NKG2D-ligands: Putting everything under the same umbrella can be misleading.

NKG2D is a key receptor for the activation of immune effector cells, mainly Natural Killer cells and T lymphocytes, in infection, cancer and autoimmune diseases. Since the detection of ligands for NKG2D in sera of cancer patients is, in many human models, indicative of prognosis, a large number of studies have been undertaken to improve understanding of the biology regulating this receptor and its ligands, with the aim of translating this knowledge into clinical practice. Although it is becoming clear that the NKG2D system can be used as a tool for diagnosis and manipulated for therapy, some questions remain open due to the complexity associated with the existence of a large number of ligands, each one of them displaying distinct biological properties. In this review, we have highlighted some key aspects of this system that differ between humans and mice, including the properties of NKG2D, as well as the genetic and biochemical complexity of NKG2D-ligands. All of these features affect the characteristics of the immune response exerted by NKG2D-expressing cells and are likely to be important factors in the clearance of a tumour or the development of autoimmunity. Implementation of more global analyses, including information on genotype, transcription and protein properties (cellular vs released to the blood stream) of NKG2D-ligands expressed in patients will be necessary to fully understand the links between this system and disease progression.

The role of zinc in the binding of killer cell Ig-like receptors to class I MHC proteins.

The binding of killer cell Ig-like Receptors (KIR) to their Class I MHC ligands was shown previously to be characterized by extremely rapid association and dissociation rate constants. During experiments to investigate the biochemistry of receptor-ligand binding in more detail, the kinetic parameters of the interaction were observed to alter dramatically in the presence of Zn(2+) but not other divalent cations. The basis of this phenomenon is Zn(2+)-induced multimerization of the KIR molecules as demonstrated by BIAcore, analytical ultracentrifugation, and chemical cross-linking experiments. Zn(2+)-dependent multimerization of KIR may be critical for formation of the clusters of KIR and HLA-C molecules, the "natural killer (NK) cell immune synapse," observed at the site of contact between the NK cell and target cell.

Interaction between the human NK receptors and their ligands.

NK cells are physiologically important in a number of contexts: mediating immunity against viruses, intracellular bacteria and parasites, and in anti-tumour immune responses. Moreover, in addition to these overtly immune protective functions, NK cells also mediate a variety of homeostatic functions, particularly in the regulation of haematopoesis and they may have an important role to play in the maintenance and development of placentation; certainly NK cells are a major component of the lymphocyte population of the decidua. The behaviour of the NK cell in these various situations is regulated by a large number of distinct receptors that transmit positive and negative signals. The balance of these signals determines whether the NK cell does nothing or is activated to proliferate, kill or secrete a wide range of cytokines and chemokines. In this review the structure and function of a number of molecules found on the NK cell surface are discussed, particular emphasis being placed on the molecular details of the recognition of target cell classical class I HLA molecules by Killer cell Immunoglobulin-like Receptors (KIR) and the binding of the non-classical class I molecule HLA-E to the heterodimer formed by the association of CD94 with various members of the NKG2 proteins.

Definition of polymorphic residues on killer Ig-like receptor proteins which contribute to the HLA-C binding site.

Killer cell immunoglobulin-like receptors (KIR) bind HLA class I proteins in an allele- and locus-specific manner. This report describes the use of transfectants expressing recombinant chimeric proteins, comprising the extracellular portions of KIR molecules and the transmembrane and cytoplasmic tails of CD3-zeta, to create an in vitro system in which signaling is readily measured and that preserves the specificity of the KIR / HLA-C interaction. The identity of the amino acid residues on the KIR molecule important for binding to the HLA protein is not well understood; although some KIR2D residues involved in HLA-C recognition have been identified, their relative importance and whether other amino acids contribute to binding was unclear. This novel system was used to study, by site-directed mutagenesis, the role of various amino acids in KIR binding to HLA-C ligand. The data presented here show that while multiple polymorphic residues contribute to the HLA-C binding site on KIR proteins, two clusters of polymorphic residues define the group allotype specificity of HLA-C binding to a KIR2D molecule.

