Ly49 Natural Killer Cell Receptors Research Articles

Irradiation and bone marrow reconstitution affect the functional Ly49 natural killer cell repertoire in rats.

Total body irradiation (TBI) is part of the preconditioning regimen for allogeneic bone marrow transplantation (alloBMT) and the procedure is associated with treatment-related toxicity and delayed immune reconstitution. Natural killer (NK) cells develop and acquire functional competence in close interaction with stromal bone marrow cells that are considered relatively radioresistant compared to the hematopoietic compartment. We thus undertook a study to assess the effect of TBI on the reconstitution of class I MHC-specific Ly49 NK cell receptors in a rat model of alloBMT. In rats subjected to TBI alone or followed by MHC-matched BMT, the irradiation conditioning induced a skewing of the Ly49 repertoire. Specifically, the activating Ly49s3bright subset exhibited increased frequency and receptor density which correlated with augmented alloreactivity relative to untreated control rats. Our results highlight the plasticity of NK cells and indicate that ionizing radiation (IR) affects the stromal compartment and as a consequence the maturation and functional properties of bone marrow-derived NK cells. These changes lasted throughout the 6 months observation period, showing that irradiation induces long term effects on the generation of the NK cell receptor repertoire.

A Positive Cooperativity Binding Model between Ly49 Natural Killer Cell Receptors and the Viral Immunoevasin m157: KINETIC AND THERMODYNAMIC STUDIES

Natural killer (NK) cells discriminate between healthy and virally infected or transformed cells using diverse surface receptors that are both activating and inhibitory. Among them, the homodimeric Ly49 NK receptors, which can adopt two distinct conformations (backfolded and extended), are of particular importance for detecting cells infected with mouse cytomegalovirus (CMV) via recognition of the viral immunoevasin m157. The interaction of m157 with activating (Ly49H) and inhibitory (Ly49I) receptors governs the spread of mouse CMV. We carried out kinetic and thermodynamic experiments to elucidate the Ly49/m157 binding mechanism. Combining surface plasmon resonance, fluorescence anisotropy, and circular dichroism (CD), we determined that the best model to describe both the Ly49H/m157 and Ly49I/m157 interactions is a conformational selection mechanism where only the extended conformation of Ly49 (Ly49*) is able to bind the first m157 ligand followed by binding of the Ly49*/m157 complex to the second m157. The interaction is characterized by strong positive cooperativity such that the second m157 binds the Ly49 homodimer with a 1000-fold higher sequential constant than the first m157 (∼10(8) versus ∼10(5) M(-1)). Using far-UV CD, we obtained evidence for a conformational change in Ly49 upon binding m157 that could explain the positive cooperativity. The rate-limiting step of the overall mechanism is a conformational transition in Ly49 from its backfolded to extended form. The global thermodynamic parameters from the initial state (backfolded Ly49 and m157) to the final state (Ly49*/(m157)2) are characterized by an unfavorable enthalpy that is compensated by a favorable entropy, making the interaction spontaneous.

Distinct Conformations of Ly49 Natural Killer Cell Receptors Mediate MHC Class I Recognition in Trans and Cis

Certain cell-surface receptors engage ligands expressed on juxtaposed cells and ligands on the same cell. The structural basis for trans versus cis binding is not known. Here, we showed that Ly49 natural killer (NK) cell receptors bound two MHC class I (MHC-I) molecules in trans when the two ligand-binding domains were backfolded onto the long stalk region. In contrast, dissociation of the ligand-binding domains from the stalk and their reorientation relative to the NK cell membrane allowed monovalent binding of MHC-I in cis. The distinct conformations (backfolded and extended) define the structural basis for cis-trans binding by Ly49 receptors and explain the divergent functional consequences of cis versus trans interactions. Further analyses identified specific stalk segments that were not required for MHC-I binding in trans but were essential for inhibitory receptor function. These data identify multiple distinct roles of stalk regions for receptor function.

