Inhibition of natural killer cell activation signals by killer cell immunoglobulin-like receptors (CD158).

The killer cell immunoglobulin-like receptor (KIR) family includes receptors that bind to HLA class I molecules on target cells and inhibit natural killer (NK)-cell cytotoxicity, and receptors such as KIR3DL7 with no known ligand and function. Inhibitory KIR recruit the tyrosine phosphatase SHP-1 to block signals transduced by any one of a number of activation receptors. Inhibition of overall protein tyrosine phosphorylation by SHP-1 during binding of KIR to MHC class I on target cells is selective, suggesting that a limited number of substrates are dephosphorylated by SHP-1. We have chosen to study KIR inhibition as it occurs during binding of KIR to MHC class I on target cells, despite the technical limitations inherent to studies of processes regulated by cell contact. KIR binding to MHC class I on target cells inhibits tyrosine phosphorylation of the activation receptor 2B4 (CD244) and disrupts adhesion of NK cells to target cells. Inhibition of proximal events in NK activation may increase the availability of NK cells by liberating them from non-productive interactions with resistant target cells. As the receptors and the signaling pathways that induce NK cytotoxicity are not fully characterized, elucidation of the inhibitory mechanism employed by KIR may provide insight into NK activation.

Natural killer cells and mast cells from gp49B null mutant mice are functional.

Immune responses are controlled by a combination of positive and negative cellular signals. Effector cells in the immune system express inhibitory receptors that serve to limit effector cell expansion and to protect the host from autoreactivity. gp49B is a receptor of unknown function that is expressed on activated mast cells and natural killer (NK) cells and whose cytoplasmic tail endows it with inhibitory potential. To gain insight into the function of gp49B in mice, we disrupted the gp49B gene by homologous recombination. gp49B(0) mice were born at expected ratios, were healthy and fertile, and displayed normal long-term survival rates. gp49B(0) mice showed no defect in NK or mast cell development. Furthermore, NK and mast cells from the gp49B(0) mice showed activation properties in vitro similar to those of cells isolated from wild-type mice. Therefore, gp49B is not critical for the development, expansion, and maturation of mast cells and NK cells in vivo. The healthy status of gp49B(0) mice makes them suitable for testing the role of gp49B in immune responses to infectious agents.

Enhanced insulin secretion and improved glucose tolerance in mice lacking CD26.

A subset of prolyl oligopeptidases, including dipeptidyl-peptidase IV (DPP IV or CD26, EC ), specifically cleave off N-terminal dipeptides from substrates having proline or alanine in amino acid position 2. This enzyme activity has been implicated in the regulation of the biological activity of multiple hormones and chemokines, including the insulinotropic peptides glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Targeted inactivation of the CD26 gene yielded healthy mice that have normal blood glucose levels in the fasted state, but reduced glycemic excursion after a glucose challenge. Levels of glucose-stimulated circulating insulin and the intact insulinotropic form of GLP-1 are increased in CD26(-/-) mice. A pharmacological inhibitor of DPP IV enzymatic activity improved glucose tolerance in wild-type, but not in CD26(-/-), mice. This inhibitor also improved glucose tolerance in GLP-1 receptor(-/-) mice, indicating that CD26 contributes to blood glucose regulation by controlling the activity of GLP-1 as well as additional substrates. These data reveal a critical role for CD26 in physiological glucose homeostasis, and establish it as a potential target for therapy in type II diabetes.

Direct binding and functional transfer of NK cell inhibitory receptors reveal novel patterns of HLA-C allotype recognition.

Cytotoxicity of human NK cells is under negative control of killer cell Ig-like receptors (KIR) specific for HLA class I. To determine the specificity of five KIR containing two Ig domains (KIR2D), direct binding of soluble recombinant KIR2D to a panel of HLA class I transfectants was assayed. One soluble KIR2D, derived from an inhibitory receptor with a long cytoplasmic tail (KIR2DL1), bound to HLA-C allotypes containing asparagine 77 and lysine 80 in the heavy chain, as expected, since these allotypes inhibit lysis by NK cells expressing KIR2DL1. Surprisingly, another KIR2D (KIR2DL2), which inhibits NK lysis of cells expressing HLA-C molecules with serine 77 and asparagine 80, bound to HLA-C allotypes carrying either amino acid motif. Expression of the KIR2DL receptors in NK cells using recombinant vaccinia viruses confirmed these patterns of recognition, and identified KIR2DL3 as another KIR reacting with both groups of HLA-C allotypes. Mutagenesis of amino acid 44 in KIR2DL1 and KIR2DL2 suggested this residue controls the affinity of KIR for the 77/80 motif of HLA-C molecules. Two other soluble KIR2D, derived from noninhibitory receptors with short cytoplasmic tails (KIR2DS), did not bind to any of the HLA class I allotypes tested. One of these receptors (KIR2DS2) is closely related in sequence to KIR2DL2. Substitution of tyrosine 45 with the phenylalanine conserved in other KIR was sufficient to permit specific binding of KIR2DS2 to HLA-C. These results show that KIR2DL receptors are specific for HLA-C, but that recognition of HLA-C allotypes appears more permissive than indicated by previous functional experiments.

