The Ly-49 family: regulation of cytotoxicity and cytokine production in murine CD3+ cells

The Ly-49 gene families are class I-recognizing receptors on murine NK cells. Most Ly-49 receptors inhibit NK cell lysis upon recognizing their target class I ligands. In this report we have examined the ability of Ly-49A and Ly-49G2 to regulate T cell functions on CD3+ cells, primarily the subset that also expresses NK-1.1 and/or DX5. The majority (>50%) of T cells that express Ly-49 molecules also coexpress NK-1.1 and/or DX5, although some NK-1.1- and/or DX5-/CD3+ cells express Ly-49 molecules. Lysis of target cells by IL-2-cultured T cells expressing Ly-49A and G2 was enhanced by Abs specific for Ly-49A and G2 as well as by Abs to class I (H-2Dd alpha1/alpha2). Murine T cells also were cultured in the presence of targets that express (H-2Dd) which is inhibiting for the Ly-49A and G2 receptors. These cells were examined for a coincident increase in cytokine production (IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF). Abs to Ly-49A and G2 or their respective class I ligands blocked the negative signals mediated via the Ly-49 receptors and increased IFN-gamma and granulocyte-macrophage CSF production after interaction of these T cells with H-2Dd-expressing tumor targets. Furthermore, an EL-4 T cell line expressing both Ly-49A and G2, when treated with mAb YE148 and 4D11, demonstrated reduced cytokine production and calcium mobilization. These results demonstrate for the first time that Ly-49 class I binding receptors, previously thought to be restricted to mouse NK cells, can mediate important physiological functions of T cell subsets.     

Expression of different members of the Ly-49 gene family defines distinct natural killer cell subsets and cell adhesion properties

The murine Ly-49 antigen belongs to a family of type II transmembrane molecules containing lectin-like domains. The original member of this family, Ly-49A, has been demonstrated to be expressed by a subpopulation of natural killer (NK) cells, bind certain class I major histocompatibility complexes (MHC), and act as a negative regulator of lytic activity. The expression patterns and functional activities of the other Ly-49s, however, is unknown. We extended the study of this family by isolating cDNAs encoding two new Ly-49 molecules. The reactivity of these and previously identified Ly-49 molecules with NK antibodies was tested in a COS cell expression system. YE1/32 and YE1/48 bound Ly-49A specifically, and 5E6 reacted only with Ly-49C. Three-color flow cytometric analysis demonstrated Ly-49A and Ly-49C expression defines complex, but distinct subsets within NK1.1+ cells. Some NK1.1-CD3+ as well as NK1.1-CD3- cells expressing Ly-49A or C were also detected. Analysis of MHC congenic strains of mice demonstrated that YE1/32+ and YE1/48+ NK cells are not deleted, as has been shown with the Ly-49A mAb A1. Furthermore, COS cells transfected with Ly-49A bound H-2d and H-2k cell lines, whereas Ly-49C transfectants bound H-2d, H-2k, H-2b, and H-2s. The antibodies 5E6 and 34-1-2S (anti-class I MHC) inhibited the binding of Ly-49C to an H-2s cell line. These results imply that the NK cell antigens Ly-49A and C bind to different repertoires of class I MHC molecules.     

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

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

Effects of N- and L-type calcium channel antagonists and (+/-)-Bay K8644 on nerve-induced catecholamine secretion from bovine perfused adrenal glands

