Murine natural killer cell activation receptors - Wiley Online Library

Hamish R. C. Smith Azza H. Idris Wayne M. Yokoyama

Murine natural killer cell activation receptors

Authors’ addresses

Summary: Natural killer (NK) cells express two types of receptors involved in target recognition: inhibitory receptors for target cell MHC class I molecules and activation receptors. While there has been significant progress in understanding the inhibitory receptors, less is known about the activation receptors. Detailed analysis of several mouse NK-cell activation receptors provides insight into the physiologic relevance of these receptors in the innate immune response.

Hamish R. C. Smith1, Azza H. Idris2, Wayne M. Yokoyama1, 1 Howard Hughes Medical Institute, Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St Louis, Missouri, USA. 2 Immunobiology Center, Mount Sinai School of Medicine, New York, USA.

Introduction Correspondence to:

Wayne M. Yokoyama Howard Hughes Medical Institute Division of Rheumatology Department of Medicine St Louis MO 63110 USA Fax: 1 314 362 9257 e-mail: [email protected] Acknowledgements

We gratefully acknowledge past and present members of our laboratory for their work on NK-cell receptors and particularly Hubert Chuang for his recent efforts on Qa-1 receptors mentioned in this manuscript. Work in the Yokoyama laboratory is supported by grants from the National Institutes of Health. H.R.C.S. is supported by a training grant from NIH. W.M.Y. is an investigator of the Howard Hughes Medical Institute.

Immunological Reviews 2001 Vol. 181: 115–125 Printed in Denmark. All rights reserved

Copyright C Munksgaard 2001

Immunological Reviews ISSN 0105-2896

Mouse and human natural killer (NK) cells utilize highly related receptor recognition systems to mediate inhibition by target cell MHC class I molecules (1). In mice, most of the currently defined NK-cell receptors belong to the C-type lectin superfamily and are encoded in the NK gene complex (NKC) on distal mouse chromosome 6 (2, 3). While it is still controversial whether these receptors bind carbohydrates (4– 6), they nevertheless bear a structural relationship to molecules that bind carbohydrates in a Ca2π-dependent manner (7), and they are expressed as disulfide-linked dimers with type II integral membrane topology. Mouse NK cells recognize MHC class I and related molecules with inhibitory Ly-49 and CD94/NKG2A receptors (8). Whereas Ly49 molecules bind classical MHC class I molecules, CD94/NKG2A receptors bind the non-classical MHC class I molecule, Qa-1, that in turn binds peptides derived from the leader sequences of classical MHC class I molecules (9). Although it does not appear that human NK cells express Ly49 homologs, they do express CD94/NKG2 receptors that are encoded in the human NKC on syntenic chromosome 12p13 and bind the Qa-1 ortholog HLA-E (10). Similar to Qa-1, HLA-E presents leader sequences from classical HLA class I molecules. In addition, the specificity of human NK cells is controlled in part by killer inhibitory receptors (KIR), type I membrane proteins belonging to the Ig superfamily, that bind specific HLA class I molecules (11). The precise orthologs of KIR in mice have

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Smith et al ¡ Mouse NK-cell activation receptors

not been clearly identified, but highly related gp49B molecules are expressed on activated mouse NK cells and can inhibit their function (12–14). Thus, mouse and human NK cells express both structural types of inhibitory receptors, C-type lectin-like with type II membrane orientation and Ig superfamily members with type I orientation. Despite their opposing membrane orientations, both structural types of inhibitory receptors mediate their effects through a conserved signaling mechanism (11). These receptors contain cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that become phosphorylated upon receptor engagement. Phosphorylated ITIMs recruit and activate the cytoplasmic SHP-1 tyrosine phosphatase that is thought to then dephosphorylate substrates that are involved in NK-cell activation pathways. The inhibitory receptors explain in part the missing self hypothesis, that NK cells survey tissues for normal expression of MHC class I that inhibits their function and that they are released from inhibition due to downregulation of MHC class I on diseased tissues (15). However, the absence of MHC class I expression alone is insufficient to permit target killing in all cases. Probably the most notable examples are the observations that mouse NK cells generally do not kill human targets and vice-versa, even when the targets lack MHC class I expression (16). Furthermore, it had been noted (see below) that NK cells could be activated when certain surface molecules were cross-linked with monoclonal antibodies (mAbs). Therefore, the tworeceptor hypothesis was proposed, suggesting that NK-cell effector functions are triggered when activation receptors bind target cell ligands and inhibitory receptors detect no or low levels of MHC class I (17). It is now clear that NK-cell activation receptors are critical for regulating NK-cell specificity for target cells. By contrast to the already detailed understanding of NK-cell inhibitory receptors, however, much less is understood concerning such activation receptors. A number of features of the low affinity IgG receptor, FcgRIII (CD16) provide general insight into NKcell activation receptors. One of the first major NK-cell receptors to be characterized, FcgRIII is the only Fc receptor defined on NK cells and mediates antibody-dependent cellular cytotoxicity (ADCC) (18–20). Human leukocytes may express either of two alternative forms, FcgRIIIA and FcgRIIIB. Human neutrophils express the glycosylphosphatidylinositol-linked FcgRIIIB form whereas NK cells express only the transmembrane FcgRIIIA molecule (which is the only form, termed FcgRIIIa, present in mouse), indicating that NK cells may express specific receptor isoforms. FcgRIII is coupled to two distinct immunore-

