparison of the Ly49P^NOD sequence with Ly-49A^NOD shows an overall amino acid identity of 82.5%, and 92.1% for the CRD region (Fig. 1A). In addition to the full-length Ly-49P we have designated P1, there are two other major PCR products amplified with Ly-49P cloning primers from NOD IL-2-activated NK cells. These smaller products are apparent splice variants of Ly-49P encoding in-frame transcripts containing the complete CRD region. Using the gene organization of Ly-49A as a reference (47), one Ly-49P variant lacks exon 4, while the other omits exons 3 and 4 (Fig. 1A). We designate the three Ly-49P splice variants by size as P1, P2, and P3. The first amino acid residue encoded by exon 5 of the P2 splice variant is predicted to differ from P1 and P3. This difference is a common result when two or more different RNA upstream donor sites are spliced to the same acceptor site. This occurs in transcripts of many genes, including CD94 and NKG2B (48, 49). In the case of P2, fusion of the first base of the codon ATT (Ile) at the end of exon 3 to the second and third bases of the codon GGC (Gly) at the beginning of exon 5 forms the codon AGC (Ser) without interruption of the established reading frame. For the P3 splice variant, the first base of the codon GTG (Val) at the end of exon 2 is fused to the second and third bases of the codon GGC (Gly) at the beginning of exon 5 to reform the glycine codon at the start of the CRD, again without interruption of the established frame. We have been unable to clone a Ly-49P-related cDNA from IL-2-activated B6 NK cell RNA with primers used for cloning Ly-49P^NOD. It appears that Ly-49P does not exist in the B6 genome, is not expressed, or is divergent in sequence from Ly-49P^NOD.

View larger version (33K): [in this window] [in a new window]   FIGURE 1. Alignment of predicted Ly-49A, -D, and -P amino acid sequences from different mouse strains. A, Amino acid sequences of Ly-49A alleles from C57BL/6, BALB/c, and NOD strains are aligned with NOD Ly-49P sequences. Exon assignments are based on the genomic organization of C57BL/6 Ly-49A. The ITIM sequence is boxed with dotted lines, and the putative transmembrane region is shown in boldface. B, NOD and 129/J Ly-49P sequences are aligned with Ly-49D alleles of NOD and C57BL/6 strains. The arginine in the transmembrane region is shown in boldface. In both A and B, dashes indicate sequence identity, asterisks indicate sequence gaps, and potential N-linked glycosylation sites are boxed with solid lines. The nucleotide sequences are available under the GenBank accession numbers AF218077 (Ly-49ANOD), AF074456 (Ly-49ANOR), AF218080 (Ly-49P1NOD), AF218081 (Ly-49P2NOD), AF218082 (Ly-49P3NOD), AF074458 (Ly-49P1NOR), AF074461 (Ly-49P2NOR), AF074462 (Ly-49P3NOR), AF218078 (Ly-49DNOD), and AF218079 (Ly-49DNOR).