Most proteins encoded by members of the Ly-49 gene family are class I-recognizing receptors on murine natural killer (NK) cells. Class I recognition by Ly-49 receptors usually results in inhibition of NK cell lysis of target cells. However, NK cells function not only in a lytic capacity, but also can mediate cytokine production. In this report we have demonstrated the ability of Ly-49A and Ly-49G2 to inhibit production of cytokines by NK cells by showing that specific antibodies against these gene products stimulate cytokine production. Murine NK cells were cultured in the presence of P815 (H2-Dd), and supernatants were analyzed for the production of interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and granulocytemacrophage colony-stimulating factor (GM-CSF). NK cell populations were sorted for Ly-49A+ or Ly-49G+ subsets, and these subsets were analyzed for their ability to alter cytokine induction by target cell interaction. In the presence of target cells expressing the appropriate class I molecules, Ly-49A and G2 were found to inhibit cytokine induction by NK cells. Examination of mRNA for IFN-gamma and GM-CSF indicated that Ly-49 receptors increased mRNA levels of NK cells. These results demonstrate that class I binding of these NK receptors can inhibit production of important physiological cytokines, in addition to the regulation of cytotoxic activity.     

Impaired MHC class I (H-2Dd)-mediated protection against Ly-49A+ NK cells after amino acid substitutions in the antigen binding cleft

The MHC class I molecule H-2Dd (Dd) acts as a ligand for the inhibitory NK cell receptor Ly-49A. We have constructed altered Dd molecules by site-directed mutagenesis, replacing residues with the corresponding amino acids from the Db molecule, which fails to inhibit via Ly-49A. Mutations at positions 73 and 156 (DdS73WD156Y) impaired the protective effect of the Dd molecule, as evaluated by testing lymphoma cells transfected with the mutant gene for sensitivity to killing by Ly-49A+ NK cells in vitro and rejection by NK cells in vivo. The altered residues form a hydrophobic ridge across the floor of the antigen binding cleft. A mutation in the alpha helix of the alpha2 domain, facing the solvent and without direct contact with the peptide (DdA150S) had no effect. Dd recognition by Ly-49A+ NK cells is considered to be peptide dependent, but not peptide specific. Our results indicate that alterations of residues buried in the antigen binding cleft can induce changes in peptide binding patterns and/or conformational changes in the Dd molecule that make the trimolecular complex less permissive for inhibition of Ly-49A+ NK cells.     

Role of conserved glycosylation site unique to murine class I MHC in recognition by Ly-49 NK cell receptor

The recognition of class I MHC molecules on target cells by the Ly-49 family of receptors regulates NK cytotoxicity. Previous studies have suggested that carbohydrates are involved in the recognition of class I MHC by Ly-49, although their precise role remains unclear. Here, we examined the role of asparagine-linked carbohydrates of the murine class I MHC in the binding to Ly-49A and Ly-49C. We have generated H-2Dd mutants that lack the highly conserved glycosylation sites at amino acid residues 86 in the alpha1 domain and 176 in the alpha2 domain, respectively. These mutant Dd cDNAs were transfected into leukemic cell lines, and the binding of the transfected cells to COS cells expressing Ly-49A or Ly-49C, as well as their susceptibility to lysis by Ly-49A+ NK cells, was examined. Only the mutation of the alpha2 domain glycosylation site significantly reduced the binding of Dd to Ly-49A and Ly-49C. Cells expressing Dd with the mutation at this site were partially resistant to killing by Ly-49A+ NK cells. These results suggest that, while carbohydrates linked to residue 176 seem to function as a part of the ligand structure for the Ly-49 family of NK receptors, there are additional structural features involved in this recognition. This glycosylation site is highly conserved among murine class I MHC but is not found among those of other species, suggesting that its role is unique to the murine immune system. It further suggests that murine class I MHC and Ly-49 gene families may have evolved in concert.     