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ceptor tyrosine-based activation motif (ITAM)-containing signaling chains, FceRIg (mouse and human) and CD3z (human), which are required for optimal expression and signal transduction of the ligand-binding chain (21, 22). NK cells also express the ITAM-containing DAP12 molecule, but it is not apparently associated with FcgRIII (19), highlighting the restricted coupling of these signaling chains to ligand-binding chains. Nevertheless, ligand binding results in tyrosine phosphorylation of the ITAMs, which then leads to recruitment and activation of Syk family tyrosine kinases ZAP-70 and Syk itself (23), though, in some instances, predominantly Syk is activated (24). By contrast to T and B cells, which respectively express only ZAP-70 or Syk molecules, NK cells express both molecules and they appear to be redundant in function. A deficiency of either signaling molecule individually does not significantly alter the capacity of NK cells to mediate natural killing or ADCC. Whereas this was directly studied in ZAP-deficient mice, NK cells from bone marrow chimeric mice were needed to study Syk deficiency, since it is lethal in the perinatal period. Reconstitution of interleukin (IL)-2Rg chain-deficient mice, lacking NK cells, with Sykdeficient bone marrow permitted development of NK cells that demonstrated natural killing (25). Detailed analysis of NK cells from a ZAP, Syk double knock-out mouse should be revealing, as should dissection of NK cells that bear deficiencies in one or more of the three ITAM-containing signaling chains. Nevertheless, NK cells express redundant pathways for tyrosine kinase activation, a theme that is also reinforced in terms of ligand-binding chains (see below). Whereas the potential complexities of CD16 activation may be instructive, CD16 itself is generally not thought to be involved in natural killing, since CD16ª NK cells still kill tumors (26–28). Hence, there has been significant interest in defining other potential NK-cell activation receptors. A number of other activation receptors were first defined by capacity to trigger NK-cell functions when cross-linked by specific mAbs in an assay termed redirected lysis or reverse ADCC (1). For example, when mouse NK cells are incubated with the human B-cell lymphoma Daudi, minimal killing occurs, presumably due to species differences in recognition. However, mAbs against NK1.1 (NKR-P1C), CD69, and Ly-6 can trigger killing (16). This is dependent on the capacity of Fc portion of the mAb to bind the Fc receptor on the target, which presumably provides cross-linking and bridging effects. Indeed, rat mAbs bind Fc receptors poorly and the rat anti-Ly-6 mAb does not trigger by itself; an anti-rat Ig preparation is required to stimulate killing with anti-Ly-6 in this assay. Also, interference with binding to the Fc receptor on


the target prevents activation. This can be accomplished by protein A or anti-target cell Fc receptor, as long as the Fc portion of the anti-Fc receptor does not bind CD16 on the NK cells to trigger ADCC or specifically bind the FcgRIII on NK cells and trigger reverse ADCC. Alternatively, digestion of the Fc portion of the triggering anti-NK-cell receptor may result in loss of ability to trigger redirected lysis. Interestingly, several activation receptors are not expressed on resting NK cells but their expression can be induced upon activation through other means; these receptors include CD69 and Ly-6. Whether these receptors are involved in the enhanced potency and broader target range of IL-2-activated NK cells remains to be determined. Finally, numerous activation receptors were initially described during cDNA cloning of molecules related to the inhibitory receptors and to analysis of homologs of NK-cell receptors in other species (29, 30). These include members of the C-type lectin superfamily, such as Ly49D, Ly49H, and CD94/NKG2C, as well as the Ig superfamily, including 2B4, p46, and NKp44. Many activation receptors were found to be highly related to the inhibitory receptors but do not contain cytoplasmic ITIMs. Instead, like FcgRIII, these receptors contain charged residues in their transmembrane domain that facilitate interaction with the ITAM-containing signaling chains, including CD3z, FceRIg, and DAP12. Less is known about the recently described DAP12 molecule (31, 32). Nevertheless, its features are highly reminiscent of the other two signaling chains in that DAP12 is required for optimal surface expression of several of the activation receptors as well as signal transduction, analogous to the functions of CD3z and FceRIg for the T-cell receptor/CD3 complex and FceRI, respectively. Importantly, DAP12 serves these functions for some NK-cell receptors (Ly49D, Ly49H, CD94/NKG2C) but not others, again supporting the redundancy and selectivity of activation receptor systems on NK cells. Triggering through the activation receptor presumably then leads to ITAM tyrosine phosphorylation, recruitment of Syk family tyrosine kinases, and downstream activation events, leading to target killing by exocytosis of granules containing perforin and granzymes, and cytokine secretion. Recent studies indicate that another NKC-encoded activation receptor, NKG2D, has a function that is distinct from the aforementioned receptors. NKG2D, present on both human and mouse NK cells, binds ligands that may be upregulated in response to environmental stimuli that are only vaguely understood at the moment (3, 33–35). However, expression of the human ligands MHC class I chain-related (MIC) A and MICB is enhanced by heat shock whereas the

