Mouse Natural Killer Research Articles

The Heterodimeric Assembly of the CD94-NKG2 Receptor Family and Implications for Human Leukocyte Antigen-E Recognition

The CD94-NKG2 receptor family that regulates NK and T cells is unique among the lectin-like receptors encoded within the natural killer cell complex. The function of the CD94-NKG2 receptors is dictated by the pairing of the invariant CD94 polypeptide with specific NKG2 isoforms to form a family of functionally distinct heterodimeric receptors. However, the structural basis for this selective pairing and how they interact with their ligand, HLA-E, is unknown. We describe the 2.5 A resolution crystal structure of CD94-NKG2A in which the mode of dimerization contrasts with that of other homodimeric NK receptors. Despite structural homology between the CD94 and NKG2A subunits, the dimer interface is asymmetric, thereby providing a structural basis for the preferred heterodimeric assembly. Structure-based sequence comparisons of other CD94-NKG2 family members, combined with extensive mutagenesis studies on HLA-E and CD94-NKG2A, allows a model of the interaction between CD94-NKG2A and HLA-E to be established, in which the invariant CD94 chain plays a more dominant role in interacting with HLA-E in comparison to the variable NKG2 chain.

RCAI-8, 9, 18, 19, and 49–52, conformationally restricted analogues of KRN7000 with an azetidine or a pyrrolidine ring: Their synthesis and bioactivity for mouse natural killer T cells to produce cytokines

Conformationally restricted analogues of KRN7000, an α- d-galactosyl ceramide, were synthesized to examine their bioactivity for mouse natural killer (NK) T cells to produce cytokines. RCAI-8, 9, 51, and 52 are the analogues with a pyrrolidine ring, and RCAI-18, 19, 49, and 50 are those with an azetidine ring. RCAI-18 was shown to be a potent inducer of cytokine production by mouse NKT cells, while RCAI-51 was a moderately active inducer.

Identification of bidirectional promoters in the human KIR genes

Although the class I MHC receptors expressed by human and mouse natural killer (NK) cells have distinct molecular origins, they are functional analogues that are expressed in a variegated pattern. The murine Ly49 class I receptors contain bidirectional promoters that have been proposed to control the probabilistic expression of these genes. Whether similar elements are present in the human killer Ig-like receptor (KIR) genes is a fundamental question. A detailed analysis of the 2 kb intergenic region separating the KIR2DL4 gene and the adjacent KIR3DL1 gene revealed that additional promoter elements exist in the human KIR genes. Remarkably, the previously characterized KIR3DL1 proximal promoter possesses bidirectional promoter activity that maps to an 88 bp DNA fragment containing CREB, AML, Sp1 and Ets transcription factor binding sites. Individual KIR genes and alleles possess bidirectional promoters with distinct properties. Analysis of KIR(+)and KIR(-) NK cells and NK precursors indicates that reverse transcripts from the bidirectional promoter are found in cells that lack KIR protein expression, but are not present in mature KIR-expressing NK cells, suggesting that reverse transcription from the proximal promoter blocks gene activation in immature NK and precursor cells.

226: Differential requirement for NKG2D in the rejection of haplomismatched and MHC-I deficient bone marrow

