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

seminars in

IMMUNOLOGY, Vol 7, 1995: pp 89–101

A family of murine NK cell receptors specific for target cell MHC class I molecules Wayne M. Yokoyama, Brian F. Daniels*, William E. Seaman*, Rosemarie Hunziker†, David H. Margulies† and Hamish R.C. Smith

function indicate that the expression of certain target cell MHC class I molecules correlates with resistance to NK cell-mediated activity.4-9 As a teleological explanation for this observation, it has been suggested that NK cells may survey tissues for normal expression of MHC class I molecules.10,11 If MHC class I expression is absent or abnormal (‘missing self’), such as in tumorigenesis or infection, NK cells may be released from the inhibitory influence of MHC class I molecules then kill the target. This may be mediated by NK cell receptors specific for MHC class I molecules on the targets.12 The Ly-49 cell surface molecule appears to determine mouse NK cell specificity and target cell MHC class I-associated resistance to lysis. In this review, we will discuss the structure, function, and molecular genetics of this molecule and its family of related molecules as well as discuss its relationship to other cell surface molecules expressed by rodent and human NK cells.

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. Key words: C-type lectin / Ly-49 / MHC class I / NK cell receptor

IN CONTRAST TO lymphocytes involved in specific (acquired) immunity, i.e. T and B lymphocytes, natural killer (NK) cells possess an apparently innate ability to respond to tumors and pathogens.1-3 Although this ability is generally considered to be non-specific and non-MHC restricted, recent studies have challenged these notions. NK cells appear to have a mechanism to specifically discriminate between their targets because they do not lyse all tumor or infected cells and they generally do not kill normal cells. The lytic activity of heterogeneous NK cell populations is clearly not restricted (at least as defined by MHC restriction for T cells) by expression of specific MHC molecules on target cells (1-3). As reviewed elsewhere in this monograph, however, several recent in-vitro and in-vivo studies of NK cell

NK cell receptors specific for target cell MHC class I molecules: the Ly-49 family of molecules Although the molecular basis for NK cell specificity remains incompletely understood, a major clue to understanding NK cell recognition has been provided by the recently defined relationship between MHC class I molecules on target cells and natural killing. Targets expressing MHC class I molecules are more resistant to natural killing than targets that do not express MHC class I molecules. Mechanisms can now be postulated to account for this correlation.10-12 Although this subject is extensively discussed elsewhere in this monograph, it is useful to briefly mention a modification of a model first described as ‘effector inhibition’.10,11 In this model, NK cells have a receptor specific for MHC class I molecules that inhibits natural killing when it engages its ligand on target cells. In the modified model, NK cells may have two types of receptors. In addition to the inhibitory

From the Howard Hughes Medical Institute, Department of Medicine, Mount Sinai Medical Center, New York, NY 10029, *Department of Veteran's Affairs Medical Center, San Francisco, CA 94121, †Laboratory of Immunology, National Institutes of Health, Bethesda, MD 20892, USA ©1995 Academic Press Ltd 1044-5323/95/020089 + 13$8.00/0

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W. M. Yokoyama et al that Ly-49A itself may belong to a family of molecules that may play a recognition role in NK cell function.

receptor, the second receptor may be capable of activating NK cells.12 In the absence of MHC class I inhibition, the NK cell may be stimulated through the activation receptor if it contacts its putative ligand on the target cell. This model implies that there are at least two levels of specificity regulating NK cell cytotoxicity. A target will be killed by a given NK cell only if the target does not appropriately display the MHC class I molecules for which the NK cell has specific inhibitory receptors, and if the target expresses the specific ligand for the activation receptor. This model is supported by studies of Ly-49 that appears to function as an inhibitory NK cell receptor with specificity for target cell MHC class I molecules. Now that several molecules highly related to Ly-49 have been described, the original Ly-49 molecule has been termed by Ly-49A and each succeeding homologue has been designated with a subsequent letter13,14 (see later). In this section, we will review the evidence supporting the role of Ly-49A in recognition of target cell MHC class I molecules and the known characteristics of other members of the Ly-49 family.

Functional studies demonstrating MHC specificity of Ly-49A + NK cells To determine a function for Ly-49A, studies were designed to examine whether Ly-49A + NK cells might exhibit properties that differed from those displayed by Ly-49A– NK cells.22 IL-2-activated NK cells from C57BL/6 mice were separated according to Ly-49A expression with mAb A1 (anti-Ly-49A).16 Both subpopulations lacked expression of CD3, CD4, CD8 and sIg, and were similar in expression of other NK cell surface markers, and functional capacity. This included ability to lyse YAC-1, and to mediate ADCC, antibody-induced redirected lysis, and lectin-facilitated lysis22. Thus, the Ly-49A + and Ly-49A– IL2-activated NK cells were phenotypically similar except for the expression of Ly-49A. Despite equivalent functional capacities, Ly-49A + and Ly-49A– effector cells differed in their ability to kill a panel of murine tumor cell targets22. This apparent specificity correlated with the MHC haplotype of the targets. Whereas syngeneic tumor cells (H-2b) were equally lysed by both effector cell subsets, target cells derived from H-2d or H-2k haplotypes were preferentially resistant to spontaneous lysis by Ly-49A + cells. Resistance also required the expression of MHC class I molecules on the targets. The resistant targets were not killed by Ly-49A + effector cells even when stimulated by other mechanisms including ADCC, lectin-mediated, or mAb-induced redirected lysis.22-24 This suggested that a specific MHC class I molecule could confer global resistance to lysis by Ly-49A + effector cells. To address this possibility, a susceptible tumor cell line (H-2b) was transfected with cDNA constructs encoding H-2Dd, Kd, or Ld, or with vector DNA alone because heterozygous (i.e. H-2d/b) targets displayed the resistant phenotype.22 Only the H-2Dd transfectants became preferentially resistant to natural killing by Ly-49A + effector cells. In contrast, transfection and expression of the other constructs had no effect on susceptibility to lysis by Ly-49A + effector cells. The gene-transferred resistance to natural killing by Ly-49A + effector cells also extended to resistance to other lysis by other mechanisms including ADCC.22-24 Thus, expression of H-2Dd by the target cell globally inhibited Ly-49A + NK cell cytolytic activity.