mouse ligands are induced by retinoic acid, perhaps then leading to NK-cell activation only in pathologic circumstances. Interestingly, NKG2D is not coupled to any of the previously mentioned signaling chains. Instead NKG2D is coupled to the DAP10 molecule, which is also required for full expression (36). DAP10 does not contain cytoplasmic ITAMs and instead contains a site that can potentially recruit phosphatidyl inositol 3-kinase, thereby providing a means for co-stimulation, perhaps analogous to the role of CD28 on T cells. The complexity of NK-cell activation receptors is evident. The repertoire of such receptors is also expanding as current research focuses on defining these receptors and their contribution to NK-cell function. Many are described in greater detail in the current issue of Immunological Reviews. In this review, we will focus on our laboratory’s studies of these activation receptors and discuss lessons learned from study of these receptors that provide an overall framework with which to understand NK-cell function in the broader context.

Ly49D: the gene product of the Chok locus

The Ly49D molecule was first identified in experiments designed to clone molecules that were highly related to the original Ly49 molecule (37). By hybridization to the original Ly49 cDNA, later renamed Ly49A, several cDNAs were cloned from a library produced from IL-2-activated NK cells. These cDNAs did not represent allelic forms since they were all cloned from a single inbred mouse strain, C57BL/6. In this analysis, the Ly49D cDNA was most unusual because its cytoplasmic domain was least related to the other Ly49 sequences. Indeed, our colleagues. L. Mason and J. Ortaldo produced a mAb (clone 12A8) specific for Ly49D (and Ly49A) and showed that the Ly49D molecule is an activation receptor at a time when Ly49 receptors were generally thought to be inhibitory receptors (38). In parallel studies, our laboratory was also dissecting a genetic locus termed Chok, which was responsible for regulating killing of Chinese hamster ovary (CHO) targets (39). Chok was originally recognized by accident. Whereas the initial experiment was designed to determine if any of P. Stanley’s CHO glycosylation mutants (40) demonstrated differences in susceptibility to killing, perhaps due to the differential action of lectin-like receptors, we determined that the parental CHO line and the mutants were all susceptible to IL-2-activated NK cells derived from C57BL/6 animals. However, none of the CHO lines were killed by NK cells from BALB/c. This was not due to IL-2 activation differences, since freshly isolated Immunological Reviews 181/2001

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NK cells displayed a similar phenotype. From analysis of NK cells from several other inbred mouse strains, we determined that the capacity to kill was unrelated to H-2 differences or expression of other NK-cell receptors known at the time, including NK1.1 and 2B4. The (C57BL/6¿BALB/c) F1 hybrid NK cells were not informative, since the phenotype was intermediate. Furthermore, the capacity to kill was also apparent in vivo, since C57BL/6 mice could eliminate adoptively transferred CHO cells whereas BALB/c animals could not. In vitro killing and in vivo elimination were also found to be perforin dependent, suggesting that this strain-dependent killing capacity was mediated through the action of an NK-cell receptor involved in target recognition. A major clue to the nature of the putative receptor came from results of genetic mapping studies (39, 41). Analysis of recombinant inbred mice generated from C57BL/6 and BALB/c progenitor strains revealed a tentative linkage with the NKC on distal mouse chromosome 6. This was verified by the use of congenic mice that were generated in the analysis of a murine cytomegalovirus (MCMV) resistance locus (Cmv1) that was also linked to the NKC. Congenic BALB/c animals with the C57BL/6 haplotype of the NKC (BALB.B6Cmv1r) gained the capacity to kill, whereas the reciprocal congenic C57BL/6 genetic background with the BALB/c haplotype of the NKC (B6.BALB-Cmv1s) lost CHO killing activity. Inasmuch as the NKC encompasses a genetic region that contains clusters of highly related genes encoding NK-cell receptors (2, 42), it was then possible to postulate the nature of the gene product of the Chok locus. Since the NKC encodes both activation and inhibitory receptors, two hypotheses were considered to explain such a receptor. One possibility was that Chok could encode an activation receptor expressed by C57BL/6 NK cells; BALB/c NK cells either failed to express this receptor or expressed an allelic form that may not recognize CHO cells. Alternatively, Chok could encode an inhibitory receptor expressed on BALB/c NK cells, and C57BL/6 NK cells either did not express this receptor or expressed an allele that did not react with CHO cells. In either event, the receptor was presumably a C-type lectin-like receptor with type II integral membrane orientation, since the NKC encodes receptors with these features. To distinguish between these possibilities, we sought to trigger BALB/c NK cells to kill CHO cells (43). Our reasoning was that in the absence of an activation receptor, this should be easily achieved, whereas the effect of a BALB/c inhibitory receptor would not permit triggering because inhibitory receptor action is generally difficult to overcome. We initially attempted to find mAbs reactive with CHO cells in order to