Mouse Natural Killer (NK) cells mediate rejection of MHC-mismatched or MHC-deficient bone marrow allografts. Counteracting activating and inhibitory signals regulate NK cell activity. The ability of F1 mice to reject parental bone marrow (BM) cells (hybrid resistance) has been attributed to the presence in the recipient of a subset of NK cells that lack inhibitory receptors for the MHC class I antigens expressed by the donor cells. Evidence supporting the “missing self” hypothesis of hybrid resistance was provided by demonstration that MHC class-I deficient donor BM is rejected by otherwise syngeneic recipients. We have previously demonstrated a role for NKG2D, an activating NK cell receptor, in rejection of Balb/c (H-2d) parental BM by (Balb/c x C57Bl/6; H-2b/d) F1 (CB6F1) recipient mice. NKG2D ligands are expressed on the regenerating myeloid BM cells in Balb/c and NOD mice but not substantially on the regenerating C57Bl/6 BM cells. Moreover, the rejection of Balb/c BM by CB6F1 recipients was blocked by a neutralizing anti-NKG2D monoclonal antibody. The purpose of this study was to determine whether NKG2D blockade was sufficient to prevent rejection of MHC-I deficient BM. Beta-2 microglobulin deficient (B2M-/-) Balb/c or NOD bone marrow was transplanted into irradiated CB6F1 mice that had been pretreated with neutralizing antibodies to NKG2D. In contrast to results with wild-type Balb/c or NOD BM whose rejection was prevented by anti-NKG2D, B2M-/- donor cells were rejected despite NKG2D blockade. Furthermore, syngeneic MHC class I+ Balb/c recipients were able to reject B2M-/- Balb/c donors despite NKG2D blockade. These results suggest that rejection of MHC class I-bearing BM requires activation via NKG2D, whereas MHC class I-deficient BM elicit a sufficiently strong NK response that augmentation by NKG2D signaling is not essential for the rejection. Therefore, the hybrid resistance model in which MHC class I-bearing BM are used for transplantation may better reflect the situation in human hematopoietic stem cell transplantation. Mouse Natural Killer (NK) cells mediate rejection of MHC-mismatched or MHC-deficient bone marrow allografts. Counteracting activating and inhibitory signals regulate NK cell activity. The ability of F1 mice to reject parental bone marrow (BM) cells (hybrid resistance) has been attributed to the presence in the recipient of a subset of NK cells that lack inhibitory receptors for the MHC class I antigens expressed by the donor cells. Evidence supporting the “missing self” hypothesis of hybrid resistance was provided by demonstration that MHC class-I deficient donor BM is rejected by otherwise syngeneic recipients. We have previously demonstrated a role for NKG2D, an activating NK cell receptor, in rejection of Balb/c (H-2d) parental BM by (Balb/c x C57Bl/6; H-2b/d) F1 (CB6F1) recipient mice. NKG2D ligands are expressed on the regenerating myeloid BM cells in Balb/c and NOD mice but not substantially on the regenerating C57Bl/6 BM cells. Moreover, the rejection of Balb/c BM by CB6F1 recipients was blocked by a neutralizing anti-NKG2D monoclonal antibody. The purpose of this study was to determine whether NKG2D blockade was sufficient to prevent rejection of MHC-I deficient BM. Beta-2 microglobulin deficient (B2M-/-) Balb/c or NOD bone marrow was transplanted into irradiated CB6F1 mice that had been pretreated with neutralizing antibodies to NKG2D. In contrast to results with wild-type Balb/c or NOD BM whose rejection was prevented by anti-NKG2D, B2M-/- donor cells were rejected despite NKG2D blockade. Furthermore, syngeneic MHC class I+ Balb/c recipients were able to reject B2M-/- Balb/c donors despite NKG2D blockade. These results suggest that rejection of MHC class I-bearing BM requires activation via NKG2D, whereas MHC class I-deficient BM elicit a sufficiently strong NK response that augmentation by NKG2D signaling is not essential for the rejection. Therefore, the hybrid resistance model in which MHC class I-bearing BM are used for transplantation may better reflect the situation in human hematopoietic stem cell transplantation.

Erratum: A thymic pathway of mouse natural killer cell development characterized by expression of GATA-3 and CD127

Nature Immunology 7, 1217–1224 (2006); corrected after print 3 November 2006 In the version of this article initially published, the symbols in the key of Figure 7a are incorrect. The filled ovals are Splenic; the filled diamonds are Thymic. The error has been corrected in the HTML and PDF versions of the article.

Functional subsets of mouse natural killer cells

Human natural killer (NK) cells can be divided into two phenotypically distinct functional subsets based on their cell surface expression of CD56 (CD56(bright) and CD56(dim)). As mouse NK cells do not express CD56, comparable mouse NK cell subsets have proven difficult to identify. Recently, we have found that mouse NK cells can be subdivided by the expression of CD27. The CD27(hi) and CD27(lo) mouse NK cell subsets show some intriguing similarities to but also some distinct differences from the human CD56 NK cell subsets in terms of their function and phenotype. Extending our knowledge of mature NK cell heterogeneity between the species will be critical to further our understanding of the pathological role of NK cells in immune responses.

IL-7 and the thymus dictate the NK cell 'labor market'

The identification of a functionally distinct thymus-dependent lineage of mouse natural killer cells demonstrates the diversity of the natural killer cell population.