The A1 and YE1/48 Abs recognize Ly-49P^NOD

The A1 Ab was previously reported to recognize a polymorphic epitope expressed only by the B6 allele of Ly-49A, since it was shown not to react with other known alleles of Ly-49A or other known Ly-49 family members (24). Since the noninhibitory Ly-49P^NOD possesses high sequence identity with Ly-49A, particularly in the CRD, where the A1 Ab is predicted to bind Ly-49A (24), the reactivity of the A1 Ab and that of a second Ly-49A-specific Ab, YE1/48, with Ly-49P^NOD were examined. We expressed Ly-49A^B6, Ly-49A^NOD, or Ly-49P^NOD in COS cells for Ab staining. Ly-49A^B6 reacted with the A1 and YE1/48 Abs, as did Ly-49A^NOD and Ly-49P^NOD (Fig. 2A). There was some expression of Ly-49P detected with both Abs when Ly-49P is expressed in the absence of DAP12; however, coexpression of DAP12 substantially augments Ly-49P expression, as indicated by enhance staining with both Abs (Fig. 2A). No staining of COS cells with the A1 Ab was observed in the absence of Ly-49A or Ly-49P expression or with the expression of DAP12 alone (data not shown). Coexpression of DAP12 was necessary for optimal expression of Ly-49P^NOD (Fig. 2A), similar to Ly-49P¹²⁹ (26). Thus, the A1 Ab does not exclusively react with the B6 allele of Ly-49A, but also recognizes the NOD allele of Ly-49A and a noninhibitory/putative activating receptor, Ly-49P.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Ly-49PNOD is recognized by the A1 and YE1/48 Abs. A, FACS analysis of COS cells transfected with Ly-49AB6, Ly-49ANOD, or Ly-49PNOD alone or with murine DAP12, using the A1 or YE1/48 Abs (shaded), or the isotype controls Abs MK-D6 or 2.4G2 (unshaded). Binding was detected with fluorescein-coupled rat anti-mouse or goat anti-rat Ab, respectively. B, FACS analysis of RNK-16 (upper panels) and Ly-49P transfected RNK-16 clones 1B9 (second panels), 1A3 (third panel), and 1G4 (bottom panel) using the Abs A1 or G28 (shaded) or the isotype control B27 M1 (unshaded).

Cross-linking of Ly-49P¹²⁹ results in transmembrane signaling, including tyrosine phosphorylation and Ca²⁺ flux (26), suggesting that it can function as an activating receptor. We were interested in the ability of Ly-49P to activate NK cytotoxicity. To this end, we transfected Ly-49P^NOD into the heterologous rat NK leukemia RNK-16, which displays NK activity (10). RNK-16 has been used successfully to demonstrate the NK-activating function of transfected Ly-49D (17) and 2B4S, an activating form of an Ig-domain containing receptor expressed by murine NK cells (50). This experimental system avoids the complication of other mouse activating and/or inhibitory receptor coexpression and cross-reaction of the A1 Ab with Ly-49 receptors other than Ly-49P. We show three Ly-49P transfectant clones that were positive for A1 Ab staining (1B9, 1A3, and 1G4), while RNK-16 was negative (Fig. 2B), indicating that the transfectants express Ly-49P^NOD. These transfectant clones expressed different mean densities of Ly-49P (Fig. 2B). Transfectant 1A3 expressed quite a high density of Ly-49P, suggesting that possessing only one predicted N-linked carbohydrate attachment site is not a limitation for proper folding and cell surface expression of this molecule. RNK-16 cells and transfectants constitutively express the rat CD8 molecule, as indicated by G28 Ab staining (Fig. 2B, right panels, and data not shown).

To determine whether Ly-49P can function as an NK activating receptor, we examined the ability of Ly-49P transfectants to perform rADCC. The rat myeloma YB2/0 expresses Fc receptors and can be rendered susceptible to lysis when bound to an Ab directed against heterologous NK-activating receptors expressed on RNK-16 cells by transfection (17). YB2/0 is normally sensitive to RNK-16 lysis (Fig. 3A); however, upon transfection of RNK-16 with mouse noninhibitory NK receptors such as Ly-49D (17) and Ly-49P^NOD (Fig. 3B), cytotoxicity is reduced substantially. This may be due to the heterologous activating receptor competing successfully for a limiting signaling component, such as the rat DAP12, with endogenous rat activating receptor(s) responsible for YB2/0 recognition and lysis. An Ab directed against Ly-49P, but not rat CD8, stimulated rADCC of the FcR-bearing YB2/0 cells by the Ly-49P RNK-16 transfectant 1B9 (Fig. 3B). In contrast, the A1 Ab was unable to enhance the cytotoxicity of untransfected RNK-16 cells even at lower E:T cell ratios where cytotoxicity is limiting (Fig. 3A). An additional isotype control Ab, F23.1 also did not affect lysis of YB2/0 by either effector cell (Fig. 3). These results indicate that Ly-49P can transmit stimulating signals resulting in NK-mediated cytotoxicity and can be considered an NK-activating receptor.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. Ly-49P transfected RNK-16 mediate reverse ADCC with the A1 Ab. RNK-16 (A) and the 1B9 Ly-49P RNK transfectant (B) were incubated with 5 µg/ml of A1, G28, or F23.1 or with medium as indicated for 15 min before addition of FcR-bearing YB2/0 target cells and 4-h cytotoxicity assay. The data represent the mean of triplicate wells ± SD.