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perform ADCC whereby sensitized CHO cells would then become susceptible to FcgRIII-mediated killing by NK cells. However, we could not find any mAbs except those against MHC class I molecules, specificities that might obscure interpretations. We explored 2,4,6-trinitrophenyl (TNP) modification of CHO cells so as to permit use of anti-TNP mAbs, but such manipulation of CHO cells markedly increased spontaneous 51Cr release in cytotoxicity assays. Fortunately, we conceived of a much simpler approach that employed the laboratory phenomenon of lectin-facilitated killing. Using the plant lectin concanavalin A, we were able to readily trigger BALB/c NK cells to kill CHO cells, consistent with the C57BL/6 activation receptor hypothesis. Based on this premise, we immunized BALB/c mice with NK cells from the NKC congenic (BALB.B6-Cmv1r) in order to produce mAbs against C57BL/6-derived NKC-encoded receptors (43). The mAbs were screened for 1) capacity to block killing of CHO cells by C57BL/6 NK cells; 2) staining of C57BL/6 but not BALB/cderived NK cells; and 3) capacity to activate C57BL/6 NK cells. Several candidate hybridoma wells were identified. One (mAb 4E4) was cloned and used for extensive analysis. Purified mAb 4E4 blocked CHO killing by NK cells from C57BL/6 as well as BALB.B6-Cmv1r-derived NK cells, indicating that it was reactive with a putative NK-cell activation receptor encoded in the NKC from C57BL/6 (43). This important finding excluded the possibility that the mAb reacted with another molecule, encoded outside the NKC, that might modulate natural killing, such as an adhesion receptor. Moreover, the mAb recognized a subset of C57BL/6-derived NK cells and was not reactive with any other splenic population or with BALB/c-derived NK cells. Finally, the mAb was able to trigger NK cells to kill an otherwise non-susceptible NKcell target, human Daudi cells, in redirected lysis assays. Thus, we concluded that the mAb recognized the Chok gene product. The mAb immunoprecipitated a disulfide-linked dimeric molecule. This finding along with the NK-cell subset reactivity suggested that the mAb recognized a member of the Ly49 family, which are known to be expressed by subsets of NK cells. Indeed, the mAb reacted with cells transfected with Ly49D cDNA but not any other Ly49 transfectant. Thus, the anti-Chok mAb was specific for Ly49D (43). To determine that Ly49D was responsible for CHO killing and rule out the possibility that the anti-Chok mAb crossreacted with Ly49D, we infected BALB/c IL-2-activated NK cells with a recombinant Ly49D-vaccinia virus construct (43). This conferred upon BALB/c NK cells the capacity to kill CHO cells whereas a vaccinia vector control did not. Transfected


CHO killing was blocked by mAb 4E4, demonstrating specific transfer of killing capacity. Taken together, these data indicate that the predominant C57BL/6 receptor capable of recognizing CHO and activating natural killing is Ly49D.