Hematopoietic cells and radioresistant host elements influence natural killer cell differentiation

Radioresistant host elements mediate positive selection of developing thymocytes, whereas bone marrow-derived cells induce clonal deletion of T cells with receptors that are strongly autoreactive. In contrast to T cell development, little is known about the elements governing the natural killer (NK) cell repertoire, which, similar to the T cell repertoire, differs between individuals bearing different major histocompatibility complex (MHC) phenotypes. We have used murine bone marrow transplantation models to analyze the influence of donor and host MHC on an NK cell subset. We examined the expression of Ly-49, which is strongly expressed on a subpopulation of NK cells of H-2b mice, but not by NK cells of H-2a mice, probably because of a negative effect induced by the interaction of Ly-49 with Dd. To evaluate the effect of hematopoietic cell H-2a expression on Ly-49 expression of H-2b NK cells, we prepared mixed allogeneic chimeras by administering T cell-depleted allogeneic (B10.A, H-2a) and host-type (B10, H-2b) marrow to lethally irradiated B10 mice, or by administering B10. A marrow to B10 recipients conditioned by a nonmyeloablative regimen. Expression of H-2a on bone marrow-derived cells was sufficient to downregulate Ly-49 expression on both H-2a and H-2b NK cells. This downregulation was thymus independent. To examine the effect of H-2a expressed only on radioresistant host elements, we prepared fully allogeneic chimeras by administering B10 bone marrow to lethally irradiated B10.A recipients. B10 NK cells of these fully allogeneic chimeras also showed downregulation of Ly-49 expression. The lower level of H-2a expressed on H-2b x H-2a F1 cells induced more marked downregulation of Ly-49 expression on B10 NK cells when presented on donor marrow in mixed chimeras than when expressed only on radioresistant host cells. Our studies show that differentiation of NK cells is determined by interactions with MHC molecules expressed on bone marrow-derived cells and, to a lesser extent, by MHC antigens expressed on radioresistant host elements.     

Mantle cell lymphomas lack expression of p27Kip1, a cyclin-dependent kinase inhibitor

The TCRs expressed on T lymphocytes recognize foreign peptides bound to MHC molecules. This reactivity is the basis of specific immune response to the foreign Ag. How such specificities are generated in the thymus is still being debated. Signals generated through TCR upon interaction with self MHC-peptide complexes are critical for maturation of the CD4+ helper and CD8+ cytotoxic subsets. We have observed maturation of CD4+ but not CD8+ T cells in Ly-6A.2 transgenic MHC null mice. Since there can be no interactions with MHC molecules in these mice, these CD4+ cells must express the T cell repertoire that exists before positive and negative selection. Interestingly, despite an absence of selection by MHC molecules, the CD4+ cells that mature recognize MHC molecules at a frequency as high as in CD4+ cells in normal mice. These results demonstrate that: 1) the germline sequences encoding TCRs are biased toward reactivity to MHC molecules; and 2) CD4+ cells as opposed to CD8+ cells have distinct lineage commitment signals. These results also suggest that signals originating from Ly-6 can promote or substitute for signals generated from TCR that are required for positive selection. Moreover, this animal model offers a system to study T cell development in the thymus that can provide insights into mechanisms of lineage commitment in developing T cells.     

The murine nonclassical class I major histocompatibility complex-like CD1.1 molecule protects target cells from lymphokine-activated killer cell cytolysis

Classical class I major histocompatibility complex (MHC) molecules, as well as the nonclassical class I histocompatibility leukocyte antigen (HLA)-E molecule, can negatively regulate natural killer (NK) cell cytotoxicity through engagement of NK inhibitory receptors. We show that expression of murine (m)CD1.1, a nonpolymorphic nonclassical MHC class I-like molecule encoded outside the MHC, protects NK-sensitive RMA/S target cells from adherent lymphokine-activated killer cell (A-LAK) cytotoxicity. Passage of effector cells in recombinant interleukin (rIL)-2 enhanced protection by mCD1.1, suggesting an expansion of relevant A-LAK population(s) or modulation of A-LAK receptor expression. Murine CD1. 1 conferred protection from lysis by rIL-2-activated spleen cells of recombination activating gene (Rag)-1(-/-) mice, which lack B and T cells, demonstrating that mCD1.1 can protect RMA/S cells from lysis by NK cells. An antibody specific for mCD1.1 partially restored A-LAK lysis of RMA/S.CD1.1 transfectants, indicating that cell surface mCD1.1 can confer protection from lysis; therefore, mCD1.1 possibly acts through interaction with an NK inhibitory receptor. CD1.1 is by far the most divergent class I molecule capable of regulating NK cell activity. Finally, mCD1.1 expression rendered RMA/S cells resistant to lysis by A-LAK of multiple mouse strains. The conserved structure of mCD1.1 and pattern of mCD1.1 resistance from A-LAK lysis suggest that mCD1.1 may be a ligand for a conserved NK inhibitory receptor.     

