Cloning and characterization of a novel activating Ly49 closely related to Ly49A.

The majority of the known Ly49 family members have been isolated from either C57BL/6 (B6) or BALB/c mice. Interestingly, the anti-Ly49 Ab reactivities observed in 129/J mice are different from those of B6 mice. Furthermore, immunoprecipitation of 129/J NK cell lysates with YE1/32 and YE1/48, Abs specific for the inhibitory Ly49A in B6, resulted in detection of the activation-associated DAP12 molecule. These results indicated a need for a more detailed study of this strain. Therefore, a cloning strategy was devised to isolate Ly49 cDNAs from 129/J mice. An immunoreceptor tyrosine-based inhibitory motif-containing, Ly49D-related clone was discovered that we have named Ly49O, and one immunoreceptor tyrosine-based inhibitory motif-lacking, Ly49A-related clone was discovered that we have named Ly49P. No anti-Ly49 mAb reacted with Ly49O, whereas the molecule encoded by the Ly49P cDNA was found to react with YE1/32 and YE1/48. Ly49P was found to associate with mouse DAP12, and Ab-mediated cross-linking of Ly49P resulted in mouse DAP12 phosphorylation and Ca2+ mobilization, indicating that Ly49P is a competent activation receptor. Ly49P, therefore, represents a novel member of the Ly49 activating receptor subfamily.     

Activation receptor-induced tolerance of mature NK cells in vivo requires signaling through the receptor and is reversible

NK cell responses are determined by signals received through activating and inhibitory cell surface receptors. Ly49H is an NK cell-specific activating receptor that accounts for the genetic resistance to murine CMV (MCMV). The Ly49H receptor has been shown to interact with two adaptor proteins (DAP12 and DAP10). In the context of MCMV infection, interaction of m157 (the MCMV-encoded ligand for Ly49H) with Ly49H results in activation of Ly49H-expressing NK cells. Chronic exposure of Ly49H with m157, however, induces tolerance in these same cells. The mechanism of this tolerance remains poorly understood. Using a transgenic mouse model, we demonstrate that induction of tolerance in Ly49H(+) NK cells by chronic exposure to m157, in vivo, requires signaling through the Ly49H adaptor protein DAP12, but not the DAP10 adaptor protein. Furthermore, mature Ly49H-expressing NK cells from wild-type mice can acquire a tolerant phenotype by 24 h posttransfer into a transgenic C57BL/6 mouse that expresses m157. The tolerant phenotype can be reversed, in vivo, if tolerant NK cells are transferred to mice that do not express the m157 protein. Thus, continuous activating receptor engagement can induce a transient tolerance in mature NK cells in vivo. These observations provide new insight into how activating receptor engagement shapes NK cell function and has important implications in how NK cells respond to tumors and during chronic viral infection.     

Ly49D-mediated ITAM signaling in immature thymocytes impairs development by bypassing the pre-TCR checkpoint

Activating and inhibitory NK receptors regulate the development and effector functions of NK cells via their ITAM and ITIM motifs, which recruit protein tyrosine kinases and phosphatases, respectively. In the T cell lineage, inhibitory Ly49 receptors are expressed by a subset of activated T cells and by CD1d-restricted NKT cells, but virtually no expression of activating Ly49 receptors is observed. Using mice transgenic for the activating receptor Ly49D and its associated ITAM signaling DAP12 chain, we show in this article that Ly49D-mediated ITAM signaling in immature thymocytes impairs development due to a block in maturation from the double negative (DN) to double positive (DP) stages. A large proportion of Ly49D/DAP12 transgenic thymocytes were able to bypass the pre-TCR checkpoint at the DN3 stage, leading to the appearance of unusual populations of DN4 and DP cells that lacked expression of intracellular (ic) TCRβ-chain. High levels of CD5 were expressed on ic TCRβ(-) DN and DP thymocytes from Ly49D/DAP12 transgenic mice, further suggesting that Ly49D-mediated ITAM signaling mimics physiological ITAM signaling via the pre-TCR. We also observed unusual ic TCRβ(-) single positive thymocytes with an immature CD24(high) phenotype that were not found in the periphery. Importantly, thymocyte development was completely rescued by expression of an Ly49A transgene in Ly49D/DAP12 transgenic mice, indicating that Ly49A-mediated ITIM signaling can fully counteract ITAM signaling via Ly49D/DAP12. Collectively, our data indicate that inappropriate ITAM signaling by activating NK receptors on immature thymocytes can subvert T cell development by bypassing the pre-TCR checkpoint.     