Lessons from genetic analysis of Chok

The Chok studies highlight the genetic approach to dissection of NK-cell function. Particularly when genetics can isolate a functional effect to a specific chromosomal interval, the genetic approach is very powerful because it is possible to neutralize the effect of other genes and their products, thereby eliminating other molecules from consideration. It is always possible that the isolated genetic region may encode more than one molecule that is involved in the phenomenon of interest due to tight genetic linkage; this seems to be a theoretical concern that is rarely encountered. Although the genetic approach requires significant commitment and time to execute successfully, recent advances in high resolution genetic maps and genome sequences will provide enhanced efficiency in this methodology. There are a number of lessons we have gleaned from this type of analysis for Chok and Ly49D. Genetic linkage to the NKC was helpful to support initial hypotheses that an NKcell receptor was responsible for the phenomenon and led to the consideration of activation and inhibitory receptor possibilities. However it proved difficult to further isolate the relevant gene by extensive breeding strategies to identify informative recombinations that would aid genetic mapping. Two reasons for this are the large physical size of the NKC (currently estimated to be ∂4.5 megabases) and the dense packing of potentially relevant genes (44). Also, multiple recombinations occurred within a small physical region, a recombination ‘‘hotspot’’, hindering development of further information from intensive breeding even when recombinants could be identified (data not shown). Finally, it is important to emphasize that genetic linkage analysis, once a relevant area is identified, is best suited to rule out candidate genes rather than precisely pinpoint the gene of interest. In pure positional cloning attempts, geneticists can take advantage of a number of characteristics anticipated for the candidate gene and its product to evaluate relevance of genes within the pinpointed interval. In the case of Chok, we hypothesized early on that its gene product would be a cell surface molecule expressed in NK cells, and that it would display allelic differences between C57BL/6 and BALB/c. However, in the NKC, there are many genes that fit these criteria since the region turned out to be replete with genes for NK-cell

receptors (45). It was also evident from the earliest studies on this region that there were likely to be multiple alleles for these genes, since it was relatively easy to identify restriction fragment length polymorphic variants for the first two genes (Ly49 and Nkrp1) that were mapped to this region (46, 47). In short, there were too many candidate genes to be evaluated by a sequence-based strategy that otherwise aids pure positional cloning approaches. The candidate gene approach, however, can benefit from use of existing mAbs against molecules that are possibly involved. Before embarking on production of a novel mAb, we used mAb 4E5, previously produced by Mason and Ortaldo against Ly49D to attempt blocking of CHO killing. This attempt was unsuccessful (39). However, the experiment did not have a positive control, since this mAb had not been previously shown to block natural killing against any target. Upon identification of the role of Ly49D in CHO killing, the experiment was repeated, and it became evident that the effective concentration (specific activity in functional assays) of the protein A-purified mAb 4E5 preparation was much lower than evident by OD280 determinations and its capacity to stain NK cells. This was unfortunate, since recognition of this situation would have obviated the effort to produce the anti-Chok mAb 4E4 (which by chance has a similar designation). On the other hand, production of mAb 4E4 in the manner described did allow for the interpretation that the only relevant molecule for CHO killing is Ly49D (or a highly related molecule with cross-reactive epitope) since the immunization and screening approaches were otherwise unbiased. Having produced a mAb with exquisite specificity for Ly49D and capacity to completely block CHO killing, it was reasonable to conclude that there was unlikely to be any other molecule on wild-type C57BL/6-derived NK cells that could mediate CHO killing unless it very closely resembled Ly49D. Moreover, preexisting mAbs against candidate genes that were made for other purposes may not have functional effects due to recognition of epitopes that are not involved in ligand binding. For example, a mAb made to detect expression of a cell surface molecule may not block binding to its ligand, and if the functional assay was utilized in screening for candidate genes, misleading information may be obtained. Nevertheless, an immunologically based approach can be very rewarding. In the case of Chok, we made use of congenic strains to limit the production of antibodies against only NKC-encoded molecules that must differ between the relevant inbred strains. Importantly, we first had to determine if Chok was an activation receptor or an inhibitory receptor (39). Analysis of F1 hybrid NK cells was not informative since these Immunological Reviews 181/2001

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cells displayed an intermediate phenotype. The elimination of the inhibitory receptor hypothesis was significant because the reciprocal immunization protocol (C57BL/6 against BALB/c NKC) would have been necessary in order to produce a mAb that would block the action of an inhibitory receptor. Hence, given the difficulty in producing such mAbs, it was critical to proceed with the immunologically based approach only when we were reasonably assured that the appropriate immunization protocol was used. Recent studies in our laboratory suggested that the Ly49D gene is not expressed in BALB/c so that C57BL/6-derived Ly49D was immunogenic in BALB/c (H. Furukawa, W. M. Yokoyama, unpublished observations). Presumably, however, if the candidate molecule is expressed in functionally distinct allelic forms, as defined by the genetic approach, a mAb should still be produced against the relevant epitope. Thus, the immunological approach is a valuable complement to the genetic analysis. A drawback to the mAb approach is that a mAb could be produced that cross-reacts with the functional epitope on several related molecules. It is therefore necessary to ascertain the correct interpretations by gene transfer or deletion experiments. In the case of Chok, the mAb approach was followed by experiments demonstrating that gene transfer of Ly49D was sufficient to confer killing of CHO cells (43). It is still possible that there is another mAb 4E4-reactive receptor that also has the capacity to kill CHO cells. However, in DAP12 knock-out mice, Ly49D expression is downregulated (48). CHO killing is intermediate and is not blocked by mAb 4E4, whereas wild-type C57BL/6 killing of CHO is nearly completely blocked by mAb 4E4. These findings suggest that in the absence of DAP12, another molecule, unrelated to Ly49D, DAP12, or mAb 4E4 reactivity, may substitute for Ly49D. Recent studies, based primarily on mAb effects, also suggest that CHO cells may be killed by other potential NK-cell receptors and that inhibitory Ly49 molecules also may recognize these targets (49). The nature of the receptors suggested by these last two studies remains to be determined. Nonetheless, the mAb approach complements the powerful information that is provided by the genetic mapping approach.