Molecular Architecture of the Major Histocompatibility Complex Class I-binding Site of Ly49 Natural Killer Cell Receptors

Natural killer (NK) cells play a vital role in the detection and destruction of virally infected and tumor cells during innate immune responses. The highly polymorphic Ly49 family of NK receptors regulates NK cell function by sensing major histocompatibility complex class I (MHC-I) molecules on target cells. Despite the determination of two Ly49-MHC-I complex structures, the molecular features of Ly49 receptors that confer specificity for particular MHC-I alleles have not been identified. To understand the functional architecture of Ly49-binding sites, we determined the crystal structures of Ly49C and Ly49G and completed refinement of the Ly49C-H-2K(b) complex. This information, combined with mutational analysis of Ly49A, permitted a structure-based classification of Ly49s that we used to dissect the binding site into three distinct regions, each having different roles in MHC recognition. One region, located at the center of the binding site, has a similar structure across the Ly49 family and mediates conserved interactions with MHC-I that contribute most to binding. However, the preference of individual Ly49s for particular MHC-I molecules is governed by two regions that flank the central region and are structurally more variable. One of the flanking regions divides Ly49s into those that recognize both H-2D and H-2K versus only H-2D ligands, whereas the other discriminates among H-2D or H-2K alleles. The modular design of Ly49-binding sites provides a framework for predicting the MHC-binding specificity of Ly49s that have not been characterized experimentally.

Structural elucidation of the m157 mouse cytomegalovirus ligand for Ly49 natural killer cell receptors.

Natural killer (NK) cells express activating and inhibitory receptors that, in concert, survey cells for proper expression of cell surface major histocompatibility complex (MHC) class I molecules. The mouse cytomegalovirus encodes an MHC-like protein, m157, which is the only known viral antigen to date capable of engaging both activating (Ly49H) and inhibitory (Ly49I) NK cell receptors. We have determined the 3D structure of m157 and studied its biochemical and cellular interactions with the Ly49H and Ly49I receptors. m157 has a characteristic MHC-fold, yet possesses several unique structural features not found in other MHC class I-like molecules. m157 does not bind peptides or other small ligands, nor does it associate with beta(2)-microglobulin. Instead, m157 engages in extensive intra- and intermolecular interactions within and between its domains to generate a compact minimal MHC-like molecule. m157's binding affinity for Ly49I (K(d) approximately 0.2 microM) is significantly higher than that of classical inhibitory Ly49-MHC interactions. Analysis of viral escape mutations on m157 that render it resistant to NK killing reveals that it is likely to be recognized by Ly49H in a binding mode that differs from Ly49/MHC-I. In addition, Ly49H+ NK cells can efficiently lyse RMA cells expressing m157, despite the presence of native MHC class I. Collectively, our results show that m157 represents a structurally divergent form of MHC class I-like proteins that directly engage Ly49 receptors with appreciable affinity in a noncanonical fashion.

Structural and functional aspects of the Ly49 natural killer cell receptors

Natural killer cells are part of the first line of innate immune defence against virus‐infected cells and cancer cells in the vertebrate immune system. They are called ‘natural’ killers because, unlike cytotoxic T cells, they do not require a previous challenge and preactivation to become active. The Ly49 NK receptors are type II transmembrane glycoproteins, structurally characterized as disulphide‐linked homodimers. They share extensive homology with C‐type lectins, and they are encoded by a multigene family that in mice maps on chromosome 6. A fine balance between inhibitory and activating signals regulates the function of NK cells. Inhibitory Ly49 molecules bind primarily MHC class I ligands, whereas the ligands for activating Ly49 molecules may include MHC class I, but also interestingly MHC class I‐like molecules expressed by viruses, as is the case for Ly49H, which binds the m157 gene product of murine cytomegalovirus. In this study, we review the function and X‐ray crystal structure of the Ly49 NK cell receptors hitherto determined (Ly49A, Ly49C and Ly49I), and the structural features of the Ly49/MHC class I interaction as revealed by the X‐ray crystal structures of Ly49A/H‐2Dd and the recently determined Ly49C/H‐2Kb.

