Ligand interactions by activating and inhibitory Ly-49 receptors.

Natural killer (NK) cells express families of homologous receptors, members of which either activate or inhibit NK cells. We demonstrate that mouse Ly-49D is an activating receptor for the MHC antigen H2-Dd, which is also a ligand for the related inhibitory receptor Ly-49A. To compare and contrast their interactions with class I MHC ligand, we studied each of these receptors expressed in a rat NK-cell hne, RNK-16, for their capacity to recognize wild-type or mutated H2-Dd. Our studies with Ly-49A reveal that functional interaction with H2-Dd depends on residues in the floor of the H2-Dd peptide-binding groove. The recent co-crystal of Ly-49A with H2-Dd indicates that these are not contact residues, thus they may contribute to allelic specificity through conformational changes in H2-Dd. We found that structural requirements for functional recognition of H2-Dd by Ly-49D differ markedly from those for recognition by Ly-49A. We note that H2-Dd expression on certain target cells is not sufficient to activate lysis mediated by Ly-49D, though the additional requirements for functional interaction are not yet identified. Here we review recent studies of Ly-49 receptor ligand specificities and their molecular basis. The functions of these related receptors with opposing functions and shared allospecificity remains unclear.

Cloning and characterization of a novel mouse myeloid DAP12-associated receptor family.

The presence of a negatively charged residue in the transmembrane domain of DAP12 precludes its cell surface expression in the absence of a partner receptor containing a positive charge in its transmembrane domain. We utilized this property of DAP12 to screen a BALB / c macrophage cDNA library for novel molecules that induce cell surface expression of DAP12. By this method, we cloned a cell surface receptor with a single Ig (V) domain, a transmembrane lysine residue, and a short cytoplasmic domain. By homology screening of BALB / c macrophage libraries, we identified a second cDNA for a highly homologous receptor. These receptors appear to be the mouse orthologues of a recently identified human cDNA, TREM-2, so we have designated the receptors as mouse TREM-2a and TREM-2b. By Northern blotting, transcripts for TREM-2 were found in each of three macrophage cell lines but not in a variety of other hematopoietic cell lines. We further demonstrate that TREM-2a is associated with endogenous DAP12 in macrophage cells, and cross-linking of TREM-2a on the surface of macrophages leads to the release of nitric oxide. Our studies define TREM-2 as a receptor family in mouse macrophages and demonstrate the capacity of these receptors to activate macrophage function through DAP12.

Activating Ly-49D and inhibitory Ly-49A natural killer cell receptors demonstrate distinct requirements for interaction with H2-D(d).

The activating Ly-49D receptor and the inhibitory Ly-49A receptor mediate opposing effects on natural killer (NK) cell cytotoxicity after interaction with the same major histocompatibility complex ligand, H2-D(d). To compare Ly-49D and Ly-49A interactions with H2-D(d), we created mutations in H2-D(d) and examined the functional ability of these mutants to activate lysis through Ly-49D or to inhibit lysis through Ly-49A. Specific single amino acid changes in either the H2-D(d) alpha(1) helix or the alpha(2) helix abrogated Ly-49D-mediated cytotoxicity, but these changes had no significant effect on Ly-49A-dependent inhibition. Each of three alpha(2) domain mutations in the floor of the peptide binding groove reduced functional recognition by either Ly-49D or Ly-49A, but all three were required to fully abrogate inhibition by Ly-49A. Our studies indicate that Ly-49D/H2-D(d) interactions require distinct determinants compared with Ly-49A/H2-D(d) interactions. These differences have important implications for the integration of activating and inhibitory signals in NK cells.

Natural killer cells and natural killer T cells.

NK cells are important in protecting against viral infections, and they may regulate the immune response. They are activated by hematopoietic blasts and pose a barrier to bone marrow transplantation. They are also abundant in the pregnant uterine decidua, although their role there is unknown. NK cells are normally inhibited from responding to host cells by inhibitory receptors that recognize self class I MHC antigens. There is evidence that NK cells may be important in the regulation of autoimmunity, but there is even stronger evidence that NKT cells regulate autoimmunity. The mechanisms by which these cells are activated and by which they regulate other cells are now being understood at the molecular level.

Natural killing of xenogeneic cells mediated by the mouse Ly-49D receptor.