Structure of the inhibitory receptor for human natural killer cells resembles haematopoietic receptors.

Abnormal cells deficient in class I major histocompatibility complex (MHC) expression are lysed by a class of lymphocytes called natural killer (NK) cells. This lysis provides a defence against pathogens and tumour cells that downregulate MHC expression to avoid an MHC-restricted, T-cell immune response. Normal cells escape lysis because their MHC molecules are recognized by NK-cell inhibitory receptors, which inhibit lysis. Several such inhibitory receptor families have been described in humans and mice. In the human killer-cell inhibitory receptor family, individual p58 members are specific for a subset of class I human leukocyte antigen (HLA)-C molecules. The human p58 natural killer-cell inhibitory receptor clone 42 recognizes HLA-Cw4, -Cw2 and -Cw6, but not HLA-Cw3, -Cw2, -Cw7 or -Cw8, which are recognized by p58 killer-cell inhibitor receptor clone 43. We have determined the X-ray structure of the p58 NK-cell inhibitory receptor clone 42 at 1.7-A resolution. The structure has tandem immunoglobulin-like domains positioned at an acute, 60-degree angle. Loops on the outside of the elbow between the domains form a binding site projected away from the NK-cell surface. The topology of the domains and their arrangement relative to each other reveal a relationship to the haematopoietic receptor family, with implications for the signalling mechanism in NK cells.

Negative signaling pathways of the killer cell inhibitory receptor and Fc gamma RIIb1 require distinct phosphatases.

Inhibition of natural killer (NK) cells by the killer cell inhibitory receptor (KIR) involves recruitment of the tyrosine phosphatase SHP-1 by KIR and is prevented by expression of a dominant negative SHP-1 mutant. Another inhibitory receptor, the low affinity Fc receptor for immunoglobulin G (IgG) (Fc gamma RIIb1), has been shown to bind SHP-1 when cocross-linked with the antigen receptor on B cells (BCR). However, coligation of Fc gamma RIIb1 with BCR and with Fc epsilon RI on mast cells leads to recruitment of the inositol 5' phosphatase SHIP and to inhibition of mast cells from SHP-1-deficient mice. In this study, we evaluated the ability of these two inhibitory receptors to block target cell lysis by NK cells, and the contribution of SHP-1 and SHIP to inhibition. Recombinant vaccinia viruses encoding chimeric receptors and dominant negative mutants of SHP-1 and SHIP were used for expression in mouse and human NK cells. When the KIR cytoplasmic tail was replaced by that of Fc gamma RIIb1, recognition of HLA class I on target cells by the extracellular domain resulted in inhibition. A dominant negative mutant of SHP-1 reverted the inhibition mediated by the KIR cytoplasmic tail but not that mediated by Fc gamma RIIb1. In contrast, a dominant negative mutant of SHIP reverted only the inhibition mediated by the Fc gamma RIIb1 tail, providing functional evidence that SHIP plays a role in the Fc gamma RIIb1-mediated negative signal. These data demonstrate that inhibition of NK cells by KIR involves primarily the tyrosine phosphatase SHP-1, whereas inhibition mediated by Fc gamma RIIb1 requires the inositol phosphatase SHIP.

Natural killer cell receptors.

The specificity in the recognition of hematopoietic target cells by natural killer cells is primarily provided by inhibitory receptors and several such receptors have been identified in the past year. Surprisingly, the recognition of MHC class I molecules by inhibitory receptors on human natural killer cells involves two different types of receptors, one with Ig domains (killer cell inhibitory receptor), and another with C-type lectin domains (CD94-NKG2). Mouse natural killer cells recognize MHC class I molecules through the C-type lectin Ly49 receptors but also express a receptor, of unknown ligand specificity, that is related to the killer cell inhibitory receptor.