Ly49H: a related mouse NK-cell activation receptor

The Ly49H cDNA was first reported by the Takei group in efforts to further define the Ly49 family of molecules (50). Like Ly49D, it was recognized that Ly49H was significantly different in sequence from other Ly49 receptors in the cytoplasmic domain, and contains a charged amino acid residue

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in the transmembrane segment (51). However, in the extracellular domain, Ly-49H is highly similar to the inhibitory Ly-49C and Ly-49I receptors. This structural relationship is also evident between Ly-49D and the Ly-49A inhibitory receptor. The extracellular domains are highly similar, although the cytoplasmic and transmembrane segments differ and confer opposing cellular functions. It is now appreciated that Ly49H can be structurally grouped with Ly49D due to its charged residue in the transmembrane domain and absence of ITIMs. The identification of DAP12 and demonstration that Ly49H requires DAP12 for optimal expression and functional activation further supports this relationship (52). However, it is noteworthy that both Ly49D and Ly49H are each significantly more similar in the extracellular domain to a Ly49 inhibitory receptor than to each other. These differences imply that there will be greater differences between the putative ligands for Ly49D and Ly49H than between an activating receptor and its related inhibitory Ly49 molecule. Hence, like Ly49D, Ly49H is an NK-cell activation receptor, but the similarity of the roles served by these two receptors in vivo remains to be elucidated. Further analysis of Ly49H was significantly aided by production of a mAb specific for Ly49H (51). To generate this mAb, the cDNA encoding the extracellular domain of Ly49H was first fused in frame to the cDNA for the transmembrane and cytoplasmic domains of Ly49A, creating a chimeric Ly49 molecule that does not require any associated molecules for cell surface expression. Transfection and expression of this chimeric Ly49 cDNA resulted in a stable C57BL/6 Ly49Hexpressing cell line that was used to immunize BALB/c mice. Although we have not explored the exact nature of the polymorphism of Ly49H between these strains, we were unable to detect Ly-49H transcripts by PCR amplification of cDNA derived from BALB/c using different sets of primers (unpublished observations). We therefore chose to exploit this polymorphism of Ly49 molecules between C57BL/6 and BALB/c with the aim of generating an antibody that was allospecific for Ly49H (51). The resulting mAb, clone 3D10, did not react with cells transfected with cDNAs for any of the known expressible Ly49 molecules in flow cytometric analysis. Since these studies were performed, several other Ly49 cDNAs were identified from C57BL/6 (53). However, only Ly49J is expressed as a full-length transcript for a functional protein (54), and it is less similar to Ly49H in the extracellular domain than Ly49C or Ly49I. Furthermore, Ly49J is an inhibitory receptor, based on its cytoplasmic ITIMs. Additional evidence supporting the specificity of mAb 3D10 for Ly-49H came from the demonstration that Ly-49H staining was se-


verely reduced in DAP-12-deficient mice generated by the Lanier lab (48). These data suggest that mAb 3D10 does not cross-react with any known or heretofore undiscovered Ly49 inhibitory molecules. However, mAb 3D10 recognizes an epitope on Ly49H independent of DAP-12, since the mAb reacts with cell lines transfected with the chimeric Ly49A-Ly49H construct (51). Thus, mAb 3D10 specifically binds the Ly49H receptor. The availability of an anti-Ly49H mAb permitted direct activation of NK cells through Ly49H (51). These studies demonstrated that cross-linking of Ly49H is sufficient to stimulate target cell killing in redirected lysis assays. In addition, immobilized anti-Ly49H triggers production of the cytokines granulocyte–macrophage colony-stimulating factor and interferon-g in vitro. Although Ly49H resembles Ly49D in terms of activation function, Ly49H is not involved in activating NK cells to kill CHO cells. Anti-Ly49H mAb has no effect on CHO killing, whereas the anti-Ly49D mAb blocks CHO killing completely and does not react with Ly49H (51). Finally, Ly49H expression appears to be more dependent on DAP12 expression than Ly49D because the anti-Ly49H mAb does not react with DAP12-deficient NK cells whereas Ly49D expression is only mildly affected (48). Thus, the structurally and functionally similar Ly49D and Ly49H family members must exhibit distinct ligand specificities.