A number of inhibitory Ly-49 family members have been shown to recognize class I molecules as ligands, including Ly-49A, -C, and –G, in their apparent role of inhibiting NK cells unless there is reduction of self-MHC expression (7, 8, 9). Much less is understood regarding the ligand specificity and role of activating Ly-49 members. Ly-49D is the only activating Ly-49 subfamily member demonstrated to recognize a class I MHC molecule (17). Since Ly-49P, like Ly-49D, is an activating Ly-49 receptor for which there are serological reagents, we attempted to determine whether Ly-49P also recognizes class I MHC molecules as ligands. We first compared the ability of RNK-16 cells and three Ly-49P transfectants to lyse NK-susceptible Con A T cell blast target cells from five different inbred mouse strains, BALB/c, B6, B10.BR, B10.D2, and B10.S (Fig. 4A). Lysis of all five strains was negligible for RNK cells, but each of the Ly-49P transfected RNK-16 cells lysed BALB/c and B10.D2 Con A-activated blasts, and the transfectant with the highest Ly-49P expression, 1A3, also lysed B10.BR blasts, but to a much lesser extent (Fig. 4A). No lysis of C57BL/6 or B10.S Con A blasts was observed. Taken together, these results suggest that Ly-49P may recognize an H-2^d product and possibly an H-2^k product.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. MHC dependence of Ly-49P-activated RNK-16 lysis of Con A-activated T cell blasts. RNK-16 cells or individual Ly-49P transfectant clones of RNK-16, 1B9, 1A3, or 1G4 were assayed for cytotoxicity against Con A blasts from various mouse strains that include H-2 congenics. Con A-activated T cell blast targets were generated from spleen cells isolated from BALB/c, C57BL/6, B10.BR, B10.D2, and B10.S (A); BALB/c, BALB.B10, and BALB.K (B); or B10.D2, NOD, NOR, B10.BR, and C57BL/6 (C) for 4-h cytotoxicity assays with the indicated effector cells. The data presented represent the mean of triplicate wells ± SD.

The foregoing results suggested that Ly-49P triggers RNK-16-mediated lysis of Con A blasts by recognizing H-2D^d. To more directly assess this possibility we attempted to block lysis mediated by the Ly-49 RNK transfectants, 1B9 and 1A3, by including the A1 Ab, which recognizes Ly-49P, or the 34-5-8S Ab, which recognizes the 1/2 domain of H-2D^d expressed by the Con A blast targets, in the cytolytic assays. Soluble PA was used to bind the Fc portion of the Abs to prevent ADCC. The A1 Ab, but not one directed against rat CD8 expressed by the RNK-16 cells, was able to completely block lysis of B10.D2 Con A blast targets by 1B9 (Fig. 5A) and 1A3 (Fig. 5B), indicating that Ly-49P is the receptor responsible for activating lytic potential of RNK-16 cells. In a similar fashion, the 34-5-8S Ab, but not an isotype control Ab 11-4.1, completely blocked lysis of the B10.D2 Con A blasts by a representative Ly-49P transfectant, 1B9 (Fig. 5C). Similar effects were observed with 34-5-8S, but not 11-4.1, using the 1A3 transfectant and B10.D2 Con A blasts (data not shown). No effect of soluble protein A alone was observed in the assays (Fig. 5). These results suggest that Ly-49P recognizes H-2D^d, possibly at a location within its 1/2 domains, as has been reported for Ly-49A and Ly-49D (10, 17, 51, 52).