An NK cell subpopulation expresses Ly-49A The Ly-49A molecule was initially identified as a disulfide-linked dimer with 44 kD subunits expressed by the murine T-cell tumor, EL4.15,16 Subsequent to the cloning of a cDNA encoding Ly-49A,17,18 its gene was found to be genetically linked to the gene encoding the NK1.1 antigen19 (see later), suggesting that the expression of these molecules may be coordinated. Although Ly-49A can be detected on rare populations of T cells and after activation with high concentrations of IL-2 (F.M. Karlhofer and W.M. Yokoyama, unpublished observations), Ly-49A was readily found to be constitutively expressed by a distinct subpopulation of CD3–NK1.1 + splenocytes.19 The structural similarity of Ly-49A to NKR-P1 (NK1.1)19–21 (see later), genetic linkage of its gene to NKR-P120 (see later), and selective Ly-49A expression on NK cells suggested that Ly-49A might also be functionally active on NK cells. Moreover, the Ly-49A gene appeared to display significant polymorphism at two levels. On Southern blot analysis of mouse genomic DNA, the Ly-49A cDNA hybridized to multiple bands consistent with several cross-hybridizing genes (see later) within an inbred mouse strain.17,18 Between strains, allelic polymorphism was suggested by the presence of several different restriction fragment length polymorphic (RFLP) patterns.19 The apparent polymorphism of the Ly-49 genes suggested 90

Ly-49 family of NK cell receptors decreased expression of Ly-49A in the H-2d haplotype. Ly-49A expression was also examined in B6.tDD and B6.tDQ mice transgenic for soluble forms of Dd. Both mice were derived by microinjection of constructs encoding chimeric molecules consisting of Dd with Q10, a soluble MHC class I molecule.27 The B6.tDD strain expresses a Dd/Q10 chimeric molecule with the α1/α2 and α3 domains derived from Dd and the hydrophilic region derived from Q10 replacing the transmembrane region. The B6.tDQ strain expresses a chimeric molecule with the α1/α2 domains derived from Dd and the Q10-α3 domain and hydrophilic region. The soluble chimeric molecules bind Dd-specific peptides and β2-microglobulin and fold correctly because they are recognized by α1/α2-domain specific antibodies that recognize only correctly folded Dd molecules.28 Moreover, these molecules can stimulate Dd-restricted T cells. Ly-49A was normally expressed in mice transgenic for either soluble Dd construct.25 Therefore, the in-vivo expression indicates that the decreased expression of Ly-49A requires membrane-bound H-2Dd, consistent with the interpretation of Ly-49A + NK cell specificity experiments done in in vitro. Several possible mechanisms could account for decreased expression of Ly-49A on NK cells in H-2Ddbearing hosts. Ly-49A molecules could complex with H-2Dd during synthesis and/or intracellular trafficking, diminishing cell surface expression of Ly-49A. However, in bone marrow chimeric mice, Sykes et al reported that donor C57BL/10 NK cells do not express Ly-49A when they matured in irradiated H-2Dd-bearing hosts [B10.A (H-2Dd)].29 In contrast, there was normal expression of Ly-49A on donor [C57BL/10 (H-2Db)] NK cells in syngeneic recipients. (C57BL/10 mice normally express Ly-49A on NK1.1 + cells whereas B10.A do not.) Bone marrow elements could also down-regulate Ly-49A expression. These data demonstrate the influence of host radioresistant and bone marrow elements on Ly-49A expression in vivo and exclude the possibility that Ly-49A is complexed with H-2Dd during protein synthesis or intracellular trafficking with resultant diminished expression of Ly-49. The data strongly support the hypothesis that the effect is due to a specific interaction between the Ly-49A and H-2Dd molecules expressed on the surface of NK cells and host elements, respectively. How do host elements modulate Ly-49A expression in vivo and what are the consequences? Although not yet fully understood, several possibilities should be

This MHC class I gene transferred resistance was reversible with mAbs directed against either Ly-49A or the α1/α2 domains of H-2Dd.22,23 Control mAbs, including an mAb specific for the H-2Dd-α3 domain, did not affect target lysis. The effects of the anti-Ly49A and anti-α1/α2 domain mAbs were not due to redirected lysis or ADCC, respectively, because F(ab')2 fragments were comparable to intact mAbs. Thus, the mAbs appeared to specifically block the interaction of Ly-49A with α1/α2 domains of H-2Dd.22 These data suggested that, upon engagement, Ly-49A may transmit ‘negative’ signals, consistent with the two receptor model for NK cell specificity for MHC class I molecules.