Structural and functional aspects of the Ly49 natural killer cell receptors

Natural killer cells are part of the first line of innate immune defence against virus-infected cells and cancer cells in the vertebrate immune system. They are called 'natural' killers because, unlike cytotoxic T cells, they do not require a previous challenge and preactivation to become active. The Ly49 NK receptors are type II transmembrane glycoproteins, structurally characterized as disulphide-linked homodimers. They share extensive homology with C-type lectins, and they are encoded by a multigene family that in mice maps on chromosome 6. A fine balance between inhibitory and activating signals regulates the function of NK cells. Inhibitory Ly49 molecules bind primarily MHC class I ligands, whereas the ligands for activating Ly49 molecules may include MHC class I, but also interestingly MHC class I-like molecules expressed by viruses, as is the case for Ly49H, which binds the m157 gene product of murine cytomegalovirus. In this study, we review the function and X-ray crystal structure of the Ly49 NK cell receptors hitherto determined (Ly49A, Ly49C and Ly49I), and the structural features of the Ly49/MHC class I interaction as revealed by the X-ray crystal structures of Ly49A/H-2Dd and the recently determined Ly49C/H-2Kb.

Hybrid resistance by mouse NK cells in vitro.

AbstractThe study of hybrid resistance was greatly aided by the development of an in vitro assay by Chadwick and Miller (1). The assay allows one to test for the presence of negative or positive signaling in natural killer (NK) cells, and the ability of monoclonal antibodies (MAbs) or F(ab′)2 reagents to block those signals. There is already good evidence for negative signals between Ly49 NK cell receptors and their specific class I antigens, e.g., Ly49A and H2-Dd (2). Thus, the inability of H2b Ly49A purified NK cells to kill H2d target cells can be reversed by adding MAb reagents that bind to either Ly49A or H2-Dd. We and others have extended these early findings to the Ly49C and I subsets of NK cells, and have recently detected positive signaling from D8 (H2b, Dd transgene) to purified H2b Ly49D+ NK cells that can be blocked by antibodies or their F(ab′)2 fragments to Ly49D. The assay can also be used to study signaling properties of fetal and neonatal NK cells lacking Ly49 receptors, which suggest that inhibitory non-Ly49 NK receptors are expressed on such NK cells and that these receptors can receive negative signals from class I molecules (3,4). The stimulation of bone marrow NK precursor cells with various cytokines, including IL-15, leads to generation of lytic NK1.1+Ly49- cells whose function is similar to that of fetal NK cells, i.e., they can lyse YAC-1 tumor cells and class I deficient cells, but cannot lyse normal lymphoblasts and lack Ly49 receptors (5). These two types of in vitro systems allow the analysis of factors necessary for various stages in NK cell differentiation and positive and negative signaling to NK cells at different stages of maturation.KeywordsNatural KillerNatural Killer CellPhorbol Myristate AcetateNatural Killer Cell FunctionHybrid ResistanceThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

A new monoclonal antibody reactive with several Ly49 NK cell receptors mediates redirected lysis of target cells.

We produced a novel hamster monoclonal antibody (MAb), 14B11, that recognizes the majority of mouse natural-killer (NK) cells. Transfection studies demonstrated that 14B11 MAb binds a subset of Ly49 receptors, including three putative inhibitory receptors, Ly49F, I, and C. No binding to Ly49A, B, D, or G was detected. In addition, 14B11 was shown to bind the putative activating receptor Ly49H, which required co-transfection of the signaling molecule DAP12 for detectable cell surface expression. Thus, 14B11 is the first reported MAb to bind Ly49H and F. At the functional level, 14B11 MAb enhanced the lysis by IL-2 activated NK cells of an FcR+ target cell line (Daudi), but not an FcR- target cell (EL-4). Because F(ab')2 fragments of 14B11 failed to enhance lytic activity, the enhancement of lysis by intact antibody is apparently due to "redirected lysis," in which stimulatory receptors on the NK cell are bridged by antibody to Fc receptors on the target cell. Cell separation experiments demonstrated that the 14B11-dependent redirected lysis was markedly increased using NK cell populations that had been depleted of Ly49F,+ I,+ or C+ NK cells. Because such depletions are expected to enrich for Ly49H+ NK cells, these results suggest that the enhancement of lysis mediated by 14B11 MAb may be due to stimulation of the activating Ly49H receptor. In conjunction with other anti-Ly49 MAbs, the 14B11 MAb will be useful in further studies of Ly49 receptor function and specificity.