View larger version (35K): [in this window] [in a new window]   FIGURE 5. Activation of RNK-16 cytotoxicity mediated by Ly-49P recognition of Con A-activated T cell blasts is blockable by A1 and 34-5-8S Abs. Cytotoxicity against B10.D2 Con A blast target cells by RNK-16 Ly-49P transfectant clones 1B9 (A), and 1A3 (B) was determined in the presence of medium, PA, or PA plus A1 or G28 (anti-rat CD8) control Ab. Abs were preincubated with PA (2 µg/10 µg of mAb) for 30 min before addition to effector cells. Effector cells were then incubated with the mAbs and PA for 15 min before the cytotoxicity assay. The PA and mAbs remained in the wells throughout the cytotoxicity assay with target cells. The final concentration of Ab was 5 µg/106 effector cells. C, Cytotoxicity against B10.D2 Con A blast targets by the RNK-16 Ly-49P transfectant 1B9 was determined in the presence of medium, PA, or PA plus 34-5-8S (anti-H-2Dd 1/2 domain) shown as 34-5 or 11-4.1(anti-H-2Kk) isotype control Ab. Abs were preincubated with PA as described for A and B, then target cells were incubated with the mAbs and PA for 15 min before the cytotoxicity assay. The final concentration of Ab was 5 µg/ml. The PA and mAbs remained in the wells throughout the cytotoxicity assays. The E:T cell ratio was 50:1 for all the assays. The 51Cr release assays were 4 h. Data are the means of triplicate wells ± SD.

Recognition of the H-2D^d 12 domain by Ly-49P stimulates cytotoxic function in RNK-16 cells

The previous Ab-blocking experiments suggested that the 1/2 domain of H-2D^d is recognized by Ly-49P. To directly assess this possibility we transfected rat YB2/0 cells with genes encoding either wild-type H-2D^d or a class I chimera referred to as K^b/D^d, in which the 3 domain of D^d remains, but the 1 and 2 domains of K^b are substituted for the same domains of D^d for use as targets of Ly-49P transfected RNK-16. Two D^d and two K^b/D^d transfectant clones were shown to express comparable densities of class I using the 34-2-12S Ab, which recognizes an epitope on the D^d 3 domain shared by both molecules (Fig. 6, upper panels). Expression of the K^b 1/2 domain on the K^b/D^d chimeric YB2/0 transfectants is confirmed by Y3 Ab staining (Fig. 6, lower panels). A substantial increase in lysis is observed with the wild-type D^d YB2/0 transfectants over that observed with YB2/0 using a representative Ly-49P transfectant of RNK-16, 1B9 (Fig. 7). No increase in lysis over that found with YB2/0 was detected using the YB2/0 transfected with the K^b/D^d chimeric gene (Fig. 7). These results indicate that Ly-49P can activate NK-mediated lysis as a consequence of interacting with the 1/2 domain of H-2D^d. This specificity is shared with the inhibitory Ly-49A receptor and the activating receptor Ly-49D.

View larger version (33K): [in this window] [in a new window]   FIGURE 6. Expression of H-2Dd and an H-2Kb12/Dd3 chimera on rat YB2/0 cell transfectants. The rat myeloma YB2/0 and YB2/0 transfected with Dd, clones 5D3 and 3B2, or a chimeric Kb 12/Dd3, clones 1C12 and 6G9, were analyzed by FACS following incubation with 34-2-12S (anti-Dd3 domain, shaded) or an isotype control Ab, 14-4-4S (anti-IEk, unshaded), and an FITC-coupled rat anti-mouse IgG (upper panels). YB2/0 and Kb/Dd chimera transfectants were also analyzed in a similar manner with the Y3 mAb (anti-H-2Kb, shaded) and an isotype control, B27 M1 (anti-HLA-B27, unshaded; lower panels).