Co-expression of NK-cell activation receptors

In marked contrast to individual T and B cells, which express a single clonotypically defined receptor, an individual NK cell may express multiple different receptors simultaneously, whether inhibitory or activation-type receptors. For the inhibitory Ly49 receptors, although initially attributed to ‘‘allelic exclusion’’ (55), the apparent monoallelic expression of individual Ly49 inhibitory receptors is now thought to be due to a stochastic mechanism (56). When the co-expression of Ly-49 inhibitory molecules on single cells is analyzed by the multiplication rule for independent probabilities, expression of a given Ly49 inhibitory molecule appears to be independent of any other inhibitory Ly49 (56). Thus, the percentage of NK cells expressing two distinct inhibitory Ly-49 molecules can be readily predicted from the product of their frequencies in the total NK-cell population. The co-expression of Ly49 activation receptors, however, does not appear to follow this distribution (51). If an NK cell expresses Ly49H, it is more likely to express Ly49D and viceversa. For example, approximately 68% of Ly49Hπ NK cells express Ly49D, but only 40% of Ly49Hª NK cells express

Ly49D. One can also conclude that NK cells lacking Ly49H expression are less likely to express Ly49D alone. An additional line of evidence suggesting regulation of Ly49 activation receptors is given by the lack of Qa-1 tetramer binding receptors (primarily due to inhibitory CD94/NKG2A) on NK cells that express Ly49D or Ly49H (51). For example, while about 45% of C57BL/6 NK cells binding Qa-1 tetramers express Ly49H, an average of 67% of NK cells that do not bind Qa-1 tetramers do express Ly49H. A similar result was observed in flow cytometric analysis of Ly49D expression with Qa-1 tetramers. And although 75% of total NK cells express either Ly49D or Ly49H or both, only 54% of NK cells binding Qa-1 tetramers co-express at least one Ly49 activation receptor, whereas the Qa-1 tetramerª NK-cell subset expresses Ly49D, Ly49H or both on 87% of cells. Thus, the Ly49 activation NK-cell receptors do not strictly adhere to the ‘‘product rule’’ that otherwise suggests stochastic mechanisms for receptor expression, and may in fact undergo an ordered coexpression with specific NK-cell receptors to generate certain NK-cell receptor patterns or phenotypes. Inasmuch as both Ly49D and Ly49H receptors require expression of a third molecule, DAP12 (52), the preferential expression of both activation receptors may reflect restricted expression of DAP12, for it is not known if every NK cell expresses DAP12. A subset of NK cells may preferentially express DAP12, and this skewed expression may be another ‘‘multiplier’’ in determining the stochastic expression of Ly49 activation receptors. Nevertheless, these findings suggest that, at least in the C57BL/6 strain, the co-expression of Ly49D and Ly49H on NK cells is in some way regulated or preferentially selected. Interestingly, the evidence to date suggests that Ly49D and Ly49H are NK-cell-specific activation receptors. We found Ly49D and Ly49H to be expressed only on NK1.1π CD3ª NK cells and are absent on T cells. Even in the case of NK/T cells that have been reported to express inhibitory Ly49 receptors, staining by Ly49D and Ly49H antibodies was not detected in flow cytometry. It is possible that Ly49D and Ly49H are capable of being expressed on CD3π cells after specific or chronic activation, or that the putative CD3π subsets expressing these Ly49 receptors are rare or difficult to isolate. It is currently not known what governs Ly49 receptor expression on T-cell subsets, but the absence of activation receptors could again be related to DAP12 expression and simply reflect the absence of DAP12 expression on T cells. An alternative mechanism for preferential expression of activation receptors may be related to the gene regulatory domains in the NKC. Promoter analysis of NKC-encoded recepImmunological Reviews 181/2001

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tors has been relatively unexplored, and it is likely that long range effects may also be relevant, such as that seen with locus control regions that govern globin gene expression. Whether enhancer elements operate in these receptors in any kind of preferential manner remains to be discovered as well. However, these effects are probably less important in expression of the inhibitory receptors, since there is strict adherence to the ‘‘product rule’’ for these receptors. On the other hand, the skewed expression of the activation receptors may be indicative of these other parameters affecting expression. Finally, it remains possible that NK-cell activation receptor expression may be regulated by host factors. By some sort of ‘‘education’’ analogous to T-cell education in the thymus, NKcell function may be regulated by their ligands in either a positive or a negative manner. While this issue will be difficult to address until ligands for NK-cell receptors are more fully understood, it is already possible to integrate observations from related transgenic and knock-out animals. Although NK-cell development is apparently normal in DAP12deficient mice and animals with other NK-cell signaling molecule deficiencies, the redundancy of these molecules may obscure their roles in NK-cell development even if positive signaling is required. One hint to some sort of activation receptor role in development is the capacity of DAP12-deficient NK cells to kill CHO cells at intermediate levels (48). This function is not blocked by the anti-Ly49D mAb which otherwise almost completely blocks CHO killing by wild-type NK cells. On the other hand, negative selection may also be important. In this regard, perturbation of NK-cell development may occur when the inhibitory receptors are engaged in vivo, as suggested by the observation that b2-microglobulin-deficient mice have decreased NK-cell function (57). Furthermore, unlike NK cells from otherwise syngeneic wild-type mice, NK cells from b2-microglobulin-deficient mice do not reject b2-microglobulin-deficient bone marrow. These findings have led to the proposal of the ‘‘receptor calibration’’ hypothesis to account for host MHC class I effects on NK-cell inhibitory receptors (58). Further analysis will be forthcoming as ligands for the NK-cell receptors become better understood.