Host MHC class I molecules modulate in-vivo expression of Ly-49A The in-vitro data strongly suggested that Ly-49A, on NK cells from H-2b mice, interacts with H-2Dd and an H-2k haplotype-encoded MHC class I molecule, on target cells. This suggested that NK cell expression of Ly-49A may be altered in H-2d or H-2k mice allowing confirmation, in a separate system, of these putative interactions.25 Moreover, an in-vivo system had the potential to reveal a physiological consequence of the interaction between these molecules. Ly-49A expression was analysed in a panel of C57BL/10 strains that are congenic for different MHC haplotypes. By three-color flow cytometry, splenic NK1.1 + cells from C57BL/10 mice were found to express Ly-49A but these cells were not detectable in the C57BL/10 strains with the H-2d or H-2k haplotype (B10.D2 and B10.BR, respectively).25 In contrast, the percentage of NK1.1 + splenocytes was unaffected. Moreover, Ly-49A was not detectable in (C57BL/6 3 DBA/2)F1 hybrids, B10.AKM (H-2m) and B10.S (H-2s) strains, indicating that neither a mutation in the Ly-49A gene, nor the absence of H-2b haplotype could account for altered Ly-49A expression. Further analysis of MHC recombinant mice demonstrated that Ly-49A expression appeared to be regulated by the H-2D subregion, specifically by H-2Dd and possibly H-2Dk. Genetic analysis with the available MHC-congenic mice could not distinguish H-2Dd itself from genetically linked genes. However, the availability of a C57BL/6 mouse (D8) transgenic for membrane bound Dd26 provided the opportunity to distinguish Dd from other genes because these mice lacked detectable Ly-49A + NK cells. This finding indicated that Dd expression alone was sufficient to account for 91

W. M. Yokoyama et al H-2Dd and possibly H-2Dk. Thus, these data strongly support the function of Ly-49A as an NK cell receptor specific for MHC class I molecules. While adhesion assays were used to establish the physical interaction between Ly-49A and its ligands, it should be noted that the function of Ly-49A is probably not adhesion per se. Cell binding was not demonstrable when Ly-49A was expressed at lower levels, comparable to levels expressed by NK cells. Moreover, cell adhesion alone seems inadequate to explain the global inability of Ly-49A + NK cells to lyse H-2Dd-transfected targets.22,23 Therefore, the data remain consistent with a physical interaction between Ly-49A and H-2Dd leading to an inhibitory signal in the NK cell.

considered: (1) Host MHC class I molecules could result in negative selection of NK cell clones, analogous to clonal deletion of T cell clones bearing T cell receptors (TCR) with high affinity for self-MHC molecules.30-32 (2) The proliferation of Ly-49A + cells could be inhibited by H-2Dd resulting in the presence of Ly-49A + NK cells in rarer numbers, below the ability to detect these cells by flow cytometry. (3) H-2Dd molecules could down-regulate Ly-49A gene transcription or protein synthesis. Consequently, Ly49A could be expressed at significantly lower levels on individual Ly-49A + NK cells that are present in normal numbers. (4) Finally, Ly-49A molecules may be expressed on the NK cell but in an altered conformation such that the Ly-49A molecules do not express the epitope recognized by the mAb. The first two possibilities do not appear to be likely because Ly49A expression could be detected when freshly isolated NK cells were purified from H-2Dd-bearing mice.25 In these experiments (in contrast to two color flow cytometry), Ly-49A is expressed at very low levels but on a percentage of freshly isolated cells similar to H-2Db mice. (We still cannot rule out the possibility that the molecule is resynthesized and begins to be expressed during the long isolation procedure.) The remaining possibilities cannot be distinguished with the existing data. However, the mechanism by which host MHC negatively regulates expression of putative MHC class I-specific NK cell receptors appears to be significantly different from their effect on T-cell receptors.32 Moreover, the data strongly support a direct in-vivo interaction between Ly-49A and H-2Dd with one manifestation being the decreased expression of Ly-49A.

Evidence for Ly-49A as an NK cell receptor for H-2Dd The data discussed so far can be summarized according to the type of experiments that have been performed. Physical interaction: adhesion assays A physical interaction between these molecules was demonstrated by cell–cell adhesion assays in a transfection system.33 Cell binding only occurred if one cell expressed Ly-49A by transfection and the other expressed H-2Dd also by transfection. In a separate system, Ly-49A-bearing cells could bind specifically to immunopurified H-2Dd.34 In both studies, the interaction was blocked by mAbs specific for either molecule.33,34 In-vivo interaction: modulation of Ly-49A expression In bone marrow chimeric mouse studies, Ly-49A is down-regulated by host MHC class I molecules, apparently acting via extracellular contact with Ly49A.29 This interaction can occur in the bone marrow. Genetic mapping studies with MHC-congenic mice indicated that the responsible host element is genetically linked to H-2Dd (or H-2Dk).25 This was further supported in studies of mice that are transgenic for membrane-bound H-2Dd.25

d

Physical interaction between Ly-49A and H-2D

A specific interaction between Ly-49A and H-2Dd could be predicted on the basis of transfection and mAb blocking data in the in-vitro functional experiments. Moreover, similar conclusions are possible with the in-vivo analysis utilizing congenic and MHC class I-transgenic mice. To confirm these interpretations, a physical interaction between Ly-49A and H-2Dd was sought in a cell adhesion system using transfected cells.33 Adhesion only occurred between transfected cells expressing Ly-49A at high levels and other cells expressing H-2Dd by transfection. Adhesion was blocked by mAbs specific for either Ly-49A or α1/α2 domains of H-2Dd Consistent results were also reported by Kane34 using EL4 tumor cells, expressing Ly-49 and immunopurified H-2 molecules, including