View larger version (25K): [in this window] [in a new window]   FIGURE 7. The Dd 1/2 domain is required for RNK-16 activation through Ly-49P. Lysis of YB2/0 or YB2/0 expressing H-2Dd, YB2/0.Dd1 (clone 5D3), and YB2/0.Dd2 (clone 3B2) or the chimeric Kb 1/Dd3, YB2/0.Kb/Dd1(clone1C12), and YB2/0.Kb/Dd2 (clone 6G9) was determined in a 4-h 51Cr release assay using the Ly-49P transfected RNK-16 cell line 1B9 as effector cells at the indicated E:T cell ratios. Data are expressed as the means of triplicate wells ± SD.

We have demonstrated that NOD IL-2-activated NK cells express Ly-49A and Ly-49P at the RNA level and that Ly-49P can serve as an NK-activating receptor by recognizing a class I MHC molecule. To determine whether these receptors may be expressed by NK cells of the NOD mouse, we performed three-color FACS analysis on freshly isolated NWNA and IL-2-activated NOD spleen cells. Because the NOD mouse strain does not express the NK1.1 epitope, we used the DX5 Ab to identify NK cells (3, 44). DX5⁺CD3^- cells (Fig. 8, top panels) were analyzed for staining with YE1/48 and A1 Abs (Fig. 8, middle and bottom panels). A substantial portion of freshly isolated NOD NK cells express A1 and YE1/48 recognized epitopes (Fig. 8, left panels), and two populations of YE1/48 and A1 positively stained fresh NOD NK cells apparently exist; a small population constituting 2.5–4% of the total NK cells that are strongly stained and a larger population of more weakly stained cells, most effectively seen with the YE1/48 Ab, that constitute 50–80% of the total NOD NK cells. This pattern contrasts with C57BL/6 NK cells, where 20% express A1 and YE1/48 epitopes that uniformly stain with high fluorescence intensities, similar to those observed for the smaller A1⁺ and YE1/48⁺ NOD NK subset(s) (44) (data not shown). The DX5⁺CD3^- IL-2-activated NOD NK cells (Fig. 8, right panels) show similar numbers of cells staining with the A1 and YE1/48 Abs compared with their freshly isolated NK counterparts, with perhaps one notable difference (Fig. 8). The IL-2-activated NOD NK expressing the YE1/48- and A1-recognized epitopes appear to fall into less discrete populations than fresh NOD NK cells, with increased numbers of cells demonstrating moderate to high levels of A1 and YE1/48 staining. Together, these results indicate that Ly-49A and/or Ly-49P are expressed at the surface of freshly isolated and IL-2-activated NOD NK cells. Since Ly-49A^NOD and Ly-49P^NOD are both recognized by YE1/48 and A1 Abs, the staining pattern does not allow us to readily discern which cells express Ly-49A and/or Ly-49P.

View larger version (48K): [in this window] [in a new window]   FIGURE 8. Expression of A1 and YE1/48 epitopes on freshly isolated and day 6 IL-2-activated DX5+CD3- NWNA NOD spleen cells. Fresh or IL-2-activated NWNA splenocytes were prepared from 5- to 8-wk-old female mice. FcR were blocked with normal mouse serum before incubating the cells with PerCP-coupled 2C11, PE-coupled DX5, and either fluorescein-coupled A1 or YE1/48 with a fluorescein-coupled mouse anti-rat Ab. The DX5+CD3- cells were collected in a gate and analyzed for reactivity with either A1 or YE1/48 (shaded) or the isotype control Abs G155-178 or A23-1, respectively (unshaded). Analysis of freshly prepared NWNA splenocytes is shown on the left, and NWNA splenocytes cultured in 1000 U/ml recombinant human IL-2 for 6 days are shown on the right.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