A "chimeric" C57l-derived Ly49 inhibitory receptor resembling the Ly49D activation receptor.

Ly49D is a natural killer (NK) cell activation receptor that is responsible for differential mouse inbred strain-determined lysis of Chinese hamster ovary (CHO) cells. Whereas C57BL/6 NK cells kill CHO, BALB/c-derived NK cells cannot kill because they lack expression of Ly49D. Furthermore, the expression of Ly49D, as detected by monoclonal antibody 4E4, correlates well with CHO lysis by NK cells from different inbred strains. However, one discordant mouse strain was identified; C57L NK cells express the mAb 4E4 epitope but fail to lyse CHO cells. Herein we describe a Ly49 molecule isolated from C57L mice that is recognized by mAb 4E4 (anti-Ly49D). Interestingly, this molecule shares extensive similarity to Ly49D(B6) in its extracellular domain, but its cytoplasmic and transmembrane domains are identical to the inhibitory receptor Ly49A(B6), including a cytoplasmic ITIM. This molecule bears substantial overall homology to the previously cloned Ly49O molecule from 129 mice the serologic reactivity and function of which were undefined. Cytotoxicity experiments revealed that 4E4(+) LAK cells from C57L mice failed to lyse CHO cells and inhibited NK cell function in redirected inhibition assays. MHC class I tetramer staining revealed that the Ly49O(C57L)-bound H-2D(d) and lysis by 4E4(+) C57L LAK cells is inhibited by target H-2D(d). The structural basis for ligand binding was also examined in the context of the recent crystallization of a Ly49A-H-2D(d) complex. Therefore, this apparently "chimeric" Ly49 molecule serologically resembles an NK cell activation receptor but functions as an inhibitory receptor.     

Tolerance and alloreactivity of the Ly49D subset of murine NK cells.

Class I-specific stimulatory and inhibitory receptors expressed by NK cell subsets contribute to the alloreactive potential of the self-tolerant murine NK cell repertoire. In this report, we have studied potential mechanisms of tolerance to the function of the positive signaling Ly49D receptor in mice that express one of its ligands, H2-Dd. Our results demonstrate that H2-Dd-expressing mice possess a large Ly49D+ subset of NK cells that is functionally capable of rejecting bone marrow cell (BMC) allografts in vivo and lysing allogeneic Con A lymphoblasts in vitro. Also, we show that the Ly49D receptor is responsible for the ability of H2b/d F1 hybrid mice to reject H2d/d parental BMC (hybrid resistance). Thus, deletion or anergy of Ly49D+ cells in H2-Dd+ hosts cannot explain self tolerance. Our functional studies revealed that coexpression of the Dd-specific Ly49A or Ly49G2 inhibitory receptors by Ly49D+ cells resulted in tolerance to Dd+ targets, while coexpression of Kb-specific inhibitory receptors Ly49C/I resulted in tolerance to Kb+ targets. Only in H2d/d cells did Ly49C/I dominantly inhibit Ly49D-Dd stimulation. This correlated with an increased mean fluorescence intensity of Ly49C expression, as well as an increased percentage of Ly49C+ cells in the Ly49D+A/G2- compartment. Therefore, we conclude that self tolerance of the Ly49D subset can be achieved through coexpression of a sufficient level of self-specific inhibitory receptors.     

Independent control of Ly49g alleles: implications for NK cell repertoire selection and tumor cell killing

A novel murine NK cell-reactive mAb, AT8, was generated. AT8 recognizes Ly49G from 129/J, BALB/c, and related mouse strains, but does not bind to Ly49G(B6). Costaining with AT8 and a Ly49G(B6)-restricted Ab (Cwy-3) provides the first direct evidence that Ly49G protein is expressed from both alleles on a significant proportion of NK cells from four different types of F(1) hybrid mice. The observed level of biallelic Ly49G expression reproducibly followed the product rule in both freshly isolated and cultured NK cells. Surprisingly, the percentage of NK cells expressing both Ly49G alleles could be dramatically increased in vitro and in vivo through IL-2R- and IFN receptor-dependent signaling pathways, respectively. Unexpectedly, Ly49G(B6+) NK cells in an H-2(d), but not H-2(b), background were more likely to lyse D(d+) and Chinese hamster ovary tumor cells than Ly49G(BALB/129+) NK cells. Furthermore, Ly49G(B6+) NK cells also proliferated to a higher degree in response to poly(I:C) than NK cells expressing a non-Ly49G(B6) allele in an H-2(d), but not H-2(b), background. These results suggest that Ly49G(B6) has a lower affinity for H-2D(d) than Ly49G(BALB/129), and the genetic background calibrates the responsiveness of NK cells bearing self-specific Ly49. Other H-2D(d) receptors on the different Ly49G(+) NK cell subsets were unequally coexpressed, possibly explaining the disparate responses of Ly49G(B6+) NK cells in different hybrid mice. These data indicate that the stochastic mono- and biallelic expression of divergent Ly49G alleles increases the range of MHC affinities and the functional potential in the total NK cell population of heterozygous mice.     