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

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

External and internal calibration of the MHC class I-specific receptor Ly49A on murine natural killer cells

Expression of the H-2Dd-specific inhibitory receptor Ly49A on murine NK cells is subject to MHC class I-dependent modulation in vivo. As a result, NK cells in H-2Dd-transgenic mice express low cell surface levels of Ly49A, whereas NK cells from nontransgenic C57BL/6 (B6) mice express high levels. The purpose of this study was to assess the role of MHC class I molecules on the NK cell itself vs those on surrounding cells in this calibration and to test whether the Ly49A levels are subject to regulation in mature NK cells also. Analysis of transgenic mice with mosaic expression of an H-2Dd/Ld transgene showed that MHC class I molecules on surrounding cells (external ligands) and on the NK cell itself (internal ligands) played distinct roles in the determination of Ly49A levels. External ligands were involved in down-regulation of Ly49A levels in vivo, whereas internal ligands kept the down-regulated levels of Ly49A low upon NK cell activation in vitro. Furthermore, in an experimental system based on adoptive transfer of spleen cells, receptor down-regulation of Ly49A occurred as a rapid adaptation process in mature NK cells after interaction with the H-2Dd ligand in vivo. This suggests that Ly49 levels are not fixed but can be changed in mature NK cells when they are exposed to a changed MHC class I environment.     

Altered expression of Ly-49 receptors on NK cells developing in mixed allogeneic bone marrow chimeras

Ly-49 molecules are used by NK cells to distinguish 'self' from 'non-self', but the determinants of Ly-49 expression that allow this distinction to be made are not understood. The education of NK cells for self/non-self recognition was studied in murine mixed allogeneic bone marrow chimeras, in which NK cells are of both host and donor origin. Marked alterations in Ly-49 receptor expression were observed on both host and donor NK cells developing in BALB/c --> B6 mixed chimeras. Ly-49A and Ly-49G2 expression was lower on host B6 NK cells of mixed chimeras compared to non-transplanted B6 controls. Among donor BALB/c NK cells, Ly-49C expression levels were reduced, but the proportion of Ly-49C+ cells was increased, whereas Ly-49G2 expression was up-regulated compared to non-transplanted BALB/c controls. Thus, Ly-49 expression on donor and host NK cells developing post-bone marrow transplantation evolves toward the expression pattern of the host and donor strains respectively, due to the presence of the allogeneic MHC. In vitro functional NK cell assays showed that donor NK cells in mixed chimeras were not tolerant to host antigens and that host NK cells were not tolerant to the donor. Our data are consistent with a model in which MHC expression in the environment has a dominant down-regulating effect on the expression of Ly-49 molecules that recognize those MHC molecules, regardless of whether they are self or allogeneic. This down-regulation, combined with the limited repertoire of Ly-49 molecules, may not be sufficient to allow NK cells to be tolerant of MHC antigens of a fully MHC-mismatched allogenic strain.     