McQueen et al. have described the existence of genes encoding putative activating Ly-49s designated K, L, and N (25). Ly-49K and N have highest homology to Ly-49C in the CRD, whereas the Ly-49L CRD most resembles Ly-49D (25). Ly-49K and N transcripts contain premature stop codons and/or defective alternative splices and do not encode functional protein products (53). We show here that full-length Ly-49P transcripts expressed in NOD and NOR mice can encode a viable protein product. This was also found to be the case for Ly-49P expressed in the 129 strain (26). We identified two additional alternatively spliced forms of Ly-49P in NOD/NOR NK cells. One splice variant, P2, encodes a Ly-49P protein that would lack the membrane-proximal stalk portion, but is otherwise not different from full-length Ly-49P. Transcripts encoding other NK C-type lectin receptor subunits, such as NKG2A and NKG2C, can undergo identical splicing (48, 54, 55). The smallest splice variant, P3, removes a portion of the cytoplasmic domain, the transmembrane segment, and the stalk region. This transcript encodes a protein that would probably remain cytosolic. Equivalent splice variants to P3 have been identified for several NK C-type lectin superfamily members (53). The P1, P2, and P3 messages appear to be expressed in approximately equivalent amounts judging from the intensity of PCR-amplified products (data not shown). It remains to be determined whether proteins encoded by Ly-49P2 and Ly-49P3 transcripts are expressed as stable polypeptides and what function they may perform.

Strain 129 NK cells, although expressing transcripts for Ly-49P, do not express transcripts for Ly-49A (26). In contrast, C57BL/6 strain NK cells express Ly-49A, but no transcripts for Ly-49P are found (26) (data not shown). It is not clear whether the Ly-49A gene is absent in the 129 strain or is simply silent, and the same question is pertinent for Ly-49P in the B6 strain. It could be speculated that the genes for Ly-49A and Ly-49P are found in separate mouse strains, or expression of these two genes may be mutually exclusive in a strain-specific manner. However, we were able to clone Ly-49A and Ly-49P cDNAs from both NOD and NOR strain IL-2-activated NK cells, suggesting that Ly-49A and Ly-49P genes can reside in the same genome and can be expressed at the same time. We identified two subsets of freshly isolated NOD NK cells and possibly IL-2-activated NOD NK cells that stained with the YE1/48 and A1 Abs, a small subset that stains relatively strongly and a larger subset that stains weakly. Since Ly-49A positive cells from several mouse strains stain strongly with these Abs (44), it is possible that the strongly stained NOD NK subset we have observed expresses Ly-49A. The level of YE1/48 staining observed with the weakly stained NOD NK cells reported here resembles the weak level of YE1/48 staining previously reported with 129 strain NK cells that express Ly-49P but not Ly-49A (26). By analogy, it is possible that the weakly staining NOD NK cells express Ly-49P. It is conceivable, therefore, that Ly-49A and Ly-49P may be expressed on separate fresh NOD NK subsets, but further studies will be necessary to resolve this possibility.

It has been suggested that the Ly-49P gene arose during evolution from recombination of two Ly-49 genes (26). This conclusion was based on a comparison of nucleotide sequences of Ly-49P, -A, and -D (26). An interval within exon 4 of Ly-49P¹²⁹ may have been a site of recombination between the inhibitory Ly-49A gene and an activating Ly-49 gene. In any event, Ly-49P DNA sequences exist in the mouse genome (26). Nucleotide sequences of Ly-49P^NOD transcripts are also consistent with some form of recombination (data not shown). Determining the identity of the activating Ly-49 donor gene contributing the 5' portion of the Ly-49P gene, however, may require full characterization of the activating Ly-49 gene repertoire. Activating Ly-49 genes unable to produce viable transcripts may be an additional source of such sequences for recombination (53). Recombination between Ly-49 genes may provide a mechanism for creating additional functional diversity within the Ly-49 gene family. Since Ly-49A and Ly-49P genes can be expressed by the same mouse strain, this suggests that if recombination was the origin of Ly-49P, Ly-49A gene duplication may have occurred before the recombination event. Gene conversion is another possible mechanism by which Ly-49P arose without involving Ly-49A gene duplication.