Cmv1-independent antiviral role of NK cells revealed in murine cytomegalovirus-infected New Zealand White mice

Ly49H(+) NK cells play a critical role in innate antiviral immune responses to murine CMV (MCMV). Ly49H(b6) recognition of MCMV-encoded m157 on infected cells activates natural killing required for host resistance. We show that mAb 3D10 (anti-Ly49H) recognizes comparable subsets of NK cells from New Zealand White (NZW), New Zealand Black (NZB), and C57BL/6 spleens. However, virus levels in the spleens of MCMV-infected NZW and NZB mice differed greatly. We found that MCMV replication in infected NZW spleens was limited through NK cells. Alternately, NZB mice were profoundly susceptible to MCMV infection. Although 3D10 mAb injections given before infection interfere with Cmv1-type resistance in C57BL/6 mice, similar mAb injections did not affect NZW resistance, likely because NZW NK cell receptors did not bind MCMV-encoded m157. Instead, anti-MCMV host defenses in hybrid NZ offspring were associated with multiple chromosome locations including several putative quantitative trait loci that did not overlap with H-2 or NK gene complex loci. This study revealed a novel pathway used by NK cells to defend against MCMV infection. Thus, the importance of Ly49H in MCMV infection may be shaped by other additional background genes.     

Functional consequences of natural sequence variation of murine cytomegalovirus m157 for Ly49 receptor specificity and NK cell activation

The Ly49H activating receptor on C57BL/6 (B6) NK cells plays a key role in early resistance to murine cytomegalovirus (MCMV) infection through specific recognition of the MCMV-encoded MHC class I-like molecule m157 expressed on infected cells. The m157 molecule is also recognized by the Ly49I inhibitory receptor from the 129/J mouse strain. The m157 gene is highly sequence variable among MCMV isolates, with many m157 variants unable to bind Ly49H(B6). In this study, we have sought to define if m157 variability leads to a wider spectrum of interactions with other Ly49 molecules and if this modifies host susceptibility to MCMV. We have identified novel m157-Ly49 receptor interactions, involving Ly49C inhibitory receptors from B6, BALB/c, and NZB mice, as well as the Ly49H(NZB) activation receptor. Using an MCMV recombinant virus in which m157(K181) was replaced with m157(G1F), which interacts with both Ly49H(B6) and Ly49C(B6), we show that the m157(G1F)-Ly49C interactions cause no apparent attenuating effect on viral clearance in B6 mice. Hence, when m157 can bind both inhibitory and activation NK cell receptors, the outcome is still activation. Thus, these data indicate that whereas m157 variants predominately interact with inhibitory Ly49 receptors, these interactions do not profoundly interfere with early NK cell responses.     

Ly49I NK cell receptor transgene inhibition of rejection of H2b mouse bone marrow transplants