Overexpression of natural killer T cells protects Valpha14- Jalpha281 transgenic nonobese diabetic mice against diabetes

Progression to destructive insulitis in nonobese diabetic (NOD) mice is linked to the failure of regulatory cells, possibly involving T helper type 2 (Th2) cells. Natural killer (NK) T cells might be involved in diabetes, given their deficiency in NOD mice and the prevention of diabetes by adoptive transfer of alpha/beta double-negative thymocytes. Here, we evaluated the role of NK T cells in diabetes by using transgenic NOD mice expressing the T cell antigen receptor (TCR) alpha chain Valpha14-Jalpha281 characteristic of NK T cells. Precise identification of NK1.1(+) T cells was based on out-cross with congenic NK1.1 NOD mice. All six transgenic lines showed, to various degrees, elevated numbers of NK1.1(+) T cells, enhanced production of interleukin (IL)-4, and increased levels of serum immunoglobulin E. Only the transgenic lines with the largest numbers of NK T cells and the most vigorous burst of IL-4 production were protected from diabetes. Transfer and cotransfer experiments with transgenic splenocytes demonstrated that Valpha14-Jalpha281 transgenic NOD mice, although protected from overt diabetes, developed a diabetogenic T cell repertoire, and that NK T cells actively inhibited the pathogenic action of T cells. These results indicate that the number of NK T cells strongly influences the development of diabetes.     

Alpha1/alpha2 domains of H-2D(d), but not H-2L(d), induce "missing self" reactivity in vivo--no effect of H-2L(d) on protection against NK cells expressing the inhibitory receptor Ly49G2

Introduction of the MHC class I transgene H-2Dd 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-2Dd/Ld MHC class I transgenes, and show that the alpha1/alpha2 domains of H-2Dd were necessary and sufficient to induce "missing self" recognition and to down-modulate Ly49A and Ly49G2 receptors. In contrast, transgenes containing the alpha1/alpha2 domains of H-2Ld 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 alpha1/alpha2 domains of H-2Ld on NK cell specificity was surprising, considering that both H-2Ld and H-2Dd have been reported to interact with Ly49G2. Therefore, the role of H-2Ld for protection against NK cells expressing Ly49G2 was re-investigated in a transfection system. In contradiction to earlier reports, we show that H-2Dd, but not H-2Ld, abolished killing by sorted Ly49G2+ NK cells, indicating that H-2Ld does not inhibit NK cells via the Ly49G2 receptor.     

The basis for self-tolerance of natural killer cells in beta2-microglobulin- and TAP-1- mice

Cells from mice with mutations in the genes for beta2-microglobulin (beta2m) or for TAP-1 express only low levels of MHC class I proteins on their surfaces, and are thus sensitive to attack by normal NK cells. Although NK cells are present in beta2m- mice and TAP-1(-) mice, they are completely self-tolerant. The underlying mechanism for this tolerance is unknown. It has been proposed that education processes render NK cells from these mice hypersensitive to class I-mediated inhibition, so that they can be inhibited even by the low levels of class I expressed on autologous cells. In this study, we present evidence against this hypothesis, by demonstrating that NK cells from beta2m- mice and TAP-1(-) mice fail to attack beta2m(-)TAP-1(-) double-mutant cells in both in vitro and in vivo assays. The latter cells express substantially lower levels of class I than single-mutant cells, based on serologic tests, as well as a significantly diminished sensitivity to attack by class I-specific CTL. Furthermore, the Ly-49 repertoire on NK cells derived from beta2m(-)TAP-1(-) mice is highly similar to that of either single mutant, indicating that the developmental processes that shape the Ly-49 repertoire cannot respond to the differences in class I levels among these mice. We propose that self-tolerance of NK cells in beta2m- mice and TAP-1(-) mice is likely to result from hyporesponsiveness of the cells to activating signals, or alternatively, to induction of inhibitory signaling through receptors specific for non-class I MHC ligands.     