NK lymphocytes lyse certain xenogeneic cells without prior sensitization. The receptors by which NK cells recognize xenogeneic targets are largely uncharacterized but have been postulated to possess broad specificity against ubiquitous target ligands. However, previous studies suggest that mouse NK cells recognize xenogeneic targets in a strain-specific manner, implicating finely tuned, complex receptor systems in NK xenorecognition. We speculated that mouse Ly-49D, an activating NK receptor for the MHC I ligand, H2-Dd, might display public specificities for xenogeneic target structures. To test this hypothesis, we examined the lysis of xenogeneic targets by mouse Ly-49D transfectants of the rat NK cell line RNK-16 (RNK. Ly-49D). Of the xenogeneic tumor targets tested, RNK.Ly-49D, but not untransfected RNK-16, preferentially lysed tumor cells derived from Chinese hamsters and lymphoblast targets from rats. Ly-49D-dependent recognition of Chinese hamster cells was independent of target N-linked glycosylation. Mouse Ly-49D also specifically stimulated the natural killing of lymphoblast targets derived from wild-type and MHC-congenic rats of the RT1lv1 and RT1l haplotypes, but not of the RT1c, RT1u, RT1av1, or RT1n haplotypes. These studies demonstrate that Ly-49D can specifically mediate cytotoxicity against xenogeneic cells, and they suggest that Ly-49D may recognize xenogeneic MHC-encoded ligands.

Mouse Ly-49D recognizes H-2Dd and activates natural killer cell cytotoxicity.

Although activation of natural killer (NK) cytotoxicity is generally inhibited by target major histocompatibility complex (MHC) class I expression, subtle features of NK allorecognition suggest that NK cells possess receptors that are activated by target MHC I. The mouse Ly-49D receptor has been shown to activate NK cytotoxicity, although recognition of MHC class I has not been demonstrated previously. To define Ly-49D-ligand interactions, we transfected the mouse Ly-49D receptor into the rat NK line, RNK-16 (RNK.mLy-49D). As expected, anti- Ly-49D monoclonal antibody 12A8 specifically stimulated redirected lysis of the Fc receptor- bearing rat target YB2/0 by RNK.mLy-49D transfectants. RNK.mLy-49D effectors were tested against YB2/0 targets transfected with the mouse MHC I alleles H-2Dd, Db, Kk, or Kb. RNK.mLy-49D cells lysed YB2/0.Dd targets more efficiently than untransfected YB2/0 or YB2/0 transfected with Db, Kk, or Kb. This augmented lysis of H-2Dd targets was specifically inhibited by F(ab')2 anti-Ly-49D (12A8) and F(ab')2 anti-H-2Dd (34-5-8S). RNK.mLy-49D effectors were also able to specifically lyse Concanavalin A blasts isolated from H-2(d) mice (BALB/c, B10.D2, and DBA/2) but not from H-2(b) or H-2(k) mice. These experiments show that the activating receptor Ly-49D specifically interacts with the MHC I antigen, H-2Dd, demonstrating the existence of alloactivating receptors on murine NK cells.

DAP12-mediated signal transduction in natural killer cells. A dominant role for the Syk protein-tyrosine kinase.

The murine Ly49 family contains nine genes in two subgroups: the inhibitory receptors (Ly49A, B, C, E, F, G2, and I) and the noninhibitory receptors (Ly49D and H). Unlike their inhibitory counterparts, Ly49D and H do not contain immunoreceptor tyrosine-based inhibitory motifs but associate with a recently described co-receptor, DAP12, to transmit positive signals to natural killer (NK) cells. DAP12 is also expressed in myeloid cells, but the receptors coupled to it there are unknown. Here we document the signaling pathways of the Ly49D/DAP12 complex in NK cells. We show that ligation of Ly49D results in 1) tyrosine phosphorylation of several substrates, including phospholipase Cgamma1, Cbl, and p44/p42 mitogen-activated protein kinase, and 2) calcium mobilization. Moreover, we demonstrate that although human DAP12 reportedly binds the SH2 domains of both Syk and Zap-70, ligation of Ly49D leads to activation of Syk but not Zap-70. Consistent with this observation, Ly49D/DAP12-mediated calcium mobilization is blocked by dominant negative Syk but not by catalytically inactive Zap-70. These data demonstrate the dependence of DAP12-coupled receptors on Syk and suggest that the outcome of Ly49D/DAP12 engagement will be regulated by Cbl and culminate in the activation of transcription factors.