Killer cell inhibitory receptors: diversity, specificity, and function.

NK cells selectively kill target cells that fail to express self-MHC class I molecules. This selective killing results from a balance between inhibitory NK receptors specific for MHC class I molecules and activating receptors that are still largely unknown. Isolation of molecular clones for the human killer cell inhibitory receptors (KIR) revealed that KIR consist of a family of molecules with Ig ectodomains and cytoplasmic tails of varying length. Soluble complexes of KIR and HLA-C molecules established that KIR recognizes and binds to its ligand as an autonomous receptor. A functional expression system in human NK clones demonstrated that a single KIR can provide both recognition of MHC class I and delivery of a dominant negative signal to the NK cell. Functional evidence has been obtained for a role of the tyrosine phosphatase SHP-1 in KIR-mediated inhibition. The presence of a conserved motif used to recruit and activate SHP-1 in the cytoplasmic tail of KIR and of the mouse Ly-49 inhibitory receptor (otherwise structurally unrelated to KIR) represents an interesting case of evolutionary convergence. Furthermore, the motif led to the identification of other receptors with inhibitory potential, including a type I Ig-like receptor shared by mouse mast cells and NK cells.

Binding of a soluble p70 killer cell inhibitory receptor to HLA-B*5101: requirement for all three p70 immunoglobulin domains.

Lysis of target cells by natural killer (NK) cells can be prevented by killer cell inhibitory receptors (KIR) specific for major histocompatibility complex class I molecules. Functional studies have identified two distinct p58 KIR, each reactive with a different group of HLA-C allotypes, and distinct p70 KIR specific for some HLA-B or HLA-A allotypes. The NK specificities for each group of HLA-C allotypes have been reproduced by direct binding of recombinant soluble p58 molecules. Here, we show that a soluble p70 KIR binds to HLA-B*5101, but not to HLA-A or HLA-C molecules. Truncated soluble forms of the HLA-B*5101-specific p70 KIR, including one with two immunoglobulin (Ig) domains reactive with a monoclonal antibody that blocks p70 KIR function, did not bind to HLA-B*5101, indicating that all three Ig domains are required for binding.

Type I transmembrane receptor with inhibitory function in mouse mast cells and NK cells.

The MHC class I-specific inhibitory receptors on human and mouse NK cells have surprisingly different structures. The mouse receptors (Ly-49) are type II transmembrane glycoproteins of the C-type lectin family, whereas the human receptors (killer cell inhibitory receptors (KIR)) belong to the Ig superfamily. This difference prompted a search for Ig-like inhibitory receptors in mice. Here we show that gp49, a mouse mast cell protein of unknown function but with sequence similarity to KIR, is expressed in NK cells. The gp49 cytoplasmic tail, containing a sequence related to an inhibitory motif shared by KIR and Ly-49, delivered a strong inhibitory signal in both human and mouse NK cells when substituted for a KIR cytoplasmic tail. These data show that Ig-like receptors with inhibitory properties exist in both species and that mouse mast and NK cells may recognize common inhibitory ligands.

A p70 killer cell inhibitory receptor specific for several HLA-B allotypes discriminates among peptides bound to HLA-B*2705.

Natural killer (NK) cells express a repertoire of killer cell inhibitory receptors (KIR) for major histocompatibility complex (MHC) class I molecules. KIR specificity for MHC class I can be broad, as in the case of a single p70 KIR that can recognize several HLA-B allotypes, including HLA-B*2705. On the other hand, recognition of MHC class I can also be highly specific, as in the case of NK clones that recognize HLA-B*2705 in a peptide-specific manner. Most NK cells express multiple KIR sequences. To determine whether the broad and specific types of HLA-B recognition by NK cells reflect the use of different receptors or a property of a single KIR we analyzed the recognition of HLA-B*2705 by the p70 KIR-11, known to recognize several HLA-B allotypes. Vaccinia virus-mediated expression of KIR-11 in NK clones resulted in inhibition by HLA-B*2705 molecules on wild type but not on target cells deficient in the transporter for antigen presentation (TAP). Two peptides (FRYNGLIHR and RRSKEITVR) loaded onto HLA-B*2705 molecules on TAP-deficient cells provided protection from lysis by NK cells expressing KIR-11 but three other B27-specific peptides did not. As the five peptides bound to HLA-B*2705 with similar stability, these data demonstrate that a single KIR specific for several HLA-B allotypes recognizes a subset of peptides bound to HLA-B*2705.