Functional specificity In cytotoxicity assays in vitro, Ly-49A + effector cells did not lyse H-2d and H-2k targets that express MHC class I molecules.22 A susceptible target was made specifically resistant by transfection and expression of H-2Dd.22 The gene transferred resistance extended to other activation pathways of NK cell cytotoxicity.22-24 92

Ly-49 family of NK cell receptors cule;47 Ly-49A could be specific for only the correctly folded Dd molecule expressed at normal levels. On the other hand, Ly-49A may interact with only specific peptides bound to Dd and not to Dd itself. Finally, Ly49A may simultaneously recognize the complex of bound peptides and MHC class I residues, in a manner analogous to the T-cell receptor. Further analysis will be required to distinguish these possibilities. The homology of Ly-49A to the C-type lectin superfamily18 suggest other possible interaction sites on Dd and/or its bound peptides. Inasmuch as several members of this superfamily bind to carbohydrate residues on glycoproteins,48 Ly-49A could recognize a carbohydrate determinant expressed only on Dd molecules. The conservation of N-linked glycosylation sites flanking MHC class I molecules may have relevance to this possibility.49 However, analysis of carbohydrate specificity in several glycoprotein systems have suggested that such interactions are not only carbohydrate specific; the protein backbone displaying the glycosidic residues may significantly influence the specificity of binding.50-51 Ly-49A could recognize a specific carbohydrate determinant only when displayed by the Dd polypeptide even though the carbohydrate could be displayed on other molecules. The apparent localizaton of Ly-49A binding to the peptide binding domain of Dd also raises the possibility that glycosylated peptides, bound to Dd are recognized by Ly-49A. Recent evidence showing that MHC molecules can display glycosylated peptides are consistent with this possibility.52,53 However, not all ligands for C-type lectin superfamily members bind in a carbohydrate-specific manner. The low affinity IgE receptor, CD23, binds IgE with no regard to glycosylation of IgE.54 Thus, the significance of the homology of Ly-49A to the C-type lectins remains to be clarified.

Resistance was reversed by mAbs directed against either Ly-49 or H-2Dd Thus, there is compelling evidence indicating that Ly-49A is an NK cell receptor for H-2Dd on target cells. The Ly-49A receptor is somewhat promiscuous because it appears to be able to interact with other MHC class I molecules, including H-2Dk.22,25,34 The result of interaction between Ly-49A and its MHC class I ligands appears to be down-regulation of NK cell cytotoxicity and its own expression in vivo. The data also indicate that Ly-49A appears to specifically interact with the α1/α2 domains of Dd There are several lines of evidence supporting this interpretation. The functional activity of Ly-49A in killing experiments, and the adhesion of Ly-49A + cells to Dd transfectants can be blocked by mAbs directed against the α1/α2 domains of Dd.22,33 In contrast, a mAb specific for the α3 domain has no activity in these experiments. In the in-vivo expression system, studies of mice with a chimeric form of Dd were also informative.25 B10.D2dm1 mice do not carry a normal Dd molecule, and instead display a chimeric molecule derived from a fusion between the Dd and Ld genes.35 The α1 and NH2 terminal half of the α2 domain of the dm1 molecule is derived from Dd while the rest of the molecule is Ld. In these mice, Ly-49A is also downregulated. Since Ld does not interact with Ly-49A in the cytotoxicity experiments and in the cell adhesion assay,22,33 these results also support the conclusion that the Ly-49A molecule interacts with the α1/α2 domains of the Dd molecule. Ly-49A, therefore, appears to bind MHC class I molecules in a manner analogous to MHC class I-restricted T-cell receptors36,37 instead of the CD8 molecule that binds to the α3 domain.38-40 This also fits well with previous observations implicating the α1/α2 domain of MHC class I in resistance. Storkus et al have localized the protective effect of HLA class I molecules to the α1/α2 domains in exon shuffle experiments.41 In addition, a single residue in a side pocket of the peptide binding cleft was shown to be critical for HLA class I protection from natural killing.42 These data suggest that MHC class I-associated peptides are involved in this protection. Support for this possibility has been shown by studies indicating that viral derived peptides appear to reverse the protective effect presumably by displacing protective ‘self-peptides’.43-46 Ly-49A binding may be influenced, therefore, by peptides bound to MHC class I. This could be in a general way because peptides are required for normal assembly and expression of a correctly folded MHC class I mole-

Relevance to Ly-49A to NK cell specificity Although there are ample data demonstrating that Ly49A is a receptor for H-2Dd, the relevance of Ly-49A to NK cell specificity has been controversial in light of the ‘missing-self’ hypothesis. The functional studies described above utilized Ly-49A + NK cells from H-2b mice; these NK cells could kill all tested tumor targets of the H-2b (self) haplotype. If Ly-49A is a putative inhibitory NK cell receptor that recognizes MHC class I molecules, as predicted by the ‘missing-self’ hypothesis, why do these cells continue to kill targets of H-2b origin? There are several potential explanations. (1) 93

W. M. Yokoyama et al 49A-H-2Dd paradigm needs further examination to ascertain its physiological relevance in view of the above possibilities, additional studies of the Ly-49 family of related molecules appear to provide further insight.