The CCR4 as a novel-specific molecular target for immunotherapy in Hodgkin lymphoma

Here, we report that tumor cells from some patients (23.8%) with Hodgkin lymphoma (HL) are positive for CC chemokine receptor 4 (CCR4). We therefore tested the chimeric anti-CCR4 monoclonal antibody (mAb), KM2760, the Fc region of which is defucosylated to enhance antibody-dependent cellular cytotoxicity (ADCC), as a novel immunotherapy for refractory HL. KM2760 demonstrated a promising antitumor activity in the CCR4-positive HL-bearing mouse model in the therapeutic setting. Although KM2760 did not induce any ADCC mediated by mouse natural killer (NK) cells, it significantly enhanced phagocytosis mediated by mouse monocytes/macrophages against the CCR4-positive HL cell line in vitro. Together with the findings that KM2760 did not exhibit any complement-dependent cytotoxicity or direct antiproliferation activity in vitro, these data indicated that KM2760 exerted its robust in vivo antitumor activity via monocytes/macrophages in mice. In the human system, KM2760 enhanced phagocytic activity mediated by monocytes/macrophages. Furthermore, it induced robust ADCC mediated by NK cells against the CCR4-positive HL cell line in vitro. Thus, it is conceivable that KM2760 would have much more potent antitumor activity in humans than in mice. Collectively, this study strongly indicates that anti-CCR4 mAb could be a novel treatment modality for patients with CCR4-positive HL.

A thymic pathway of mouse natural killer cell development characterized by expression of GATA-3 and CD127

Natural killer (NK) cell development is thought to occur in the bone marrow. Here we identify the transcription factor GATA-3 and CD127 (IL-7R alpha) as molecular markers of a pathway of mouse NK cell development that originates in the thymus. Thymus-derived CD127+ NK cells repopulated peripheral lymphoid organs, and their homeostasis was strictly dependent on GATA-3 and interleukin 7. The CD127+ NK cells had a distinct phenotype (CD11b(lo) CD16- CD69(hi) Ly49(lo)) and unusual functional attributes, including reduced cytotoxicity but considerable cytokine production. Those characteristics are reminiscent of human CD56(hi) CD16- NK cells, which we found expressed CD127 and had more GATA-3 expression than human CD56+ CD16+ NK cells. We propose that bone marrow and thymic NK cell pathways generate distinct mouse NK cells with properties similar to those of the two human CD56 NK cell subsets.

NKT cells join the war on Lyme disease

Whether natural killer T cells recognize antigens derived from dangerous pathogens remains unclear. New data demonstrate that a glycolipid from Borrelia burgdorferi, the causative agent of Lyme disease, directly stimulates mouse and human natural killer T cells.

Modulation of acute graft-versus-host disease and chimerism after adoptive transfer of in vitro-expanded invariant Vα14 natural killer T cells

Mouse natural killer T cells with an invariant Vα14-Jα18 TCR rearrangement (Vα14i NKT cells) are able to regulate immune responses through rapid and large amounts of Th1 and Th2 cytokine production. It has been reported that in vivo administration of the Vα14i NKT cell ligand, α-galactosylceramide (α-GalCer) significantly reduced morbidity and mortality of acute graft-versus-host disease (GVHD) in mice. In this study, we examined whether adoptive transfer of in vitro-expanded Vα14i NKT cells using α-GalCer and IL-2 could modulate acute GVHD in the transplantation of spleen cells of C57BL/6 mice into (B6 × DBA/2) F 1 mice. We found that the adoptive transfer of cultured spleen cells with a combination of α-GalCer and IL-2, which contained many Vα14i NKT cells, modulated acute GVHD by exhibiting long-term mixed chimerism and reducing liver damage. Subsequently, the transfer of Vα14i NKT cells purified from spleen cells cultured with α-GalCer and IL-2 also inhibited acute GVHD. This inhibition of acute GVHD by Vα14i NKT cells was blocked by anti-IL-4 but not by anti-IFN-γ monoclonal antibody. Therefore, the inhibition was dependent on IL-4 production by Vα14i NKT cells. Our findings highlight the therapeutic potential of in vitro-expanded Vα14i NKT cells for the prevention of acute GVHD after allogeneic hematopoietic stem cell transplantation.