Ly-49A and Ly-49D are receptors that recognize H-2D^d (10, 17). Ly-49P shares amino acid sequence identities with both these receptors, particularly with the CRD of Ly-49A, where there are only nine amino acid differences in this domain, which consists of 127 residues (Fig. 1). Despite being highly related in sequence to Ly-49A and -D, the specificity of Ly-49P was not assured. We have demonstrated that Ly-49P does indeed recognize H-2D^d in NK functional studies. It is possible that Ly-49P may also recognize an H-2^k product; however, should this be the case, our data suggest that it is probably with substantially lower affinity. It is worth noting that Ly-49A has been reported to recognize D^k, although less effectively than D^d (56, 57), whereas Ly-49D can recognize D^d, but does not appear to be able to interact with H-2^k products (17, 18). It is possible that Ly-49P may share the class I specificity of Ly-49A, but its ability to also recognize H-2^k products still remains to be confirmed. In our studies, Ly-49P^NOD did not recognize NOR or NOR Con A blasts, suggesting that this NK-activating receptor, or this allele of Ly-49P, does not recognize self MHC molecules in the NOD mouse.

The crystal structure of Ly-49A bound to H-2D^d has recently been resolved to 2.3Å (58). The Ly-49A CRD folds into seven ß strand and two helix secondary structure elements. Given the high degree of sequence identity between Ly-49A and Ly-49P in the CRD, it is likely that the Ly-49P CRD would form a very similar structure. In the crystal complex, Ly-49A binds H-2D^d at two sites: site 1 straddles the 1/2 domain boundary, and site 2 involves the cleft formed by the 3 domain, ß₂-microglobulin, and the bottom of the platform supporting the peptide binding groove where a portion of CD8 has also been shown to bind (58). It remains to be determined whether site 2 on D^d participates in Ly-49A binding when in trans, i.e., during an interaction with D^d expressed on a target cell. We show that the D^d 1/2 domain is required for Ly-49P recognition, which is consistent with participation of site 1. In fact, Ly-49P conserves all 12 of the Ly-49A^B6 contact residues for this site (58) (Fig. 1A). Ly-49P also conserves 24 of the 25 residues of Ly-49A that may interact with site 2 (58) (Fig. 1A).

The NOD mouse strain from which we cloned Ly-49P^NOD has immunodeficiencies and develops type I diabetes (45, 59). Adoptive transfer experiments have demonstrated that T cells are required for the induction of diabetes in the NOD mouse (reviewed in Ref. 45). NK cells as well as T cells and B cells are found in the cellular infiltrates of pancreatic insulitis in the NOD mouse, but the role of NK cells, if any, in the disease process remains unclear (60). NK cells of NOD mice have been described as functionally deficient. This characteristic is attributed as a result of observations that NOD NK cells lyse the prototype NK target cell, Yac-1, less efficiently than NK cells of other mouse strains (61, 62). Determining the extent of the NOD NK deficiency will require a comprehensive analysis of NK lytic potential and cytokine production in response to additional target cell types. We have shown that NOD mouse NK cells express viable transcripts for the NK activator molecule Ly-49P, and subsets of NOD NK cells stain with Abs reactive with Ly-49P and Ly-49A, suggesting that Ly-49P may be expressed at the cell surface of some NOD NK cells. Ly-49P and other NK activator molecules may be relevant for NOD NK recognition of targets other than Yac-1. The identification of NK activator molecules and the development of serological reagents specific for these receptors may aid in the characterization of NOD NK functional efficiency as well as in determining the contribution, if any, of NK cells to the development of diabetes in the NOD mouse.