The alpha2 domain of H-2Dd restricts the allelic specificity of the murine NK cell inhibitory receptor Ly-49A.

Mouse NK lymphocytes express Ly-49 receptors, which inhibit cytotoxicity upon ligation by specific MHC I molecules on targets. Different members of the lectin-like mouse Ly-49 receptor family recognize distinct subsets of murine H-2 molecules, but the molecular basis for the allelic specificity of Ly-49 has not been defined. We analyzed inhibition of natural killing by chimeric MHC I molecules in which the alpha1, alpha2, or alpha3 domains of the Ly-49A-binding allele H-2Dd were exchanged for the corresponding domains of the nonbinding allele H-2Db. Using the Ly-49A-transfected rat NK cell line, RNK-mLy-49A.9, we demonstrated that the H-2Dd alpha2 domain alone accounts for allelic specificity in protection of rat YB2/0 targets in vitro. We also showed that the H-2Dd alpha2 domain is sufficient to account for the allele-specific in vivo protection of H-2b mouse RBL-5 tumors from NK cell-mediated rejection in D8 mice. Thus, in striking contrast to the alpha1 specificity of Ig-like killer inhibitory receptors for human HLA, the lectin-like mouse Ly-49A receptor is predominantly restricted by the H-2Dd alpha2 domain in vitro and in vivo.

Mouse Ly-49A interrupts early signaling events in natural killer cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase.

The lytic activity of natural killer (NK) cells is inhibited by the expression of class I major histocompatibility complex (MHC) antigens on target cells. In murine NK cells, Ly-49A mediates inhibition of cytotoxicity in response to the class I MHC antigen H-2Dd. In this report, we studied the function of mouse Ly-49A in both the rat NK cell tumor line, RNK-16, transfected with Ly-49A cDNA, and in primary NK cells. We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation. We also show that Ly-49A directly associates with the cytoplasmic tyrosine phosphatase SHP-1, and that Ly-49A function is impaired in NK cells from SHP-1 mutant viable motheaten mice and from SHP-1-deficient motheaten mice. Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor. These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

Divergent functions of lectin-like receptors on NK cells.

NK cells express a superfamily of surface proteins that share a common structure: dimeric type II integral membrane proteins whose extracellular domains have structural features of C-type (calcium-dependent) lectins. These receptors are encoded in a single genetic region called the NK complex (NKC). The NKC encompasses several families of genes, including Ly-49 (in mice and rats), NKR-P1 (in mice, rats, and humans). NKG2 (in humans and rats), and CD94 (in humans). Different NKC receptors have been shown to activate or to inhibit NK function, and different receptors within the same family can have opposing functions. In this review, we discuss the molecular pathways by which NK cells are activated, and the mechanisms by which inhibitory receptors interrupt activation. By studying the inhibitory receptor Ly-49A, we have demonstrated that inhibition utilizes the cytoplasmic phosphatase, SHP-1, which binds to a motif in the receptor cytoplasmic domain, termed an immunoreceptor tyrosine-based inhibitory motif (ITIM). In this regard, the lectin-like receptors are functionally similar to the immunoglobulin-like killer inhibitory receptors (KIRs) on human NK cells. The presence of an ITIM generally correlates with inhibitory activity among NKC lectin-like receptors, as demonstrated by the human NKG2 receptor family. Lanier and his colleagues have recently shown that NKG2 receptors can form heterodimers with the invariant lectin-like receptor CD94. Selective association of CD94 with different NKG2 receptors may explain functional differences for CD94 in different NK clones.

An autosomal dominant locus, Nka, mapping to the Ly-49 region of a rat natural killer (NK) gene complex, controls NK cell lysis of allogeneic lymphocytes.