The initial observations concerned a small panel of H-2b tumor targets. Correa et al extended the initial observations by demonstrating that two other H-2bderived targets, an EL-4 subline, and R8.15, were efficiently lysed by Ly-49A + NK cells from H-2b mice.24 However, RMA and another EL-4 subline were relatively resistant to lysis by the Ly-49A + effector cells, suggesting that the susceptibility of H-2b targets to killing by Ly-49A + effector cells is not a general rule. That the resistance of the H-2b targets is due to Ly-49A or H-2b-encoded MHC class I molecules has not been determined but this observation suggests that (2) the susceptible H-2b targets may possess a mechanism that enhances their susceptibility even when MHC class I molecules are expressed. For example, these cells may express high levels of the ligand for the putative activation receptor. (3) Another possibility is that the susceptible H-2b targets express MHC class I molecules that are incapable of providing the resistant phenotype, i.e. they may lack the appropriate expression of the relevant H-2b MHC class I molecule. H-2Kb and H-2Db expression was apparently normal by flow cytometry analysis with specific mAbs22 but other H-2b class I molecules may be more important. (4) Alternatively, the susceptible H-2b-derived targets could express the relevant MHC class I molecules but these molecules manifest a deficiency in the ability of these molecules to inhibit killing. This deficiency could include MHC class I-bound peptide molecules if Ly49A recognizes only a subset of MHC class I molecules bound to specific peptides. Another MHC class I deficiency could be abnormality in specific MHC class I-associated carbohydrate residues. (5) Ly-49A + NK cells may be recognizing an allotypic determinant on H-2Dd that mimics its physiologic (H-2b-encoded) MHC class I ligand. This allotypic determinant could bind Ly-49A with a higher avidity than self MHC class I molecules. Certainly, T-cell receptors specific for a given Ag/self-MHC molecular complex can also react with determinants on non-self (allotypic) MHC molecules.55,56 Moreover, much of the original basis of modern immunology made use of reactivity against allotypic determinants such as those involved in tissue transplantation. In this sense, then, the Ly-49A-H-2Dd paradigm is important because it permits the identification of the molecules that participate in MHC class I-associated target cell resistance. (6) Finally, the least likely possibility is that Ly-49A is constitutively expressed in H-2b mice but has no functional relevance to NK cell specificity in these mice. This seems teleologically unlikely since 20% of the NK cells in these mice express this molecule. Although the Ly-

The polymorphic Ly-49 family of molecules TCRs are highly polymorphic paralleling the polymorphism of its ligands–peptides and peptide-binding domains of MHC class I molecules — whereas CD8 molecules are minimally polymorphic and so are α3 domains of MHC class I molecules. Therefore, the apparent localization of Ly-49A binding to the α1/α2 domains of Dd raised the possibility that Ly-49A may be similarly polymorphic. One conceivable hypothesis is that Ly-49A may belong to a family of highly related molecules displaying significant polymorphism. Moreover, Ly49A– NK cells could express other molecules, perhaps structurally related to Ly-49A, that endow similar function. When the original Ly-49A cDNA was isolated, Southern blot analysis revealed multiple bands,17,18 and several distinct, RFLP variants among inbred mouse strains.19 These studies strongly suggested that Ly-49A belongs to a family of molecules that are encoded by cross-hybridizing genes having several allelic forms but it remained possible that the Ly-49A gene was comprised of multiple, small exons, or that some of the cross-hybridization species represented pseudogenes. Recently, two cDNAs were isolated by cross-hybridization with the Ly-49A cDNA from a cDNA library constructed from (C57BL/ 6 3 CBA)F1 hybrid mouse lung cells.13 The two additional molecules, Ly-49B and Ly-49C, are similar in size and are 52% and 64% identical, respectively, to the Ly-49A polypeptide sequence. These molecules may represent an allelic (CBA) form of Ly-49A (originally cloned from a C57BL/6-derived cell line), or the CBA or C57BL/6 alleles of entirely new Ly-49 family members because C57BL/6 and CBA mice display different RFLP variants for the Ly-49 genes.19 Further detailed analysis should elucidate the relationship of these molecules but these data helped establish that Ly-49A belongs to a family of related molecules. To examine the Ly-49 family without the complication of allelic forms, other cDNAs encoding Ly49A-related molecules have been obtained from a single, inbred mouse strain (C57BL/6).14 By crosshybridization with the Ly-49A cDNA or PCR amplification, six cDNAs were identified that were related to 94

Ly-49 family of NK cell receptors

Ly-49-related molecules as NK cell receptors

the Ly-49A cDNA and were verified by examination of multiple cDNAs derived from IL-2-activated CD3– NK cells. The cDNAs fell into four groups according to nucleotide homology and identity of the deduced polypeptides. Since none of these cDNAs appeared to be allelic forms of Ly-49B or C, the four new groups were designated Ly-49D through Ly-49G. At the nucleotide level, the new cDNA sequences are 77–88% identical to Ly-49A with the alignments being maintained throughout the sequences. The Ly-49G group contained three sequences, Ly-49G.1, 2 and 3, that are identical to each other except for distinct DNA segments that are inserted or deleted. These cDNAs may represent alternative splicing of single mRNA species. All of the new cDNAs contain open reading frames encoding polypeptides that are homologous to the Ly-49A polypeptide (66–79% identical). As expected, the deduced proteins have similar structural features, including general overall and domain size, type II integral membrane orientation, conserved cysteines and external C-type lectin homology. Finally, these molecules cannot be allelic variants of each other because they were cloned from a single inbred mouse strain. Thus, the Ly-49related molecules form a distinct family of highly related molecules within the superfamily of C-type lectins. All exons of the Ly-49A gene have been identified by genomic cloning.13,57 However, overlapping contiguous clones have not been reported possibly because the gene is larger than the 23 kb inserts that can be accommodated in lambda phage cloning vectors. The upper limit of the size of the Ly-49A gene is estimated to be 30 kb by Southern blot analysis of restricted genomic DNA but the cross-hybridizing Ly49 family members may have genes with similar restrictions sites, complicating definitive analysis. Until the physical linkage can be determined by careful sequence analysis of relevant lambda or cosmid clones, the full Ly-49A gene remains incompletely characterized. Genomic analysis of the other Ly-49 genes has been limited to examination of individual exons of distinct genes. Wong et al described five partial, C57BL/6-derived, genomic sequences representing individual exons related to Ly-49A.13 Sequence alignment indicates that the L10 and U4 genomic clones contain exons for the Ly-49G gene whereas the U10 genomic clone contains an exon of the Ly-49F gene.14 Thus, limited genomic analysis has demonstrated that the Ly-49-related molecules are derived from distinct genes.