Molecular analyses of the interactions between human NK receptors and their HLA ligands.

NK cell cytotoxicity is regulated by the action of multiple families of receptors. The interactions of these receptors with their ligands control different activating/inhibiting signal pathways and it is the balance of these signals which determines the behavior of the NK cell. The major described inhibitory pathways begin either with the recognition of a target cell classical class I HLA molecule by a killer cell immunologlobulin-like receptor (KIR) or the binding of the non-classical class I molecule HLA-E to the CD94/NKG2-A heterodimer. Activating counterparts to these inhibitory NK receptors have also been described and this review focuses on the molecular details of the binding of the inhibitory and activating receptors to their HLA ligands.

Kinetics and peptide dependency of the binding of the inhibitory NK receptor CD94/NKG2-A and the activating receptor CD94/NKG2-C to HLA-E.

The lytic function of human natural killer (NK) cells is markedly influenced by recognition of class I major histocompatibility complex (MHC) molecules, a process mediated by several types of activating and inhibitory receptors expressed on the NK cell. One of the most important of these mechanisms of regulation is the recognition of the non-classical class I MHC molecule HLA-E, in complex with nonamer peptides derived from the signal sequences of certain class I MHC molecules, by heterodimers of the C-type lectin-like proteins CD94 and NKG2. Using soluble, recombinant HLA-E molecules assembled with peptides derived from different leader sequences and soluble CD94/NKG2-A and CD94/NKG2-C proteins, the binding of these receptor-ligand pairs has been analysed. We show first that these interactions have very fast association and dissociation rate constants, secondly, that the inhibitory CD94/NKG2-A receptor has a higher binding affinity for HLA-E than the activating CD94/NKG2-C receptor and, finally, that recognition of HLA-E by both CD94/NKG2-A and CD94/NKG2-C is peptide dependent. There appears to be a strong, direct correlation between the binding affinity of the peptide-HLA-E complexes for the CD94/NKG2 receptors and the triggering of a response by the NK cell. These data may help to understand the balance of signals that control cytotoxicity by NK cells.

Differential binding to HLA-C of p50-activating and p58-inhibitory natural killer cell receptors.

Natural killer (NK) cell cytotoxicity is regulated in large part by the expression of NK cell receptors able to bind class I major histocompatibility complex glycoproteins. The receptors associated with recognition of HLA-C allospecificities are the two-domain Ig-like molecules, p50 and p58 proteins, with highly homologous extracellular domains but differing in that they have either an activating or inhibitory function, respectively, depending on the transmembrane domain and cytoplasmic tails that they possess. We have compared the binding to HLA-Cw7 of an inhibitory p58 molecule, NKAT2, the highly homologous activating p50 molecule, clone 49, and a second activating p50 molecule, clone 39, which has homologies to both NKAT1 and NKAT2. NKAT2 binds to HLA-Cw7 with very rapid association and dissociation rates. However, the p50 receptors bind only very weakly, if at all, to HLA-C. The molecular basis of this difference is analyzed, and the functional significance of these observations is discussed.

Kinetics of interaction of HLA-C ligands with natural killer cell inhibitory receptors.

The recognition of HLA-C molecules by specific inhibitory receptors is a crucial step in the regulation of natural killer (NK) cell function. Using soluble, recombinant HLA-C molecules and NK inhibitory receptors (NKIR, members of the immunoglobulin superfamily), we show that HLA-C binds to NKIR molecules with extremely fast association and dissociation rates, among the fastest of the immune system interactions so far studied. These kinetics may be essential for the biological function of NK cells, i.e., to facilitate the rapid immunosurveillance of cells for absent or diminished expression of class I MHC proteins.

The class I MHC homologue of human cytomegalovirus inhibits attack by natural killer cells.

