Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors.

Mouse NK cells express at least seven inhibitory Ly49 receptors. Here we employ a semiquantitative cell-cell adhesion assay as well as class I/peptide tetramers to provide a comprehensive analysis of specificities of Ly49 receptors for class I MHC molecules in eight MHC haplotypes. Different Ly49 receptors exhibited diverse binding properties. The degree of class I binding was related to the extent of functional inhibition. The tetramer studies demonstrated that neither glycosylation nor coreceptors were necessary for class I binding to Ly49 receptors and uncovered peptide-specific recognition by a Ly49 receptor. The results provide a foundation for interpreting and integrating many existing functional studies as well as for designing tests of NK cell development and self-tolerance.     

Role of Qa-1(b)-binding receptors in the specificity of developing NK cells

NK cells acquire the ability to recognize MHC class I molecules during development. Studies with Qa-1(b) tetramers (Qa-1 tetramers) showed that nearly all NK1.1(+) cells from newborn C57BL/6 mice express Qa-1-binding receptors. Cytotoxic activity of these cells is fully inhibited by Qa-1 ligands on target cells. In contrast, neither receptors for H-2K(b) nor H-2D(b) were observed on NK1.1(+) cells from newborn mice. After birth, frequencies of Qa-1 tetramer(+)/ NK1.1(+) cells gradually decrease as the number of Ly49(+) /NK1.1(+) cells increases. Cell transfer studies showed that Qa-1 tetramer(+) cells from newborn mice do not lose expression of Qa-1 receptors, but that they further acquire expression of Ly49 molecules. Acquisition of Qa-1-binding receptors appears largely independent of host MHC class I molecules, as observed in studies using beta2-microglobulin-deficient (beta2m(-/-)) mice as well as K(b)/ D(b-/-) and K(b)/D(b)/beta2m(-/-) mice. The present results suggest that Qa-1-binding receptors play an important role in the specificity of developing NK cells, and suggest that these cells rely mainly on inhibitory receptors specific for non-classical MHC class I molecules to maintain self tolerance during the first weeks of life.     

Variable MHC class I engagement by Ly49 natural killer cell receptors demonstrated by the crystal structure of Ly49C bound to H-2K(b)

The Ly49 family of natural killer (NK) receptors regulates NK cell function by sensing major histocompatibility complex (MHC) class I. Ly49 receptors show complex patterns of MHC class I cross-reactivity and, in certain cases, peptide selectivity. To investigate whether specificity differences result from topological differences in MHC class I engagement, we determined the structure of the peptide-selective receptor Ly49C in complex with H-2K(b). The Ly49C homodimer binds two MHC class I molecules in symmetrical way, a mode distinct from that of Ly49A, which binds MHC class I asymmetrically. Ly49C does not directly contact the MHC-bound peptide. In addition, MHC crosslinking by Ly49C was demonstrated in solution. We propose a dynamic model for Ly49-MHC class I interactions involving conformational changes in the receptor, whereby variations in Ly49 dimerization mediate different MHC-binding modes.     

Interaction of the NK cell inhibitory receptor Ly49A with H-2Dd: identification of a site distinct from the TCR site

Natural killer cell function is controlled by interaction of NK receptors with MHC I molecules expressed on target cells. We describe the binding of bacterially expressed Ly49A, the prototype murine NK inhibitory receptor, to similarly engineered H-2Dd. Despite its homology to C-type lectins, Ly49A binds independently of carbohydrate and Ca2+ and shows specificity for MHC I but not bound peptide. The affinity of the Ly49A/H-2Dd interaction as determined by surface plasmon resonance is from 6 to 26 microM at 25 degrees C and is greater by ultracentrifugation at 4 degrees C. Biotinylated Ly49A stains H-2Dd-expressing cells. Competition experiments indicate that the Ly49A and T cell receptor (TCR) binding sites on MHC I are distinct, suggesting complex regulation of cells that bear both TCR and NK cell receptors.     