Natural Killer (NK) cells can recognize and kill MHC-incompatible normal bone marrow-derived cells. Presently characterized MHC-binding receptors on NK cells, including the Ly-49 family in the mouse, transmit inhibitory signals upon binding to cognate class I MHC ligands. Here we study in vivo NK-mediated lysis of normal allogeneic lymphocytes in crosses between alloreactivity-competent PVG rats and alloreactivity-deficient DA rats. NK cells from both strains are able to lyse standard tumor targets. We identify an autosomal dominant locus, Nka, that controls NK-mediated alloreactivity. Individuals carrying the dominant PVG allele in single dose were fully competent in eliminating allogeneic target cells, suggesting that Nka encodes or regulates a gene product inducing or activating alloreactivity. By linkage analysis and pulsed field gel electrophoresis, a natural killer gene complex (NKC) on rat chromosome 4 is described that contains the rat NKR-P1 and Ly-49 multigene families plus a rat NKG2D homologue. Nka maps within the NKC, together with the most telomeric Ly-49 family members, but separate from NKG2D and the NKR-P1 family. The Nka-encoded response, moreover, correlates with the expression of transcripts for Ly-49 receptors in NK cell populations, as Northern blot analysis demonstrated low expression of Ly-49 genes in DA NK cells, in contrast to high expression in alloreactivity-competent PVG, (DA X PVG)F1, and PVG.1AVI NK cells. The low Ly-49 expression in DA is not induced by MHC haplotype, as demonstrated by high expression of Ly-49 in the DA MHC-congenic PVG.1AVI strain. Finally, we have cloned and characterized the first four members of the rat Ly-49 gene family. Their cytoplasmic domains demonstrate substantial heterogeneity, consistent with the hypothesis that different Ly-49 family members may subserve different signaling functions.

NKR-P1A is a target-specific receptor that activates natural killer cell cytotoxicity.

NKR-P1A is a lectinlike surface molecule expressed on rat natural killer (NK) cells. NKR-P1A has structural and functional features of an activating NK cell receptor, but a requirement for NKR-P1A in target cell lysis has not been determined. To define the role of NKR-P1A in natural killing, we have generated a mutant of the rat NK cell line, RNK-16, lacking expression of all members of the NKR-P1 receptor family. Although these NKR-P1-deficient NK cells were able to kill many standard tumor targets, including YAC-1, they were selectively deficient in the lysis of IC-21 macrophage, B-16 melanoma, and C1498 lymphoma targets. Reexpression of a single member of the NKR-P1 family, NKR-P1A, on mutant cells restored lysis of IC-21, and killing of IC-21 targets through rat NKR-P1A was completely blocked by F(ab')2 anti-NKR-P1A. Reexpression of NKR-P1A also restored transmembrane signaling to IC-21, as assessed by the generation of inositol-1,4,5-trisphosphate. The generation of inositol-1,4,5-trisphosphate was also restored in response to B-16 targets, but both B-16 and C1498 cells remained resistant to lysis, indicating that other NK cell molecules, perhaps within the NKR-P1 family, are required for the efficient killing of these tumors. These results are the first to demonstrate that NKR-P1A is a target-specific receptor that activates natural killing.

A family of murine NK cell receptors specific for target cell MHC class I molecules.

The Ly-49A molecule is an NK cell receptor specific for MHC class I molecules on target cells. When Ly-49A engages H-2Dd, Ly-49A+ NK cells become globally incapable of killing their targets in vitro. This interaction also occurs in vivo. Ly-49A belongs to a family of highly related molecules, including Ly-49C (5E6 antigen) and LGL-1 that also determine NK cell specificity. In the NK gene complex, the Ly-49 family is genetically linked to genes encoding NKR-P1 and CD69 that are structurally related and capable of activating NK cells. Finally, Ly-49 may be related to human molecules that are selectively expressed on NK cells and influence NK cell specificity. These findings highlight the emerging significance of the Ly-49 family in NK cell activity.

Ly-49A, a receptor for H-2Dd, has a functional carbohydrate recognition domain.

Ly-49A+ murine natural killer (NK) cells cannot lyse target cells that express H-2Dd. We demonstrate a functional requirement for carbohydrate recognition by Ly-49A. Treatment of H-2Dd+ target cells with tunicamycin prevents their binding to Ly-49A+ cells and renders them susceptible to lysis by Ly-49A+ NK cells. Fucoidan, a sulfated polysaccharide, binds to Ly-49A in a calcium-dependent manner, and this binding is inhibited by monosaccharides, particularly sulfated hexoses. The inactivation of Ly-49A+ NK cells by H-2Dd+ target cells is reversed in the presence of glucose 6-SO4. These results indicate that Ly-49A has a functional carbohydrate recognition domain and that target expression of carbohydrates alters their susceptibility to natural killing.