Consequences of co-expression of NK-cell activation receptors

In attempts to understand the functions of NK-cell activation receptors in physiological conditions, it is useful to consider the role of NK cells in immune responses. NK cells are critical in controlling viral infections, particularly herpesviruses, such

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as MCMV (59). Mice with deficient NK-cell functions are susceptible to infection, and a major locus controlling MCMV replication and host survival maps to the NKC (60). NK cells appear to mediate this control, since resistant mouse strains can be made susceptible by injection of antibodies that deplete NK cells, such as anti-NK1.1 (61–63). Interestingly, the antibodies have a significant effect only when injected prior to or within 24–48 h of the viral inoculum (64). If a longer period elapses, then the anti-NK-cell antibodies have no effect. Hence, NK cells are particularly important in the context of early innate immunity against pathogens. One possible consequence of dual expression of Ly49 activation receptors is that they may synergize in activating NK cells. It is possible that both receptors may combine to trigger an NK cell when the ligand for either receptor alone is insufficient to trigger the NK cell. This may provide the NK cell with the capacity to respond earlier in the context of an immune response when ligand concentrations are still low, or in the case of a pathogen that can reduce but not completely eliminate expression of Ly49 activation receptor ligands. Either notion is consistent with the role of NK cells in the earliest phases of innate immune reactions. Detailed understanding of ligand specificity will be helpful, since this concept may need revision if NK cell activation receptors have overlapping or broad promiscuous specificities. Another possible consequence is that an individual NK cell could be fully triggered by any one of its activation receptors. In this regard, pathogens may express sufficient levels of only one ligand but could still trigger most of the NK cells in a bulk population because of the widespread expression of the cognate activation receptor. Conceptually this is important in terms of ‘‘amplification’’ strategies. Whereas individual specific immune cells, i.e. T and B cells, express only one clonotypic rearranged receptor, exposure to ligand triggers clonal expansion and activation. However, it takes several days to achieve a critical mass of enough specific T or B cells to effect a response. NK cells, by virtue of their role in early, innate immunity, do not have sufficient time to utilize this strategy. Teleologically, then, this relatively small population of cells, which in number is only ∂10% of the total T-cell pool, is capable of mounting a significant defense, involving a large fraction of the NK-cell population, because their receptors are widely expressed.

Human versus mouse Ly49 receptors

In humans, cDNA cloning thus far has identified only a single molecule, termed Ly49L, which lacks the distal portion of the


C-type lectin-like domain (65). The functional relevance of this molecule and whether it represents a polymorphic variant is still to be resolved. However, with the recent announcement of the completion of sequencing of the human genome, a first pass sequence analysis of the human NKC appears to confirm that only Ly49L is represented in the human NKC as a residual member of the Ly49 family (66). This finding also raises an interesting evolutionary issue related to the expansion of the Ly49 family and possible contraction of the Igsuperfamily NK-cell receptors in rodents and vice-versa in humans. The evolutionary pressures that resulted in this outcome are poorly understood at the moment. It is important, however, to note that detailed analysis of the Ly49 family as well as other C-type lectin-like NK-cell receptors in mice has been extremely rewarding and has provided a conceptual framework with which to understand NKcell biology. From the first description of the molecular basis for the missing-self hypothesis (67) to current studies on NK-

cell activation receptors summarized here, the paradigms gleaned from understanding the role of Ly49 receptors suggest that their continued analysis in the mouse, even if not directly translated to human Ly49 molecules, will provide further insight into NK-cell biology.

Concluding remarks

As we gain a clearer picture of the wide distribution of receptor phenotypes in the NK-cell population, we see receptors singly or in combination that are much more diverse than might have been predicted for a cell population once referred to as ‘‘null cells.’’ In fact, the diverse nature of NK-cell receptor expression suggests that this lymphocyte population might recognize a broad array of pathogens, possibly attenuating the spread of a pathogen, while the acquired arm of the immune system differentiates into an effector population, resulting in immunity.

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