It is extremely tempting to speculate that the Ly49-related molecules will display similar functional properties to Ly-49A, i.e. determine specificity for MHC class I molecules, and act as inhibitory receptors. Subsets of NK cells may display these molecules analogous to expression of Ly-49A.19 This heterogeneity may obscure delineation of NK cell specificity unless the subsets are purified before analysis. For example, among IL-2-activated NK cells, only 20% express Ly-49A. The unseparated IL-2-activated NK cells kill Dd targets almost as well as highly purified Ly49A– effector cells (F.M. Karlhofer and W.M. Yokoyama, unpublished observations). The contribution of Ly-49A to specificity could not be determined until Ly-49A + effector cells were isolated.22 In the same way, Ly-49A– effector cells now appear to lyse targets relatively indiscriminately but if it can be determined that Ly-49A– effector cells are heterogeneous with respect to other Ly-49-related molecules, similar studies on purified subsets of the Ly-49– NK cells may reveal more precise specificity. Thus, the specificity of an individual NK cell may be dictated by its expressed repertoire of Ly-49-related molecules. A definition of the role of each of these molecules will require the development of serologic reagents such as mAbs specific for each form. Such mAbs will have to be scrutinized for possible cross-reactivity with more than one of the Ly-49 family members, particularly due to the close structural relationship between the molecules. Nevertheless, it should be possible to determine, for example, if Ly-49A– NK cells can be divided into more subsets based on expression of the Ly-49-related molecules, i.e. Ly-49A– NK cells may be comprised of two distinct subsets, Ly-49E + and Ly49F. + On the other hand, NK cells may express more than one of these molecules simultaneously and NK cells may belong to overlapping subpopulations based on expression of each of the molecules. For example, Ly-49E may be expressed by both Ly-49A + and Ly49A– NK cells. Obviously, many permutations are possible with at least five and possibly seven (at the cDNA level) full-length Ly-49-related molecules, and there may be preferential expression of one molecule with another. The development of new mAbs specific for each of the Ly-49-related molecules will also permit purification of NK cell subsets for functional analysis. However, studies of mAb-purified NK cell subsets will need to consider the possible functional alteration of the Ly-49-related molecule due to the presence of the mAb. In studies of Ly-49A, NK cell 95

W. M. Yokoyama et al terms of their influence on NK cell specificity. At least two of these molecules are now known to belong to the Ly-49-family. Other molecules are structurally related to the Ly-49 family and also encoded in the NKC but may subserve a different function. Finally, several human NK cell molecules, implicated in MHC class I-associated target cell resistance, appear to be distinct molecules but one recent example is more reminiscent of Ly-49A in structure and function. In the phenomenon termed hybrid resistance, host NK cells in irradiated F1 hybrid mice reject bone marrow grafts from highly inbred parental strains.58 This process appears to involve specific recognition of parental determinants encoded in a locus genetically linked to the H-2D subregion.59 Some studies have suggested that this determinant maps centromeric of the H-2Dd gene (59) but investigations of an H-2Dd transgenic C57BL/6 mouse strongly suggest that the parental determinant can be an MHC class I molecule such as H-2Dd itself.60. However, it has been difficult to assign parental determinants to known MHC class I molecules. To address the NK cell specificity for these parental determinants, mAbs were produced to identify NK cells mediating rejection of specific donor marrow. The mAb SW5E6 reacts with a disulfidelinked homodimer (54 kD subunits) present on approximately 40% of NK cells in C57BL/6 mice.61 Expression of this antigen can be related to specificity in hybrid resistance because SW5E6 + NK cells appear to mediate rejection of bone marrow cells expressing H-2d/Hh-1d but not H-2b/Hh-1b. The in-vitro target cell specificity of this subset has not yet been described. A cDNA encoding the antigen recognized by mAb SW5E6 has been recently cloned from a CB.17-scid NK cell library by eukaryotic expression and mAb reactivity.62 (CB.17 mice are a substrain of BALB/c mice.) Interestingly, the nucleotide sequence is identical to the Ly-49C sequence obtained from (C57BL/6 3 CBA)F1 hybrid mice.13 Since BALB/c and CBA mice have the same RFLP variant for Ly-49 genes and both are distinct from C57BL/6 (19), the cloned Ly-49C sequence possibly reflects the CBA allele. Thus, a Ly-49 family member has been implicated in the specificity of NK cells in hybrid resistance. The function and specificity of Ly-49C has not been determined directly. However, the potential role of H-2D-encoded MHC class I molecules in hybrid resistance, the specificity of Ly-49A for H-2Dd, and the correlation between Ly-49C and NK cell specificity in hybrid resistance strongly support a direct role for Ly49C in determining NK cell specificity by recognition