Anti-tumor and immunomodulating effects of Pleurotus ostreatus mycelia-derived proteoglycans

Pleurotus ostreatus is one of the widely cultivated edible mushrooms. Water-soluble proteoglycan fractions from P. ostreatus mycelia were purified by alcohol-precipitation, ion exchange and followed by gel permeation (Sephadex G-100) chromatography. Three neutral fractions were found, which had polysaccharide to protein ratios 14.2, 26.4 and 18.3, respectively. These fractions were tested for in vitro and in vivo immunomodulatory and anticancer effects on Sarcoma-180-bearing mouse model. In vivo injection of proteoglycans to Sarcoma-180-bearing mice decreased the number of tumor cells and cell cycle analysis showed that most of the cells were found to be arrested in pre-G 0/G 1 phase of cell cycle. All of the three proteoglycans elevated mouse natural killer (NK) cell cytotoxicity and stimulated macrophages to produce nitric oxide. The Fourier transform infra red (FTIR) spectra suggested the presence of beta-glycosidic bond in all the fractions. Fraction I strongly interacted with glucose/mannose-specific lectin Concanavalin A (ConA), indicating the presence of large number of terminal sugar with glucose/mannose. Thus, the three neutral proteoglycans derived from the mushroom ( P. ostreatus) mycelia could be used as immunomodulators and anti cancer agents.

Mechanisms imposing the Vβ bias of Vα14 natural killer T cells and consequences for microbial glycolipid recognition

Mouse and human natural killer T (NKT) cells recognize a restricted set of glycosphingolipids presented by CD1d molecules, including self iGb3 and microbial α-glycuronosylceramides. The importance of the canonical Vα14-Jα18 TCR α chain for antigen recognition by NKT cells is well recognized, but the mechanisms underlying the Vβ8, Vβ7, and Vβ2 bias in mouse have not been explored. To study the influences of thymic selection and the constraints of pairing with Vα14-Jα18, we have created a population of mature T cells expressing Vα14-Jα18 TCR α chain in CD1d-deficient mice and studied its recognition properties in vitro and in vivo. Transgenic cells expressed a diverse Vβ repertoire but their recognition of endogenous ligands and synthetic iGb3 was restricted to the same biased Vβ repertoire as expressed in natural NKT cells. In contrast, α-GalCer, a synthetic homologue of microbial α-glycuronosylceramides, was recognized by a broader set of Vβ chains, including the biased NKT set but also Vβ6, Vβ9, Vβ10, and Vβ14. These surprising findings demonstrate that, whereas Vβ8, Vβ7, and Vβ2 represent the optimal solution for recognition of endogenous ligand, many Vβ chains that are potentially useful for the recognition of foreign lipids fail to be selected in the NKT cell repertoire.

Alcohol Abuse Enhances Neuroinflammation and Impairs Immune Responses in an Animal Model of Human Immunodeficiency Virus-1 Encephalitis

Neuroinflammatory disorders (including human immunodeficiency virus-1 encephalitis, HIVE) are associated with oxidative stress and inflammatory brain injury, and excessive alcohol use can exacerbate tissue damage. Using a murine model of HIVE, we investigated the effects of alcohol abuse on the clearance of virus-infected macrophages and neuroinflammation. Severe combined immunodeficient mice were reconstituted with human lymphocytes, and encephalitis was induced by intracranial injection of HIV-1-infected monocyte-derived macrophages (HIV-1(+) MDM). Animals were fed an ethanol-containing diet beginning 2 weeks before lymphocyte engraftment and for the entire duration of the experiment. Lymphocyte engraftment was not altered by ethanol exposure. Alcohol-mediated immunosuppression in ethanol-fed mice was manifested by a significant decrease in CD8(+)/interferon-gamma(+) T lymphocytes, a fivefold increase in viremia, and diminished expression of immunoproteasomes in the spleen. Although both groups showed similar amounts of CD8(+) T-lymphocyte infiltration in brain areas containing HIV-1(+) MDMs, ethanol-fed mice featured double the amounts of HIV-1(+) MDMs in the brain compared to controls. Ethanol-exposed mice demonstrated higher microglial reaction and enhanced oxidative stress. Alcohol exposure impaired immune responses (increased viremia, decreased immunoproteasome levels, and prevented efficient elimination of HIV-1(+) MDMs) and enhanced neuroinflammation in HIVE mice. Thus, alcohol abuse could be a co-factor in progression of HIV-1 infection of the brain.