A natural killer cell receptor specific for a major histocompatibility complex class I molecule.

Target cell expression of major histocompatibility complex (MHC) class I molecules correlates with resistance to lysis by natural killer (NK) cells. Prior functional studies of the murine NK cell surface molecule, Ly-49, suggested its role in downregulating NK cell cytotoxicity by specifically interacting with target cell H-2Dd molecules. In support of this hypothesis, we now demonstrate a physical interaction between H-2Dd and Ly-49 in both qualitative and quantitative cell-cell binding assays employing a stable transfected Chinese hamster ovary (CHO) cell line expressing Ly-49 and MHC class I transfected target cells. Binding occurred only when CHO cells expressed Ly-49 at high levels and targets expressed H-2Dd by transfection. Monoclonal antibody blocking experiments confirmed this interaction. These studies indicate that the specificity of natural killing is influenced by NK cell receptors that engage target cell MHC class I molecules.

The Ly-49 and NKR-P1 gene families encoding lectin-like receptors on natural killer cells: the NK gene complex.

Natural killer cells lyse tumor and virally infected cells in a specific manner that has not been molecularly characterized. Target cell expression of major histocompatibility complex (MHC) class I molecules is correlated with target cell resistance to natural killing. A mechanism to explain this observation is that NK cells may display two types of recognition and activation molecules that have opposing functions when bound to target cell ligands. One type of surface receptor such as the NKR-P1 molecule may activate NK activity whereas the other, represented by the mouse Ly-49 molecule, may engage target cell MHC molecules and inhibit cytotoxicity by transducing "negative" signals. NKR-P1 and Ly-49 are structurally related, and they are encoded by genetically linked loci in a chromosomal region, termed the NK gene complex (NKC), on distal mouse chromosome 6. Target cell susceptibility to natural killing may be dependent upon specific ligand-receptor interaction with these activating or inhibitory NKC-encoded molecules.

Molecular cloning of the NK1.1 antigen, a member of the NKR-P1 family of natural killer cell activation molecules.

In mice, the NK1.1 alloantigen is expressed on all NK cells and it initiates transmembrane signals that activate cytotoxicity. NK1.1 has been mapped to a chromosomal region that encodes two families of receptor-like molecules, the NKR-P1 family and the Ly-49 family. Both of these gene families encode type II integral membrane proteins whose extracellular domains are homologous with known C-type lectins. We have isolated and expressed a member of the NKR-P1 gene family (mNKR-P1.9) and have demonstrated both by immunofluorescence and by immunoprecipitation that this cDNA encodes NK1.1. These findings, which demonstrate the receptor-like structure of NK1.1, will facilitate studies regarding the role of NK1.1 in natural killing and regarding the identification of possible NK1.1 ligands.

The OX-44 molecule couples to signaling pathways and is associated with CD2 on rat T lymphocytes and a natural killer cell line.

The MRC OX-44 molecule, which is expressed on all peripheral leukocytes, identifies the subset of thymocytes capable of proliferating in response to alloantigens and lectins (Paterson, D.J., J.R. Green, W.A. Jefferies, M. Puklavec, and A.F. Williams. 1987. J. Exp. Med. 165:1). When we isolated monoclonal antibodies (mAbs) on the basis of their ability to activate the phosphatidylinositol signaling pathway in RNK-16 cells (a rat leukemia line with natural killer activity), three of the resulting mAbs recognized the OX-44 molecule. Addition of these mAbs to RNK-16 elicits protein tyrosine phosphorylation, generates inositol phosphates, and increases the concentration of cytoplasmic free calcium. These responses require the addition of intact mAb and are not observed with F(ab')2 fragments. One of these mAbs (7D2) is mitogenic for freshly isolated rat splenic T cells and synergizes with a mAb to the T cell antigen receptor in this activation. A 50-60-kD glycoprotein coprecipitates with the OX-44 molecule from RNK-16 cells and rat splenic T cells. Peptide mapping and reprecipitation studies indicate that the coprecipitating molecule is CD2. Thus, the OX-44 molecule can couple to multiple signaling pathways and associates with CD2 on both RNK-16 and rat T cells.