The NOD/SCID mouse has shown particular utility in studying human tumor growth and development as well as human hemopoiesis, since it accepts a broader spectrum of human xenografts than SCID or nude mice (59, 63). Human graft acceptance by NOD/SCID mice has been suggested to be due to the NK functional deficiency of the NOD mouse in combination with a lack of B cells and T cells related to the SCID mutation (59, 63). Mice lacking the common -chain, which is required for signaling through a number of cytokine receptors, including IL-2R and IL-15R, do not develop functional NK cells (64). Mice lacking the common -chain coupled with the SCID mutation accept various human xenografts much more readily than NOD/SCID mice (64). This difference may suggest that NOD NK cells have an ability, although possibly diminished relative to that of other strains, to reject xenografts. Since the NK-activating receptor Ly-49D of C57BL/6 origin has demonstrated xenoreactivity against cells of rat and hamster origin, albeit not against the limited number of human target cell types tested as yet (22, 23), perhaps Ly-49D, Ly-49P, or additional NK-activating Ly-49 receptors expressed by the NOD mouse are responsible for some of the residual barriers to human xenografts exhibited by the NOD/SCID mouse.

NK cells are not the only cells that express Ly-49 receptors, as some T cell subsets can also express these receptors (3). NK T (NKT) cells, for example, express TCR using a conserved TCR -chain and are regulatory T cells that rapidly secrete large quantities of IL-4 or IFN- in response to glycolipid Ags presented by the nonclassical class I-like CD1 molecules (65). As the name implies, NKT cells express various receptors normally found on NK cells, including NKRP.1 and Ly-49 molecules. Interestingly, NKT cell numbers are reduced in the diabetes-prone NOD mouse relative to other mouse strains, and manipulations that substantially increase the number of NKT cells result in protection from diabetes (66, 67). Evidence provided by Exley et al. (68) suggests that the NK receptor NKR-P1A, expressed on human NKT cells, may function as a costimulator in NKT cell activation. It remains to be determined whether mouse NKT cells express activating Ly-49 family members and what role these receptors may play in NKT cell function.

The significance of Ly-49P expression in NOD and NOR mice with respect to immune regulation is unclear. However, it is perhaps notable that some of the pancreas-infiltrating T cells of both strains of mice express Ly-49P transcripts and the A1 epitope by FACS analysis (data not shown). Whether cells of NOD or NOR mouse tissues other than splenic T cells express ligands for Ly-49P in normal or disease states remains to be determined.

	Acknowledgments

 
We thank Dr. Mary Nakamura (University of California, San Francisco, CA) for RNK-16 cells and for helpful advice in transfecting them, Dr. Andrey Shaw (Washington University, St. Louis, MO) for the BSREN expression vector, and Dr. Michael Durairaj (University of Alberta, Edmonton, Canada) for the K^b/D^d chimera cDNA. We thank Dr. John Elliott (University of Alberta) for provision of NOD and NOR mice and COS-7 cells, and Dr. Fumio Takei (Terry Fox Laboratories, Vancouver, Canada) for provision of the YE1/48 Ab.

	Footnotes

 
¹ This work was supported by operating grants from the National Cancer Institute of Canada (to K.K.) and the Medical Research Council of Canada (to P.S.). E.T.S. is supported by an Alberta Heritage Foundation for Medical Research studentship. K.P.K. and P.S. are Alberta Heritage Foundation for Medical Research senior scholars.

² Address correspondence and reprint requests to Dr. Kevin P. Kane, Department of Medical Microbiology and Immunology, 660 HMRC, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.

³ Abbreviations used in this paper: SHP-1, SH2 domain-containing tyrosine phosphatase; ⁵¹Cr, Na⁵¹CrO₄; CRD, carbohydrate recognition domain; ITIM, immunoreceptor tyrosine-based inhibitory motif; NKT cells, NK T cells; NOD, nonobese diabetic; NOR, nonobese diabetes resistant; NWNA, nylon wool nonadherent; PA, protein A; PerCP, peridinin chlorophyll protein; rADCC, reverse Ab-dependent cellular cytotoxicity; RACE, rapid amplification of cDNA ends.

Received for publication January 11, 2000. Accepted for publication May 24, 2000.

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