H-2 allele specificity of the NK cell C-type lectin-like MHC class I receptor Ly49A visualized by soluble Ly49A tetramer

Ly49A is a C-type lectin-like receptor on NK cells that recognizes MHC class I ligands, H-2D(d) and D(k). The engagement of Ly49A with the ligands inhibits activation of NK cells and protects target cells from lysis by NK cells. Here we express the extracellular region of Ly49A with an N-terminal biotinylation tag in Escherichia coli to obtain soluble Ly49A (sLy49A) after refolding. sLy49A is indistinguishable from native Ly49A expressed on NK cells serologically and in the ability to specifically bind H-2D(d) after tetramerization with R-phycoerythrin-coupled streptavidin. The fluorescently labeled tetramer of sLy49A is applied to explore MHC class I haplotype specificity of Ly49A. We demonstrate the hierarchical reactivity of Ly49A with H-2 of various alleles in the order of d > k, r > p > v > q > s > z. Reactivity of sLy49A tetramer to spleen lymphocytes from B10.QBR mice (H-2K(b), I(b), D(q), Qa-1/Tla(b)) but not from C57BL/10 mice (H-2(b)) identifies H-2D(q) and L(q) as candidates for a Ly49A ligand. Binding of sLy49A tetramer to H-2D(q)- or L(q)-transfected cell lines demonstrates that the two highly related MHC class I molecules, H-2D(q) and L(q), are ligands for Ly49A. sLy49A tetramer staining also demonstrates preferential expression of Ly49A ligand on a subset of B cells in P/J mice. These results provide the basis to examine the molecular mechanism by which Ly49A discriminates polymorphic MHC class I molecules.     

Role of interaction between Ly49 inhibitory receptors and cognate MHC I molecules in IL2-induced development of NK cells in murine bone marrow cell cultures

Murine bone marrow (BM) cell preparations lack mature cytotoxic natural killer (NK) cells, but NK cells may be induced in these cell preparations by culturing with interleukin-2 (IL2). Present study was aimed at studying the role of interactions between Ly49 molecules and major histocompatibility complex (MHC) class I molecules during IL2-induced development of mature NK cells in BM cell cultures. Addition of monoclonal antibodies (mabs) specific to class I MHC molecules of H-2b haplotype, to block any interaction of MHC I molecules with their receptors, was found to inhibit NK cell development. Mouse NK cells express several types of Ly49 molecules including Ly49C, which is an inhibitory receptor specific to MHC I molecules of H-2b haplotype. Blocking Ly49-MHC I interaction by using anti-Ly49C mab inhibited the development of cytotoxic NK cells. Addition of anti-Ly49A (no specificity for H-2b MHC I molecules) or anti-Ly49D (activating receptor specific for MHC I molecules of many H-2 haplotypes including H-2b) mabs, however, had no effect on IL2-induced NK cell development in BM cells. Mabs specific to Ly49C molecule and MHC I molecules of H-2b haplotype inhibited the development of mature NK cells from highly purified NK precursor cell population. These results indicate that specific interaction between inhibitory self-reactive Ly49 molecules and MHC I molecules may be crucial for NK cell development. We propose a model in which Ly49-MHC I interaction may have a permissive role in allowing development of only such NK cell clones that expresses at least one self-reactive inhibitory Ly49 molecule so that lysis of autologous healthy cells by mature NK cells may be avoided.     

Augmentation of NK cell-mediated cytotoxicity to tumor cells by inhibitory NK cell receptor blockers

NK cells monitor expression of MHC class I by inhibitory receptors and preferentially kill cells that lose or down-regulate MHC class I expression. One possible mechanism by which tumor cells evade NK cell killing is continued expression of appropriate MHC class I ligands to engage inhibitory receptors on NK cells. We show here that small-mol.-wt blockers against the mouse inhibitory NK cell receptor Ly49A enhance NK cell killing of such tumor cells. We identified Ly49A-binding peptides by selecting phages with the capacity to bind recombinant Ly49A expressed in Escherichia coli from a phage display random peptide library. The Ly49A-binding peptides could also bind Ly49A expressed on mammalian cells. Importantly, the Ly49A-binding peptides blocked Ly49A recognition of its MHC class I ligands H-2Dd and H-2Dk. Moreover, blockade of Ly49A by the peptides enhanced cytotoxicity of Ly49A+ NK cells towards H-2Dd-expressing tumor cells. These results clearly indicate effectiveness of small-mol.-wt blockers of inhibitory NK cell receptors in enhancing NK cell-mediated killing of tumor cells that are otherwise resistant because of MHC class I expression.     