Direct binding of a soluble natural killer cell inhibitory receptor to a soluble human leukocyte antigen-Cw4 class I major histocompatibility complex molecule.

Natural killer (NK) cells expressing specific p58 NK receptors are inhibited from lysing target cells that express human leukocyte antigen (HLA)-C class I major histocompatibility complex molecules. To investigate the interaction between p58 NK receptors and HLA-Cw4, the extracellular domain of the p58 NK receptor specific for HLA-Cw4 was overexpressed in Escherichia coli and refolded from purified inclusion bodies. The refolded NK receptor is a monomer in solution. It interacts specifically with HLA-Cw4, blocking the binding of a p58-Ig fusion protein to HLA-Cw4-expressing cells, but does not block the binding of a p58-Ig fusion protein specific for HLA-Cw3 to HLA-Cw3-expressing cells. The bacterially expressed extracellular domain of HLA-Cw4 heavy chain and beta2-microglobulin were refolded in the presence of a HLA-Cw4-specific peptide. Direct binding between the soluble p58 NK receptor and the soluble HLA-Cw4-peptide complex was observed by native gel electrophoresis. Titration binding assays show that soluble monomeric receptor forms a 1:1 complex with HLA-Cw4, independent of the presence of Zn2+. The formation of complexes between soluble, recombinant molecules indicates that HLA-Cw4 is sufficient for specific ligation by the NK receptor and that neither glycoprotein requires carbohydrate for the interaction.

Recruitment of tyrosine phosphatase HCP by the killer cell inhibitor receptor.

Cytolysis of target cells by natural killer (NK) cells and by some cytotoxic T cells occurs unless prevented by inhibitory receptors that recognize MHC class I on target cells. Human NK cells express a p58 inhibitory receptor specific for HLA-C. We report association of the tyrosine phosphatase HCP with the p58 receptor in NK cells. HCP association was dependent on tyrosine phosphorylation of p58. Phosphotyrosyl peptides corresponding to the p58 tail bound and activated HCP in vitro. Furthermore, introduction of an inactive mutant HCP into an NK cell line prevented the p58-mediated inhibition of target cell lysis. These data imply that the inhibitory function of p58 is dependent on its tyrosine phosphorylation and on recruitment and activation of HCP.

The Ig-related killer cell inhibitory receptor binds zinc and requires zinc for recognition of HLA-C on target cells.

Members of the Ig superfamily are predominantly receptors that mediate interactions between cells or provide signals to cells when binding specific ligands. Here we describe an Ig-related receptor that requires zinc for its function. Killer cell inhibitory receptors (KIR) belonging to the Ig superfamily mediate inhibition of NK cells upon recognition of HLA-C molecules on target cells. An abundance of histidine residues in the first extracellular domain of KIR, including the signature zinc binding motif HEXXH, suggested that this receptor may bind zinc. Two distinct KIR molecules that mediate recognition of HLA-Cw4 and -Cw8, respectively, bound specifically to zinc affinity columns. Furthermore, addition of the zinc chelator 1,10-phenanthroline during chromium release assays reversed the inhibition of killing by NK clones specific for HLA-Cw4 or HLA-Cw8, demonstrating that zinc is necessary for the inhibitory function of KIR. Such functionally relevant zinc binding has not been described for other members of the Ig superfamily and may represent a novel regulatory mechanism for Ag receptor-ligand interactions.

Molecular clones of the p58 NK cell receptor reveal immunoglobulin-related molecules with diversity in both the extra- and intracellular domains.

Recognition of major histocompatibility class I molecules on target cells by natural killer (NK) cells confers selective protection from NK-mediated lysis. Cross-linking of the p58 NK receptor, involved in the recognition of HLA-C alleles, delivers a negative signal that prevents target cell lysis. Molecular cloning of the p58 NK receptor reported here revealed a new member of the immunoglobulin superfamily. Five distinct p58 receptors, with sequence diversity in the immunoglobulin-related domains, were identified in a single individual. All NK clones tested expressed at least one p58 member. Three different types of transmembrane and cytoplasmic domains exist, even among receptors with closely related extracellular domains. These data revealed a repertoire of NK cells with clonally distributed p58 receptors exhibiting diversity in both extracellular and intracellular domains.