subsets were purified with the anti-Ly-49A mAb and cultured for several additional days to minimize any mAb effect.22,23 This was a critical step because the anti-Ly-49A mAb reverses the inhibitory effect of the interaction between Ly-49A and H-2Dd by blocking this interaction.22,33 If experiments were performed on the mAb-purified subsets immediately after purification, i.e. on FACS sorted cells, no functional difference would have been noted between the Ly49A + and Ly-49A– cells. For this reason, it has thus far been impossible to analyse Ly-49A subsets from freshly isolated NK cells. The development of new serologic reagents, however, may permit the use of mAbs to negatively select Ly-49A + NK cells on the basis of mAb depletion of Ly-49A– cells that may express other Ly49-related molecules. Detailed expression and functional analysis of the Ly-49 family of molecules will be aided by the development of further serologic reagents. With regard to the function of Ly-49 molecules, these molecules may be predominantly inhibitory, like Ly-49A. However, the function of these molecules may not be inhibitory or under certain circumstances, these molecules could also be capable of activation and subserve two seemingly opposing functions. The post-ligand binding function of Ly-49A, thus far, has been examined in cytotoxicity and in-vivo expression experiments. Although the data are consistent with an inhibition of cytotoxicity, a negative signaling receptor must transmit signals that could be viewed as a ‘positive’ signal at the biochemical level. In addition, the signals that turn off cytotoxicity could have other effects including activation of cytokine secretion, for example. It is noteworthy that a human molecule with features similar to Ly-49A, CD94 (see later), appears to have dual function in cytotoxicity whereas Ly-49C may have a role in direct activation. Thus, the family of Ly-49-related molecules may provide NK cells with additional molecules that bind to MHC class I on target cells. The Ly-49-related molecules could be functionally expressed by NK cells that are Ly-49A-negative. Moreover, the Ly-49-related molecules may be functionally expressed in hosts that down-regulate Ly-49A expression, such as H-2Ddbearing strains. Further characterization of the Ly49-related molecules should be helpful in understanding NK cell specificity.

Relationship of Ly-49-related molecules to other NK cell surface molecules Other NK cell surface molecules have been defined in 96

Ly-49 family of NK cell receptors homology, selective expression on a subset of NK cells, and correlation of expression with NK cell specificity. Like Ly-49A, the LGL-1 molecule is associated with NK cell specificity because LGL-1 + IL-2-activated NK cells do not lyse P815 or L5178YR target cells that are efficiently lysed by LGL-1– cells even though both effector populations have equivalent capacity to kill YAC targets.68 However, in contrast with Ly-49A, LGL1 expression is enhanced by IL-2-activation in vitro.66 Moreover, the anti-LGL-1 mAb can induce the killing of P815 targets by LGL-1 + NK cells.66 Although this could occur by blocking of an inhibitory interaction between LGL-1 and its putative target cell ligand, the anti-LGL-1 mAb may mediate its effect by inducing redirected lysis. This interpretation is supported by studies showing that the stimulatory effect was dependent on expression of Fc receptors on the targets, binding of anti-LGL-1 to these receptors, and the intact anti-LGL-1 mAb. This is the most striking contrast to Ly-49A because it raises the possibility that the Ly-49 family could have activating as well as inhibitory function on NK cells. Determination of the ligand specificity of these molecules will be helpful in further dissecting the role of these molecules in NK cell specificity. The Ly-49 family of molecules also share several unique features with the MusNKR-P1 family of molecules.20,21,69-71 Members of both families are selectively expressed on NK cells (MusNKR-P1 molecules by all NK cells and Ly-49 molecules by NK cell subsets) as disulfide-linked homodimers. Each family contains several distinct genes that are highly related within the family, such that it is difficult to distinguish the different members by hybridization conditions alone. The deduced polypeptide sequences reveal the structural relationship between the NKR-P1 and Ly-49 families because both families encode type II integral membrane proteins with homology to the C-type lectin superfamily. Finally, both gene families are genetically linked to the NK gene complex (NKC) on distal mouse chromosome 6.20 The functional, expression, structural and genetic similarities of the Ly-49 and MusNKR-P1 families are striking but it should be emphasized that their genes do not display nucleotide homology and the cDNA probes do not crosshybridize between gene families. Although MusNKRP1 molecules have been found to only activate NK cell cytotoxic activity,72-74 functional studies described above strongly suggest that Ly-49A appears to be inhibitory while other Ly-49 family members, i.e. LGL1, may be stimulatory. Further functional analysis of other members of the NKR-P1 and Ly-49 molecules