The Ly49 family of genes encode NK cell receptors that bind class I MHC Ags and transmit negative signals if the cytoplasmic domains have immunoregulatory tyrosine-based inhibitory motifs (ITIMs). 5E6 mAbs recognize Ly49C and Ly49I receptors and depletion of 5E6+ NK cells prevents rejection of allogeneic or parental-strain H2d bone marrow cell (BMC) grafts. To determine the function of the Ly49I gene in the rejection of BMC grafts, we transfected fertilized eggs of FVB mice with a vector containing DNA for B6 strain Ly49I (Ly49IB6). Ly49IB6 is ITIM+ and is recognized by 5E6 as well as Ly49I-specific 8H7 mAbs. Normal FVB H2q mice reject H2b but not H2d BMC allografts, and the rejection of H2b BMC was inhibited partially by anti-NK1.1 and completely by anti-asialo GM1, but not by anti-CD8, Abs. In FVB mice, NK1.1 is expressed on only 60% NK cells. FVB. Ly49IB6 hosts failed to reject H2d or H2b BMC, but did reject class I-deficient TAP-1-/- BMC, indicating that NK cells were functional. Nondepleting doses of anti-Ly49I Abs reversed the acceptance of H2b BMC by FVB.Ly49IB6 mice. FVB.Ly49IB6+/- mice were crossed and back-crossed with 129 mice-H2b, 5E6-, poor responders to H2d BMC grafts. While transgene-negative H2b/q F1 or first-generation back-crossed mice rejected H2b marrow grafts (hybrid resistance), transgene-positive mice did not. Thus B6 strain Ly49I receptors transmit inhibitory signals from H2b MHC class I molecules. Moreover, Ly49IB6 has no positive influence on the rejection of H2d allografts.     

Ly49h-deficient C57BL/6 mice: a new mouse cytomegalovirus-susceptible model remains resistant to unrelated pathogens controlled by the NK gene complex

Cmv1 was the first mouse cytomegalovirus (MCMV) resistance locus identified in C57BL/6 mice. It encodes Ly49H, a NK cell-activating receptor that specifically recognizes the m157 viral protein at the surface of MCMV-infected cells. To dissect the effect of the Ly49h gene in host-pathogen interactions, we generated C57BL/6 mice lacking the Ly49h region. We found that 36 h after MCMV infection, the lack of Ly49h resulted in high viral replication in the spleen and dramatically enhanced proinflammatory cytokine production in the serum and spleen. At later points in time, we observed that MCMV induced a drastic loss in CD8(+) T cells in B6.Ly49h(-/-) mice, probably reflecting severe histological changes in the spleen. Overall, our results indicate that Ly49H(+) NK cells contain a systemic production of cytokines that may contribute to the MCMV-induced pathology and play a central role in maintaining normal spleen cell microarchitecture. Finally, we tested the ability of B6.Ly49h(-/-) mice to control replication of Leishmania major and ectromelia virus. Resistance to these pathogens has been previously mapped within the NK gene complex. We found that the lack of Ly49H(+) NK cells is not associated with an altered resistance to L. major. In contrast, absence of Ly49H(+) NK cells seems to afford additional protection against ectromelia infection in C57BL/6 mice, suggesting that Ly49H may recognize ectromelia-infected cells with detrimental effects. Taken together, these results confirm the pivotal role of the Ly49H receptor during MCMV infection and open the way for further investigations in host-pathogen interactions.     

Diversity of NK cell receptor repertoire in adult and neonatal mice.

Murine NK cytotoxicity is regulated by two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2. We developed a single-cell RT-PCR method to analyze expression of all known Ly49 and NKG2A genes in individual NK cells and determined the receptor repertoires of NK cells from adult and neonatal (1-wk-old) C57BL/6 mice. In adult mouse NK cells, up to six different receptors were coexpressed in random combinations. Of 62 NK cells examined, 42 different patterns of receptor expression were observed. Most of them expressed at least one Ly49, whereas NKG2A was detected in 32% of the cells. Over 75% of them expressed Ly49C, I, or NKG2A, which are thought to recognize self-class I MHC (H-2b). Coexpression of multiple Ly49 receptors and NKG2A was stochastic. In contrast, very few neonatal NK cells expressed any Ly49, but almost 60% of them expressed NKG2A. These results demonstrate that adult NK cells are quite heterogeneous and have diverse receptor repertoires. They also suggest that the expression of NKG2A precedes Ly49 expression in NK cell ontogeny, and NKG2A is a major inhibitory receptor in neonatal NK cells.     