Identification and characterization of two related murine genes, Eat2a and Eat2b, encoding single SH2-domain adapters

Human EAT-2 (SH2D1B) and SLAM-associated protein (SAP) (SH2D1A) are single SH2-domain adapters, which bind to specific tyrosine residues in the cytoplasmic tail of six signaling lymphocytic activation molecule (SLAM) (SLAMF1)-related receptors. Here we report that, unlike in humans, the mouse and rat Eat2 genes are duplicated with an identical genomic organization. The coding regions of the mouse Eat2a and Eat2b genes share 91% identity at the nucleotide level and 84% at the protein level; similarly, segments of introns are highly conserved. Whereas expression of mouse Eat2a mRNA was detected in multiple tissues, Eat2b was only detectable in mouse natural killer cells, CD8+ T cells, and ovaries, suggesting a very restricted tissue expression of the latter. Both the EAT-2A and EAT-2B coimmunoprecipitated with mouse SLAM in transfected cells and augmented tyrosine phosphorylation of the cytoplasmic tail of SLAM. Both EAT-2A and EAT-2B bind to the Src-like kinases Fyn, Hck, Lyn, Lck, and Fgr, as determined by a yeast two-hybrid assay. However, unlike SAP, the EAT-2 proteins bind to their kinase domains and not to the SH3 domain of these kinases. Taken together, the data suggest that both EAT-2A and EAT-2B are adapters that recruit Src kinases to SLAM family receptors using a mechanism that is distinct from that of SAP.

Isolation and Characterization of a Novel Immunomodulatory α-Glucan−Protein Complex from the Mycelium ofTricholoma matsutakein Basidiomycetes

Tricholoma matsutake, a high-class edible mushroom in Japan, has been reported to have excellent biological activities, but difficulty in cultivating the fruit bodies and limited bulk availability have restricted detailed studies. We have developed a method of culturing in tanks, enabling the bulk supply of the mycelia. The preparation (CM6271) exerts modulative effects on the immune competence of mice and rats. In this study, a sodium hydroxide extract of CM6271 was defatted followed by fractionation with a combination of ion exchange chromatography and gel filtration in order to identify the components involved in the expression of the activity, and a single peak fraction (MPG-1) was obtained with reversed phase chromatography. MPG-1 was a glycoprotein (sugar:protein ratio, 94.3:5.7) with a relative molecular mass of 360 kDa, and the sugar moiety contained about 90% glucose. NMR spectra and methylation analysis revealed that the alpha-1,4-linkage was the predominant glucan linkage with alpha-1,6- and alpha-1,2-linkages in the minority. The amino acid composition in the protein moiety was rich in glutamine, alanine, asparagine, leucine, glycine, valine, serine, threonine, isoleucine, and proline. MPG-1 was resistant to degradation with amylase or protease. The oral administration of MPG-1 promoted, in a dose-dependent manner, the recovery of the mouse natural killer cell activity and serum IL-12 level that had been reduced by the loading of restraint stress. The dose of MPG-1 (25 mg/kg) required for the expression of the effect decreases to 1/12 of that of CM6271 (300 mg/kg). Furthermore, MPG-1 formed a complex with TGF-beta1 in vitro, modulating the biological activity of TGF-beta1 by binding to its active form. These results indicate that the mycelium of T. matsutake contains a novel alpha-glucan-protein complex with immunomodulatory activities.

Cytokine production and migration of in vitro-expanded NK1.1 − invariant Vα14 natural killer T (Vα14i NKT) cells using α-galactosylceramide and IL-2

Mouse natural killer T cells with invariant Vα14 rearrangement (Vα14i NKT cells) can rapidly produce both Th1 and Th2 cytokines and regulate various immune responses, such as autoimmunity and tumor immunity. In this study, we describe the phenotypical and functional characterization of in vitro-expanded mouse Vα14i NKT cells from spleen using a combination of α-galactosylceramide (α-GalCer) and IL-2. The expanded Vα14i NKT cells retained the memory/activated (CD44 +CD69 +CD62L −) and CD4 + or CD4 −8 − double negative phenotypes but modulated or lost the classical NKT cell marker, NK1.1. The expanded Vα14i NKT cells continuously released IL-4 and IFNγ and induced NK cell IFNγ production in vitro. Furthermore, the expanded Vα14i NKT cells migrated into the liver and spleen after adoptive transfer into lymphopenic SCID mice, and they were able to rapidly produce IL-4 and IFNγ after α-GalCer injection. Our findings suggest that the intrinsic characteristics of the cytokine secretion of Vα14i NKT cells were equivalent to that of in vitro-expanded Vα14i NKT cells. In vitro-expanded Vα14i NKT cells are considered to be useful for NKT cell defect-related diseases, such as autoimmunity and cancer.