Quantifying the reduction in accessibility of the inhibitory NK cell receptor Ly49A caused by binding MHC class I proteins in cis

Murine natural killer (NK) cells are inhibited by target cell MHC class I molecules via Ly49 receptors. However, Ly49 receptors can be made inaccessible to target cell MHC class I by a cis interaction with its MHC class I ligand within the NK cell membrane. It has recently been demonstrated that MHC class I proteins transfer from the target cells to the NK cell. Here, we establish that the number of transferred MHC class I proteins is proportional to the number of Ly49A receptors at the NK cell surface. Ly49A+ NK cells from mice expressing the Ly49A ligand H-2D(d) showed a 90% reduction in Ly49A accessibility compared to Ly49A+ NK cells from H-2D(d)-negative mice. The reduction was caused both by lower expression of Ly49A and interactions in cis between Ly49A and H-2D(d) at the NK cell surface. Approximately 75% of the Ly49A receptors on H-2D(d)-expressing NK cells were occupied in cis with endogenous H-2D(d) and only 25% were free to interact with H-2D(d) molecules in trans. Thus, H-2D(d) ligands control Ly49A receptor accessibility through interactions both in cis and in trans.     

Purified MHC class I molecules inhibit activated NK cells in a cell-free system in vitro

Natural killer cells have been shown to interact with MHC class I molecules via inhibitory receptors. However, it is not known whether the inhibition induced by MHC class I molecules requires other NK cell-target cell interactions. Thus, we examined whether purified MHC class I molecules alone were able to inhibit NK cell function. Purified H-2K(b) and H-2D(b) molecules inhibited the release of IFN-gamma from spleen (H-2(b))-derived lymphokine-activated killer (LAK) cell cultures stimulated by anti-NK1.1 antibody in a concentration-dependent manner. LAK cells generated from newborn mice that express low levels of MHC class I binding Ly49 inhibitory receptors were significantly less sensitive to inhibition by H-2K(b) compared to LAK cells from adult mice. Furthermore, LAK cells generated from spleen cells of Ly49C-transgenic mice were significantly more sensitive to inhibition by H-2K(b) compared to non-transgenic littermates. Taken together, the data indicate that MHC class I induced inhibition of NK cell mediated effector functions, as assessed by IFN-gamma release after NK1.1 triggering, does not require additional cell surface molecules other than MHC class I.     

Recognition of H-2K(b) by Ly49Q suggests a role for class Ia MHC regulation of plasmacytoid dendritic cell function

Ly49Q is a member of the polymorphic Ly49 family of NK cell receptors that displays both a high degree of conservation and a unique expression pattern restricted to myeloid lineage cells, including plasmacytoid dendritic cells (pDC). The function and ligand specificity of Ly49Q are unknown. Here, we use reporter cell analysis to demonstrate that a high-affinity ligand for Ly49Q is present on H-2(b), but not H-2(d), H-2(k), H-2(q), or H-2(a)-derived tumor cells and normal cells ex vivo. The ligand is peptide-dependent and MHC Ia-like, as revealed by its functional absence on cells deficient in TAP-1, beta(2)m, or H-2K(b)D(b) expression. Furthermore, Ly49Q is specific for H-2K(b), as the receptor binds peptide-loaded H-2K(b) but not H-2D(b) complexes, and Ly49Q recognition can be blocked using anti-K(b) but not anti-D(b) mAb. Greater soluble H-2K(b) binding to ligand-deficient pDC also suggests cis interactions of Ly49Q and H-2K(b). These results demonstrate that Ly49Q efficiently binds H-2K(b) ligand, and suggest that pDC function, like that of NK cells, is regulated by classical MHC Ia molecules. MHC recognition capability by pDC has important implications for the role of this cell type during innate immune responses.     

A species-specific determinant on beta2-microglobulin required for Ly49A recognition of its MHC class I ligand

The mouse inhibitory NK cell receptor Ly49A recognizes the mouse MHC class I molecule H-2D(k). The present study focuses on the species specificity of beta(2)-microglobulin (beta(2)m), an invariant component of MHC class I, in the interaction between Ly49A and H-2D(k). Transfection of the beta(2)m-defective mouse cell line R1E/TL8x.1 with human (h) beta(2)m induced cell-surface expression of H-2D(k), but failed to protect the cells from killing by Ly49A(+) NK cells. In contrast, the cells transfected with mouse (m) beta(2)m were protected from killing by Ly49A(+) NK cells. These data indicate that Ly49A distinguishes mbeta(2)m from hbeta(2)m when it recognizes the H-2D(k) complexes. To identify the species-specific determinant of beta(2)m required for Ly49A recognition of H-2D(k), we prepared a panel of mbeta(2)m mutants and tested the H-2D(k) that included each of the beta(2)m mutants for its capacity to engage Ly49A on NK cells. Ly49A failed to functionally recognize the H-2D(k) that included the mbeta(2)m with K3R and Q29G mutations. Moreover, Ly49A was able to recognize the H-2D(k) that included the hbeta(2)m with R3K and G29Q mutations. These data indicate that Lys3 and Gln29 consist of the central part of the species-specific determinant of beta(2)m required for Ly49A recognition of H-2D(k). The two residues are conserved in the mouse and the rat, in which NK cells use Ly49 family molecules as the receptors specific for MHC class I. These results suggest functional importance of beta(2)m in NK cell recognition of target cells.     