Ly49-dependent NK cell licensing and effector inhibition involve the same interaction site on MHC ligands

NK cells become functionally competent to be triggered by their activation receptors through the interaction of NK cell inhibitory receptors with their cognate self-MHC ligands, an MHC-dependent educational process termed "licensing." For example, Ly49A(+) NK cells become licensed by the interaction of the Ly49A inhibitory receptor with its MHC class I ligand, H2D(d), whereas Ly49C(+) NK cells are licensed by H2K(b). Structural studies indicate that the Ly49A inhibitory receptor may interact with two sites, termed site 1 and site 2, on its H2D(d) ligand. Site 2 encompasses the α1/α2/α3 domains of the H2D(d) H chain and β(2)-microglobulin (β2m) and is the functional binding site for Ly49A in effector inhibition. Ly49C functionally interacts with a similar site in H2K(b). However, it is currently unknown whether this same site is involved in Ly49A- or Ly49C-dependent licensing. In this study, we produced transgenic C57BL/6 mice expressing wild-type or site 2 mutant H2D(d) molecules and studied whether Ly49A(+) NK cells are licensed. We also investigated Ly49A- and Ly49C-dependent NK licensing in murine β2m-deficient mice that are transgenic for human β2m, which has species-specific amino acid substitutions in β2m. Our data from these transgenic mice indicate that site 2 on self-MHC is critical for Ly49A- and Ly49C-dependent NK cell licensing. Thus, NK cell licensing through Ly49 involves specific interactions with its MHC ligand that are similar to those involved in effector inhibition.     

Interactions of Ly49 family receptors with MHC class I ligands in trans and cis

The Ly49A NK cell receptor interacts with MHC class I (MHC-I) molecules on target cells and negatively regulates NK cell-mediated target cell lysis. We have recently shown that the MHC-I ligand-binding capacity of the Ly49A NK cell receptor is controlled by the NK cells' own MHC-I. To see whether this property was unique to Ly49A, we have investigated the binding of soluble MHC-I multimers to the Ly49 family receptors expressed in MHC-I-deficient and -sufficient C57BL/6 mice. In this study, we confirm the binding of classical MHC-I to the inhibitory Ly49A, C and I receptors, and demonstrate that detectable MHC-I binding to MHC-I-deficient NK cells is exclusively mediated by these three receptors. We did not detect significant multimer binding to stably transfected or NK cell-expressed Ly49D, E, F, G, and H receptors. Yet, we identified the more distantly related Ly49B and Ly49Q, which are not expressed by NK cells, as two novel MHC-I receptors in mice. Furthermore, we show using MHC-I-sufficient mice that the NK cells' own MHC-I significantly masks the Ly49A and Ly49C, but not the Ly49I receptor. Nevertheless, Ly49I was partly masked on transfected tumor cells, suggesting that the structure of Ly49I is compatible in principal with cis binding of MHC-I. Finally, masking of Ly49Q by cis MHC-I was minor, whereas masking of Ly49B was not detected. These data significantly extend the MHC-I specificity of Ly49 family receptors and show that the accessibility of most, but not all, MHC-I-binding Ly49 receptors is modulated by the expression of MHC-I in cis.     

Critical residues at the Ly49 natural killer receptor's homodimer interface determine functional recognition of m157, a mouse cytomegalovirus MHC class I-like protein

NK cell function is regulated by Ly49 receptors in mice and killer cell Ig-like receptors in humans. Although inhibitory Ly49 and killer cell Ig-like receptors predominantly ligate classical MHC class I molecules, recent studies suggest that their activating counterparts recognize infection. The quintessential example is resistance to the mouse CMV in C57BL/6 mice, which depends on the functional recognition of m157, a mouse CMV-encoded MHC class I-like molecule, by Ly49H, an activating NK cell receptor. We have taken advantage of the natural variation in closely related members of the Ly49C-like receptors and the availability of Ly49 crystal structures to understand the molecular determinants of the Ly49H-m157 interaction and to identify amino acid residues discriminating between m157 binding and nonbinding receptors. Using a site-directed mutagenesis approach, we have targeted residues conserved in receptors binding to m157 (Ly49H and Ly49I(129)) but different from receptors lacking m157 recognition (Ly49C, Ly49I(B6), and Ly49U). Wild-type and mutant receptors were transfected into reporter cells, and physical binding as well as functional activation by m157 was studied. Our findings suggested that the Ly49 MHC class I contact "site 2," I226, may not be involved in m157 binding. In contrast, residue Y146 and G151, mapping at the receptor homodimer interface, are likely critical for functional recognition of the m157 glycoprotein. Our combined functional and three-dimensional modeling approach suggested that the architecture of the Ly49H dimer is crucial to accessing m157, but not MHC class I. These results link Ly49 homodimerization variability to the direct recognition of pathogen products.     