Recognition and destruction of virus-infected cells by class I major histocompatibility complex (MHC) restricted cytotoxic T lymphocytes (CTL) is a central part of the immune system's attempts to control and eliminate virus infection. It is therefore not surprising that many viruses have evolved strategies to interfere with the processing and presentation of peptide antigen on class I MHC molecules (reviewed in ref. 1). These mechanisms act to prevent or reduce expression of MHC molecules at the cell surface. However, many natural killer (NK) cells are able to recognize and destroy host cells that no longer express class I MHC molecules (the 'missing self' hypothesis). Thus, any virus-infected cell that has lost cell-surface expression of MHC class I to avoid CTL attack should become susceptible to NK-cell-mediated destruction. We describe here the first example, to our knowledge, of a viral strategy to evade immune surveillance by NK cells.

Self and viral peptides can initiate lysis by autologous natural killer cells.

Natural killer (NK) cells are inhibited by specific allotypes of class I major histocompatibility complex ligands recognized by polymorphic inhibitory receptors (e.g., NKIR1 and NKIR2). NK1- and NK2-specific clones recognize two groups of HLA-C allotypes that are distinguished by a dimorphism at residue 80 in the alpha1 helix (alphaLys-80 and alphaAsn-80, respectively). "Empty" HLA-Cw7 expressed in peptide transporter-deficient cells and HLA-Cw7 loaded with several peptides each functioned as inhibitory ligands for NK2 lines and clones. However, loading of HLA-Cw7 with two other peptides derived from glutamic acid decarboxylase or coxsackie virus (each of which has been associated with autoimmune diabetes mellitus) abrogated this inhibitory recognition. Both peptides contained Lys at P8 of the epitope. Substitution of P8 with Ala or two other basic amino acids, His and Arg, resulted in peptides that were inhibitory, as were peptides with P8 Val, Glu, or Asn. The manner in which a Lys at P8 might affect recognition is discussed, together with a hypothesis for a novel mechanism by which an autoimmune disease might be initiated.

The binding site of NK receptors on HLA-C molecules.

The protection of cells expressing class I HLA molecules from NK lysis is mediated by natural killer cell inhibitory receptors (NKIR). Using site-directed mutagenesis, residues on HLA-C that determine the locus specificity (alphaVal-76), allotype group specificity (a dimorphism alphaAsn-80/Lys-80), and affinity of NKIR binding (a second pair of dimorphisms, alphaAla-73, Asp-90 or alphaThr-73, Ala-90) have been identified. Thus the "footprint" of the NKIR on the alpha1 helix of the class I MHC molecule HLA-C and its associated beta strands are similar in position to the site occupied by superantigens on and behind the alpha1 helix of the class II MHC molecule HLA-DR1, but further toward its C-terminus. The intermediate affinity binding of NKIR to HLA-C, determined by alpha73 and alpha90, has an essential role in preventing cross-reactivity and ensuring the availability of NK cells for immunosurveillance; low affinity and high affinity mutants are both physiologically impaired.

Human NK cells: their ligands, receptors and functions.

The expression, or lack thereof, of class I MHC glycoproteins has a marked influence on natural killer cell function. Cells which do not express class I MHC molecules are susceptible to lysis by NK cells, and transfection of these targets with class I MHC genes can render these cells resistant to NK attack. This inhibition of NK-killing is mediated by a novel family of receptors expressed mainly on NK cells, but also found on some T-cells. The function of these class I MHC binding receptors when expressed on T-cells is discussed also and a novel co-stimulatory activity of NKAR described. Lastly, a novel mechanism by which human cytomegalovirus evades immune surveillance by NK cells is documented.

Multiple receptors for HLA-G on human natural killer cells.