The role of an exposed loop in the alpha2 domain in the mouse MHC class I H-2Dd molecule for recognition by the monoclonal antibody 34-5-8S and the NK-cell receptor Ly49A

Natural killer (NK) cells express major histocompatibility complex (MHC) class I-specific inhibitory receptors. The region mediating the protective effect of the MHC class I molecule H-2Dd (Dd), recognized by the inhibitory receptor Ly49A, has been mapped to the alpha1/alpha2 domains. Here we have focused on an exposed loop in the N-terminal part of the alpha2 domain, which constitutes a major structural motif that differs between Dd (strong binding to Ly49A) and Db (weak binding to Ly49A at best). We mutated the residues 103, 104 and 107 in Dd to the corresponding amino acids in Db. The Dd mutant molecule retained the ability to be stabilized by a Dd-binding peptide. However, the mutation totally abolished the recognition by the conformational dependent monoclonal antibody (MoAb) 34-5-8S, known to inhibit the interaction between Dd and Ly49A. In addition, there was a marked impairment of the binding to Ly49A as evaluated by the ability of tetramers of the Dd mutant molecule to bind to Ly49A-transfected reporter cells and spleen cells. These results demonstrate that the introduced changes at positions 103, 104 and 107 directly or indirectly affect the epitopes for the MoAb 34-5-8S and the Ly49A receptor.     

α1 / α2 domains of H-2Dd,but not H-2Ld,induce “missing self” reactivity in vivo – No effect of H-2Ld on protection against NK cells expressing the inhibitory receptor Ly49G2

Introduction of the MHC class I transgene H-2D^d on C57BL / 6 (B6) background conveys NK cell-mediated “missing self” reactivity against transgene-negative cells, and down-regulates expression of the inhibitory receptors Ly49A and Ly49G2 in NK cells. We here present an analysis of transgenic mice expressing chimeric H-2D^d / L^d MHC class I transgenes, and show that the α1 / α2 domains of H-2D^d were necessary and sufficient to induce “missing self” recognition and to down-modulate Ly49A and Ly49G2 receptors. In contrast, transgenes containing the α1 / α2 domains of H-2L^d induced none of these changes, suggesting that not all MHC class I alleles in a host necessarily take part in NK cell education. The lack of effect of the α1 / α2 domains of H-2L^d on NK cell specificity was surprising, considering that both H-2L^d and H-2D^d have been reported to interact with Ly49G2. Therefore, the role of H-2L^d for protection against NK cells expressing Ly49G2 was re-investigated in a transfection system. In contradiction to earlier reports, we show that H-2D^d, but not H-2L^d, abolished killing by sorted Ly49G2⁺ NK cells, indicating that H-2L^d does not inhibit NK cells via the Ly49G2 receptor.     

MHC class I molecules on adenovirus E1A-expressing tumor cells inhibit NK cell killing but not NK cell-mediated tumor rejection