Requisite H2k role in NK cell-mediated resistance in acute murine cytomegalovirus-infected MA/My mice

Human CMV infections are a major health risk in patients with dysfunctional or compromised immunity, especially in patients with NK cell deficiencies, as these are frequently associated with high morbidity and mortality. In experimental murine CMV (MCMV) infections, Ly49H activation receptors on C57BL/6 (B6) NK cells engage m157 viral ligands on MCMV-infected cells and initiate dominant virus control. In this study, we report that MCMV resistance in MA/My relies on Ly49H-independent NK cell-mediated control of MCMV infection as NK cells in these mice do not bind anti-Ly49H mAb or soluble m157 viral ligands. We genetically compared MA/My resistance with MCMV susceptibility in genealogically and NK gene complex-Ly49 haplotype-related C57L mice. We found that MCMV resistance strongly associated with polymorphic H2k-linked genes, including MHC and non-MHC locations by analysis of backcross and intercross progeny. The H2b haplotype most frequently, but not absolutely, correlated with MCMV susceptibility, thus confirming a role for non-MHC genes in MCMV control. We also demonstrate a definite role for NK cells in H2k-type MCMV resistance because their removal from C57L.M-H2k mice before MCMV infection diminished immunity. NK gene complex-linked polymorphisms, however, did not significantly influence MCMV control. Taken together, effective NK cell-mediated MCMV control in this genetic system required polymorphic H2k genes without need of Ly49H-m157 interactions.     

<Emphasis Type="Italic">Ly49h</Emphasis> is necessary for genetic resistance to murine cytomegalovirus

Natural killer (NK) cells play critical roles in antiviral immunity. While the importance of effector mechanisms such as interferons has been demonstrated through knockout mice, specific mechanisms of how viruses are recognized and controlled by NK cells are less well defined. Previous genetic studies have mapped the resistance genes for murine cytomegalovirus (MCMV), herpes simplex virus-1 (HSV-1), and ectromelia virus to the NK gene complex on murine chromosome 6, a region containing the polymorphic Ly49 and Nkrp1 families. Genetic resistance to MCMV in C57BL/6 has been attributed to Ly49H, an activation receptor, through susceptibility of the recombinant inbred strain BXD-8 that lacks Ly49h (also known as Klra8) but derived about half of its genome from its DBA/2 progenitor. However, it remained possible that epigenetic effects could account for the MCMV phenotype in BXD-8 mice. Herein, we report the generation of a novel congenic murine strain, B6.BXD8-Klra8 ( Cmv1-del )/Wum, on the C57BL/6 genetic background to evaluate the effect of deletion of a single NK activation receptor, Ly49H. Deletion of Ly49H rendered mice much more susceptible to MCMV infection. This increase in susceptibility did not appear to be a result of a difference in NK cell expansion or interferon-gamma (IFN-gamma) production between the C57BL/6 and the B6.BXD8 strains. On the other hand, the deletion of Ly49h did not otherwise affect NK cell maturation or Ly49D expression and had no effect on susceptibility to HSV-1 or ectromelia virus. In conclusion, Ly49h is necessary for genetic resistance to MCMV, but not HSV-1 or ectromelia virus.     

TheCmv1Host Resistance Locus Is Closely Linked to theLy49Multigene Family within the Natural Killer Cell Gene Complex on Mouse Chromosome 6

Natural killer (NK) cells play important roles in controlling tumor cells and against a range of infectious organisms. Recent studies of mouse NK cell surface receptors, which may be involved in the specificity of NK cells, have shown that many of these molecules are encoded by theLy49andLy55(Nkrp1) multigene families that map to distal mouse chromosome 6. Also mapping to this NK cell gene complex (NKC) is the resistance locus,Cmv1,which is involved in genetically determined resistance to murine cytomegalovirus (MCMV). The aim of this study was to localizeCmv1more precisely in relation to other NKC loci by generating a high-resolution genetic map of the region. We have analyzed 1250 backcross mice comprising panels of 700 (BALB/c × C57BL/6J)F₁× BALB/c and 550 (A/J × C57BL/6J)F₁× A/J progeny. A total of 25 polymorphic genes or microsatellite markers were analyzed over a region of 10 map units fromD6Mit134toD6Mit59.TheCmv1phenotypes of mice recombinant in this interval were tested by infection with MCMV. The results obtained indicate that the functionally important NKC region is a tightly linked cluster of loci spanning at least 0.4 map units. Furthermore,Cmv1maps distal to, but very closely linked to, theLy49multigene family (<0.2 map units), suggesting that MCMV resistance may be conferred by MHC class I-specific NK cell receptors.