Interferon gamma production by natural killer (NK) cells and NK1.1+ T cells upon NKR-P1 cross-linking

Natural killer (NK) cells play an important role in immune response by producing interferon gamma (IFN-gamma) as well as exhibiting cytotoxic function. IFN-gamma produced by NK cells has been suggested to be involved in differentiation of T helper cells. On the other hand, the NKR-P1 molecule was recently identified as one of the important NK cell receptors, and it recognizes certain kinds of oligosaccharides on target cells and triggers NK cells for cytotoxicity. In the present study, we found that NK cells produce great amounts of IFN-gamma upon cross-linking of the NKR-P1 molecule. In contrast, stimulation of NK cells with IL-2 induced proliferation without producing IFN-gamma. Similar to NK cells, NK1.1+ T cells also produced IFN-gamma upon NKR-P1 cross-linking. NK1.1+ T cells produced IFN-gamma but not interleukin 4 (IL-4) upon NKR-P1 cross-linking, whereas they secreted both IFN-gamma and IL-4 upon T cell receptor cross-linking. These results indicate that NKR-P1 is a receptor molecule on NK and NK1.1+ T cells that induces not only cytotoxicity but also IFN-gamma production. Our findings provide a new pathway for IFN-gamma production by NK and NK1.1+ T cells through NKR-P1 molecules; it may be essential for immune regulation.     

Characterization of an associated 16-kDa tyrosine phosphoprotein required for Ly-49D signal transduction

Ly-49D is an activating receptor on NK cells that does not become tyrosine phosphorylated upon activation. This report demonstrates that immunoprecipitation of Ly-49D, following pervanadate treatment or specific Ab cross-linking, coprecipitates a 16-kDa tyrosine-phosphorylated protein (pp16). Immunoblotting experiments and data from TCR-zeta/Fc epsilonRIgamma double knockout mice confirm that pp16 is not TCR-zeta, TCR-eta, or Fc epsilonRIgamma. Association of pp16 with Ly-49D involves a transmembrane arginine since mutation to leucine (Ly-49D[R54L]) abolishes association with pp16 in transfected P815 cells. In addition, Ly-49D(R54L) transfectants fail to mediate Ca2+ mobilization following Ab cross-linking. Therefore, signaling through Ly49D on NK cells depends on association with a distinct tyrosine phosphoprotein (pp16) in a manner analogous to that of TCR and FcR. Expression of this novel signaling peptide in both the NK and myeloid lineages indicates that pp16 is likely involved in the signal transduction cascade of additional receptor families.     

Beta2-microglobulin-deficient NK cells show increased sensitivity to MHC class I-mediated inhibition, but self tolerance does not depend upon target cell expression of H-2Kb and Db heavy chains

Mice lacking beta2-microglobulin (beta2m- mice) express greatly reduced levels of MHC class I molecules, and cells from beta2m- mice are therefore highly sensitive to NK cells. However, NK cells from beta2m- mice fail to kill beta2m- normal cells, showing that they are self tolerant. In a first attempt to understand better the basis of this tolerance, we have analyzed more extensively the target cell specificity of beta2m- NK cells. In a comparison between several MHC class I-deficient and positive target cell pairs for sensitivity to beta2m- NK cells, we made the following observations: First, beta2m- NK cells displayed a close to normal ability to kill a panel of MHC class I-deficient tumor cells, despite their nonresponsiveness to beta2m- concanavalin A (Con A)-activated T cell blasts. Secondly, beta2m- NK cells were highly sensitive to MHC class I-mediated inhibition, in fact more so than beta2m+ NK cells. Thirdly beta2m- NK cells were not only tolerant to beta2m- Con A blasts but also to Con A blasts from H-2Kb-/Db- double deficient mice in vitro. We conclude that NK cell tolerance against MHC class I-deficient targets is restricted to nontransformed cells and independent of target cell expression of MHC class I free heavy chains. The enhanced ability of beta2m- NK cells to distinguish between MHC class I-negative and -positive target cells may be explained by increased expression of Ly49 receptors, as described previously. However, the mechanisms for enhanced inhibition by MHC class I molecules appear to be unrelated to self tolerance in beta2m- mice, which may instead operate through mechanisms involving triggering pathways.