Expression of adenovirus E1A gene products in tumor cells enhances NK cell lysis in vitro and NK-mediated rejection in vivo, despite increasing class I molecules on tumor cells. It is unclear why the increased expression of MHC class I molecules does not appear to confer resistance to killing by NK cells. One possibility is the unique capacity of E1A to sensitize cells to multiple NK cell killing mechanisms including perforin/granzyme, Fas ligand, tumor necrosis factor-alpha and TRAIL. To examine this issue, MCA-102-E1A tumor cells (H-2(b)) that express E1A and are NK sensitive were transfected with H-2D(d), the ligand for the NK inhibitory receptor, Ly49A. Expression of H-2D(d) molecules by MCA-102-E1A cells protected them from lysis by a Ly49A(+) NK cell clone and Ly49A(+) NK cells isolated from C57BL/6 nude mice. In contrast, NK cell-mediated rejection of MCA-102-E1A tumor cells was not inhibited by the expression of H-2D(d) molecules, nor was killing by polyclonal populations of NK cells isolated from C57BL/6-nude mice. H-2D(d) interacts with several inhibitory Ly49 receptors that are non-clonally expressed on NK cells in C57BL/6 mice: Ly49A (20% of NK cells), Ly49G2 (54% of NK cells) and Ly49C/I (47% of NK cells). Our data indicate that while E1A sensitizes cells to NK cell killing, it does not interfere with signal transduction by inhibitory NK receptors. Therefore, a small population of NK cells that do not express Ly49A, Ly49G2 or Ly49C/I inhibitory receptors are likely responsible for the rejection of MCA-102-E1A-D(d) tumor cells in vivo.     

Structure of the Ly49 family of natural killer (NK) cell receptors and their interaction with MHC class I molecules

Natural killer (NK) cells are an essential component of the innate immunity toward tumors and virally infected cells. The function of NK cells is regulated by a precise balance between inhibitory and activating signals. These signals are mediated by NK cell receptors that bind either classical MHC class I molecules or their structural relatives such as MICA, ULBP, RAE-1, and H-60. Two separate families of NK cell receptors have been identified: the immunoglobulin-like family (KIR, LIR) and C-type lectin-like family (Ly49, NKG2D, and CD94/NKG2). Here we summarize the structure of Ly49 C-type lectin-like proteins hitherto solved (Ly49A, Ly49C and Ly49I) and their interaction with MHC class I molecules as determined by the co-crystal structure of Ly49A/H-2Dd and Ly49C/H-2Kb.     

A role for the src family kinase Fyn in NK cell activation and the formation of the repertoire of Ly49 receptors

NK cell function is regulated by a dual receptor system, which integrates signals from triggering receptors and MHC class I-specific inhibitory receptors. We show here that the src family kinase Fyn is required for efficient, NK cell-mediated lysis of target cells, which lack both self-MHC class I molecules and ligands for NKG2D, an activating NK cell receptor. In contrast, NK cell inhibition by the MHC class I-specific receptor Ly49A was independent of Fyn, suggesting that Fyn is specifically required for NK cell activation via non-MHC receptor(s). Compared to wild type, significantly fewer Fyn-deficient NK cells expressed the inhibitory Ly49A receptor. The presence of a transgenic Ly49A receptor together with its H-2(d) ligand strongly reduced the usage of endogenous Ly49 receptors in Fyn-deficient mice. These data suggest a model in which the repertoire of inhibitory Ly49 receptors is formed under the influenced of Fyn-dependent NK cell activation as well as the respective MHC class I environment. NK cells may acquire Ly49 receptors until they generate sufficient inhibitory signals to balance their activation levels. Such a process would ensure the induction of NK cell self-tolerance.     

MHC-dependent shaping of the inhibitory Ly49 receptor repertoire on NK cells: evidence for a regulated sequential model.

Engagement of MHC class I-specific inhibitory receptors regulates natural killer (NK) cell development and function. Using both new and previously characterized anti-Ly49 monoclonal antibodies, we comprehensively determined expression and co-expression frequencies of four Ly49 receptors by NK cells from MHC-congenic, MHC class I-deficient, and Ly49A-transgenic mice to study mechanisms that shape the inhibitory Ly49 repertoire. All Ly49 receptors were expressed on partially overlapping subsets. Significantly, in the absence of class I MHC, several receptor pairs were co-expressed more frequently than predicted from a purely random expression model, indicating that biases independent of MHC class I underlie receptor co-expression in some cases. MHC interactions were found to inhibit Ly49 co-expression variably depending on the MHC allele and the receptor pair examined. These data extend previous evidence that interactions with MHC shape the repertoire. It was previously proposed that developing NK cells express Ly49 receptors sequentially and cumulatively, until self-MHC specific receptors are expressed and inhibit new receptor expression. Fulfilling a major prediction of this model, we found that class I recognition by a Ly49A transgene expressed by all developing NK cells equivalently inhibited expression of endogenous self-specific and nonself-specific Ly49 receptors.     