Epitope mapping of Ly-49G and G-like receptors: CK-1 antibody defines a polymorphic site of functional interaction with class I ligand

Ly-49 receptors regulate mouse natural killer cell functions. Members of the polymorphic Ly-49 multigene family recognize specific alleles of major histocompatibility complex class I (MHC I) or MHC I-like proteins. Previous studies have provided insight into the nature of Ly-49A and -C interaction with their high-affinity MHC I ligands, H-2Dd and Kb, respectively. Unlike Ly-49C, recognition of MHC I by Ly-49A is regulated in part by residues within the beta4-beta5 loop of its ectodomain. Ly-49A and -G are within the same Ly-49 subfamily, and both receptors recognize Dd. However, there have been no studies that define specific sites on Ly-49G that mediate class I MHC recognition. The Ly-49G receptors of different inbred mouse strains can differ as a result of amino acid polymorphisms within their ectodomains. In this report, we have generated a novel antibody, CK-1, which recognizes Ly-49G(B6) and a Ly-49G(B6)-like receptor, Ly-49M(nonobese diabetic), but not Ly-49G(BALB/c). By exploiting the differences within ectodomains of C57BL/6 and BALB/c Ly-49G allele products, we identified epitopes recognized by the Ly-49G-specific antibodies CK-1 and Cwy-3, whose epitopes mapped within the beta4-beta5 loop and the beta1 strand, respectively, and were nonoverlapping. Although both antibodies specifically recognized the Ly-49G(B6) ectodomain, Cwy-3 was unable to block its interaction with MHC I, and CK-1 significantly inhibited it. The importance of residues within the beta4-beta5 loop in Ly-49G recognition demonstrates that its interaction with MHC I is similar to that of Ly-49A but not Ly-49C.

Bacterial glycolipids and analogs as antigens for CD1d-restricted NKT cells.

The CD1 family of proteins binds self and foreign glycolipids for presentation to CD1-restricted T cells. To identify previously uncharacterized active CD1 ligands, especially those of microbial origin, numerous glycolipids were synthesized and tested for their ability to stimulate mouse and human natural killer T (NKT) cells. They included analogs of the well known NKT cell agonist alpha-galactosyl ceramide (alpha-GalCer), bacterial glycolipids, and variations of the self-glycolipid, sulfatide. Bacterial glycolipids, alpha-galacturonosyl-ceramides from Sphingomonas wittichii, although structurally similar to alpha-GalCer, have significant differences in the sugar head group as well as the ceramide portion. The Sphingomonas glycosphingolipids (GSLs) and sulfatide variants were shown to activate human NKT cells as measured by IL-4 and IFN-gamma secretion. Moreover, CD1d-dimer staining revealed human NKT cell reactivity toward these GSLs and to the sulfatides in a fashion comparable with alpha-GalCer. Because alpha-GalCer is a marine-sponge-derived ligand, our study here shows that bacterium-derived antigens are also able to stimulate mouse and human NKT cells.

2B4 acts as a non-major histocompatibility complex binding inhibitory receptor on mouse natural killer cells.

Natural killer (NK) cells are critical in the immune response to tumor cells, virally infected cells, and bone marrow allografts. 2B4 (CD244) is expressed on all NK cells and the ligand for 2B4, CD48, is expressed on hematopoietic cells. Cross-linking 2B4 on NK cells with anti-2B4 monoclonal antibody leads to NK cell activation in vitro. Therefore, 2B4 is considered to be an activating receptor. Surprisingly, we have found, using antibody-blocking and 2B4-deficient NK cells, that NK lysis of CD48+ tumor and allogeneic targets is inhibited by 2B4 ligation. Interferon γ production by NK cells is also inhibited. Using a peritoneal tumor clearance assay, it was found that 2B4−/− mice have increased clearance of CD48+ tumor cells in vivo. Retroviral transduction of 2B4 was sufficient to restore inhibition in 2B4−/− primary NK cells. It was found that although mature NK cells express SH2D1A, in vitro–derived NK cells do not. However, both populations are inhibited by 2B4 ligation. This indicates that 2B4 inhibitory signaling occurs regardless of the presence of SH2D1A. These findings reveal a novel role for 2B4 as a non–major histocompatibility complex binding negative regulator of NK cells.