HLA-G is the putative natural killer (NK) cell inhibitory ligand expressed on the extravillous cytotrophoblast of the human placenta. Killing of the class I negative human B cell line 721.221 by NK cells is inhibited by the expression of HLA-G. This inhibition is dependent on a high level of HLA-G expression. In the present study, the nature of the receptors that mediate the inhibition has been studied with 140 NK cell lines from two donors and 246 NK clones from 5 donors by blocking the inhibition using monoclonal antibodies against the known NK inhibitory receptors: CD158a, CD158b, and CD94. Both CD94 and the two CD158 proteins can function as receptors, although the former clearly predominates. In many cases, a combination of antibodies to these receptors is required to achieve maximal reversal of inhibition. Moreover, in at least one-third of the NK cells that are inhibited by HLA-G, these antibodies alone or in combination do not reverse inhibition, strongly suggesting the existence of a third major unidentified receptor for HLA-G.

Enhancement of class II-restricted T cell responses by costimulatory NK receptors for class I MHC proteins.

An important feature of the human immune system is the ability of T cells to respond to small quantities of antigen. Class II major histocompatibility complex (MHC)-restricted T cells that expressed a costimulatory natural killer (NK) cell receptor for class I MHC proteins were cloned. In the presence of low doses of superantigen, the proliferative response of these T cell clones was three- to ninefold greater when the T cells were costimulated by way of the NK receptor. Thus, the action of costimulatory NK receptors on T cells may play a significant role in initiating and sustaining immune responses.

Protection from natural killer cell-mediated lysis by HLA-G expression on target cells.

The outermost layer of the human placenta is devoid of classical class I human leukocyte antigens (HLA-A, HLA-B, and HLA-C) and class II proteins (HLA-DR, HLA-DQ, and HLA-DP). Although this prevents recognition by maternal T lymphocytes, the lack of class I molecules leaves these cells susceptible to attack by natural killer (NK) cells. However, trophoblast cells directly in contact with the maternal tissues express the class I molecule HLA-G, which may be involved in protecting the trophoblast from recognition by NK cells. Here evidence is provided that expression of HLA-G is sufficient to protect otherwise susceptible target cells from lysis by activated NK1 and NK2 cell lines and clones that are specific for distinct groups of HLA-C alleles. The receptors on NK cells that recognize HLA-G are also identified.

Protection from lysis by natural killer cells of group 1 and 2 specificity is mediated by residue 80 in human histocompatibility leukocyte antigen C alleles and also occurs with empty major histocompatibility complex molecules.

Recognition of major histocompatibility complex class I molecules by natural killer (NR) cells leads to inhibition of target cell lysis. Based on the capacity of different human histocompatibility leukocyte antigen (HLA)-C and HLA-B molecules to inhibit target cell lysis by NK lines and clones, three NK allospecificities have been defined: NK1 and NK2 cells are inhibited by different HLA-C allotypes and NK3 cells by some HLA-B allotypes. The NK1 and NK2 inhibitory ligands on target cells correspond to a dimorphism of HLA-C at residues 77 and 80 in the alpha 1 helix: Asn77-Lys80 in NK1 and Ser77-Asn80 in NK2 inhibitory ligands. It has been reported that protection from NK1 killers depended on the presence of the Lys residue at position 80, an upward pointing residue near the end of the alpha 1 helix (and not on Asn77), whereas inhibition of NK2 effector cells required Ser77, a residue deep in the F pocket and interacting with the peptide (and not Asn80). As part of ongoing experiments to investigate the structural requirements for NK cell inhibition by HLA-C locus alleles, we also examined the effects of mutations at residues 77 and 80 on the ability of HLA-C alleles to confer protection from NK lysis. We present data confirming that the NK1 specificity depended on Lys80 (and not on Asn77); however recognition of NK2 ligands by NK cells was also controlled by the amino acid at position 80 (Asn), and mutation of Ser77 had no effect. Furthermore, bound peptide was shown to be unnecessary for the inhibition of NK cell-mediated lysis since HLA-C molecules assembled in the absence of peptide in RMA-S cells at 26 degrees C were fully competent to inhibit NK cells specifically. The implications of these data for peptide-independent recognition of HLA-C by NK receptors are discussed.