of MHC class I molecules. In hybrid resistance, this could be mediated by direct killing of stem cells.63 Alternatively, since NK cells produce colony stimulating factors and cytokines64 that modulate bone marrow engraftment, bone marrow engraftment could be controlled by NK cells in an indirect manner. In view of the inhibitory influence of Ly-49A engagement on NK cell cytotoxicity, it is possible that 5E6 + NK cells may kill H-2d bone marrow cells but be inhibited by an MHC class I molecule in the H-2b haplotype. Hybrid resistance could be more complicated and constitutive help by NK cells could be inhibited when Ly-49C contacts its ligand on stem cells, again in concert with inhibition of NK activity. Finally, triggering through Ly-49C may release cytokines that stimulate bone marrow engraftment. Precise understanding of the physiologic ligand for Ly49C should provide mechanisms to account for hybrid resistance. The implication of one member of the Ly-49 family in hybrid resistance, raises the possibility that other Ly-49 family members may be responsible for other NK cell specificities in this system. Moreover, it is important to note that the best characterized member of this family, Ly-49A, can interact with more than one MHC class I molecule, at least Dd and Dk (see previously). If other Ly-49 family members can interact with more than one MHC class I molecule, this apparent promiscuous specificity may help to explain the heretofore difficulty in correlating parental determinants as typed in hybrid resistance assays to known MHC class I molecules as defined by conventional serology and molecular analysis. Detailed examination of the specificities of the individual Ly-49 family members should therefore help solve the perplexities of hybrid resistance. The LGL-1 antigen is recognized by a rat mAb that reacts with a subset of murine NK cells.65 Although initial studies suggested that this molecule is a monomer, more recent studies are consistent with a disulfide-linked homodimer (87 kD nonreduced, 40 kD reduced) that is difficult to reduce with 2-mercaptoethanol.66 Similar features have been described for Ly-49A.18 Recently, a cDNA encoding the LGL-1 molecule has been isolated from the CB.17-scid NK cell library. This cDNA is highly homologous to the Ly-49 family of molecules.67 Although it is distinct from any known sequences, it is most closely related to the Ly-49G sequences. The LGL-1 sequence may therefore represent a BALB/c allelic form of the Ly49G sequence from C57BL/6 mice. The LGL-1 molecule bears several features in common with Ly-49A, including overall structural 97

W. M. Yokoyama et al that appear to be selectively expressed by NK cells on Northern blot analysis. The expression of these molecules at the protein level and their function have not yet been determined. These molecules could represent the human homolog of mouse NKR-P1 or Ly-49 molecules, or be related to mouse molecules that are yet to be described. However, the apparent localization of the genes encoding the NKG2 molecules to human chromosome 1286 is compatible with the syntenic location of the NKR-P1 and Ly-49 genes in the NKC on mouse chromosome 6 to human chromosome 12p13.2 as predicted.20 Further analysis will be required to determine the relationship of the human NKG2 molecules to the mouse molecules. Finally, cDNAs encoding the human and mouse CD69 molecules have been cloned and found to encode molecules homologous to Ly-49.87-89 The CD69 molecule is expressed by hematopoietic cells soon after activation. For example, resting T cells do not express CD69 but the molecule can be detected by flow cytometry with specific mAbs within 1 hr after T cell activation with phorbol esters and other mitogens including anti-CD3.90 Anti-CD69 mAbs can stimulate lymphocyte proliferation (requires co-stimulation with phorbol ester)90-92 and NK cell killing in the redirected lysis assay.74 Genetic linkage analysis has shown that the CD69 gene can be localized to the NKC.88,93 In contrast to Ly-49 and NKR-P1, however, CD69 appears to be an inducible antigen and more broadly expressed. Nevertheless, its structural homology, genetic linkage and capacity to activate NK cells are shared with other members of the NKC. Further analysis of the NKC may reveal other genes that are functionally important on NK cells as well as hematopoietic cells.

should be determined in the near future as cDNAs encoding additional family members are now available. Studies of human NK cells have also suggested that human NK cells can recognize an allospecific determinant that maps to the MHC class I region.75,76 As discussed in detail elsewhere in this monograph, the antigens (now termed p58 molecules) recognized by mAb GL183 and mAb EB6 are expressed by subsets of human NK cells that overlap77,78 and the expression of specific p58 molecules can be correlated with the MHC-associated specificity of NK cell clones. F(ab')2 fragments of anti-p58 mAbs were capable of reversing the inhibitory influence of HLA-C molecules in transfected targets, consistent with a blocking effect of the mAbs.79,80 These studies are reminiscent of the salient features of expression and functional analysis of Ly-49A and of the anti-Ly-49A mAb blocking studies, as described above. However, the relationship of the p58 antigens to the Ly-49-related molecules remains to be defined because the p58 antigens are not disulfide-linked dimers, and their primary structures have not been reported. Nevertheless, the p58 molecules may be inhibitory NK cell receptors specific for HLA-C molecules and it will be interesting to learn their relationship to mouse Ly-49 particularly with regard to their structure, chromosomal location of their genes, and their ligand specificity. A more striking resemblance to the murine Ly-49 molecules is borne by the Kp43 (CD94) molecule. This dissulfide-linked homodimer (43 kD subunits) is expressed by a subset of human NK cells.81 The expression of CD94 on NK cell clones can be correlated with the protective effect of target cell HLA-B7 molecules.82 Transfection of C1R targets with HLA-B7 protected the target from lysis by CD94 + NK cell clones. Lysis was restored by intact anti-CD94 mAb or its F(ab')2 fragments, consistent with the recognition of HLA-B7 by CD94 and subsequent inhibition of cytotoxicity. It will therefore be of interest to determine the primary structure of the CD94 molecule and its relationship to Ly-49. Other molecules have been described that appear to be functionally important in human NK cell activity83,84 but their general properties in expression, functional or sequence analysis demonstrate that these molecules are distinct from the Ly-49 family of molecules. In contrast, cDNAs resembling the NKR-P1 and Ly-49 families of molecules85 have been isolated from human NK cell libraries by subtractive hybridization. These cDNAs encode type II integral membrane proteins with external C-type lectin domains

Acknowledgements The authors thank our colleagues, especially Drs Franz Karlhofer, Randy Ribaudo, Megan Sykes and Jay Ryan, for their contributions to the work summarized here and for helpful discussions. We thank the members of our laboratories for their continued interest and enthusiasm. This work was supported by Howard Hughes Medical Institute grants from the NIH to WMY.

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