Thymus-dependent modulation of Ly49 inhibitory receptor expression on NK1.1+gamma/delta T cells

Major histocompatibility complex (MHC) class I-specific inhibitory receptors are expressed not only on natural killer (NK) cells but also on some subsets of T cells. We here show Ly49 expression on gamma/delta T cells in the thymus and liver of beta2-microglobulin-deficient (beta2m-/-) and C57BL/6 (beta2m+/+) mice. Ly49C/I or Ly49A receptor was expressed on NK1.1+gamma/delta T cells but not on NK1.1-gamma/delta T cells. The numbers of NK1.1+gamma/delta T cells were significantly smaller in beta2m+/+ mice than in beta2m-/- mice with the same H-2b genetic background. Among NK1.1+gamma/delta T cells, the proportions of Ly49C/I+ cells but not of Ly49A+ cells, were decreased in beta2m+/+ mice, suggesting that cognate interaction between Ly49C/I and H-2Kb is involved in the reduction of the number of Ly49C/I+ gamma/delta T cells in beta2m+/+ mice. The frequency of Ly49C/I+ cells in NK1.1+gamma/delta T cells was lower in both lethally irradiated beta2m+/+ mice transplanted with bone marrow (BM) from beta2m-/- mice and lethally irradiated beta2m-/- mice transplanted with BM from beta2m+/+ mice than those in adult thymectomized BM-transplanted chimera mice. These results suggest that reduction of Ly49C/I+ NK1.1+gamma/delta T cells in beta2m+/+ mice is at least partly due to the down-modulation by MHC class I molecules on BM-derived haematopoietic cells or radioresistant cells in the thymus.     

Major histocompatibility complex genes determine natural killer cell tolerance

Murine natural killer (NK) cell subsets, as defined by expression of members of the Ly49 gene family, discriminate target cells expressing different major histocompatibility complex (MHC) class I alleles. For example, Ly49A⁺ NK cells lyse H-2^b but not H-2^d tumor target cells. The specificity arises because D^d on target cells binds to Ly49A, transducing an inhibitory signal into the Ly49A⁺ NK cells. The capacity of NK cells to discriminate allelic class I determinants raises a key issue: are NK cells self-tolerant, and if so what are the mechanisms that lead to self-tolerance? As previously reported, potentially autoaggressive Ly49A⁺ NK cells are not clonally deleted in H-2^b mice. However, IL-2- cultured Ly49A⁺ effector cells from H-2^b mice exhibit reduced lysis of H-2^b (self) concanavalin A blast target cells, compared to Ly49A⁺ effector cells from H-2^d mice. Possible mechanisms accounting for this self-tolerance are addressed in this report. Self-tolerance was not due to anergy of the cells, because the Ly49A⁺ effector cells from both types of mice lysed β2-microglobulin-deficient target cells efficiently and equivalently. These results also suggest that tolerance results from inhibition mediated by β2m-dependent H-2^b class I molecules. Significantly, blockade of Ly49A on Ly49A⁺ effector cells from H-2^b mice did not restore lysis of H-2^b target cells, suggesting that inhibition is not mediated through the Ly49A receptor. Additional experiments suggest that inhibition is also not mediated primarily through the Ly49C receptor. These results suggest that Ly49A⁺ effector cells from H-2^b mice, unlike those from H-2^d mice, express inhibitory receptors specific for H-2^b molecules that are distinct from Ly49A and Ly49C.     

Major histocompatibility complex class I-dependent skewing of the natural killer cell Ly49 receptor reportoire

Subsets of mouse natural killer (NK) cells express receptors encoded by the Ly49 gene family that recognize allelic determinants on major histocompatibility complex (MHC) class I molecules. Recognition of self class I molecules typically inhibits NK cell lytic function. The presence of NK cell subsets expressing receptors which are able to discriminate class I alleles raises the possibility that there exist mechanisms to coordinate the NK cell receptor repertoire with the class I molecules of the host. In the present study, we determined the effects of class I gene expression on the frequencies of NK cells expressing three different Ly49 receptors defined by monoclonal antibodies. We show here an MHC-dependent skewing of NK cell subsets expressing multiple Ly49 receptors with specificity for self MHC. The results provide the first evidence that the frequencies of NK cells expressing different Ly49 receptors are determined by the host's MHC molecules. The results also extend previous findings that MHC class I expression influences the cell surface levels of each Ly49 receptor, suggesting an additional mechanism by which MHC molecules may influence the effective specificity of NK cells. Models to account for self tolerance and MHC-controlled repertoire differences are discussed.