The NK cell MHC class I receptor Ly49A detects mutations on H-2Dd inside and outside of the peptide binding groove

The NK cell inhibitory receptor Ly49A recognizes the mouse MHC class I molecule H-2D(d) and participates in the recognition of missing self. Previous studies indicated that the determinant recognized by Ly49A exists in alpha1/alpha2 domain of H-2D(d). Here we have substituted polymorphic as well as conserved residues of H-2D(d) alpha1/alpha2 domain (when compared with H-2K(d), which does not interact with Ly49A). We then tested the ability of the H-2D(d) mutants to interact with Ly49A by soluble Ly49A tetramer binding and NK cell cytotoxicity inhibition assays. Individual introduction of mutations converting the H-2D(d) residue into the corresponding H-2K(d) residue (N30D, D77S, or A99F) in H-2D(d) partially abrogated the interaction between Ly49A and H-2D(d). Introduction of the three mutations into H-2D(d) completely abolished Ly49A recognition. Individual introduction of D29N or R35A mutation into the residues of H-2D(d) that are conserved among murine MHC class I severely impaired the interaction. The crystal structure of H-2D(d) reveals that D77 and A99 are located in the peptide binding groove and that N30, D29, and R35 are in the interface of the three structural domains of MHC class I: alpha1/alpha2, alpha3, and beta(2)-microglobulin. These data suggest that Ly49A can monitor mutations in MHC class I inside and outside of the peptide binding groove and imply that inhibitory MHC class I-specific receptors are sensitive to mutations in MHC class I as well as global loss of MHC class I. Our results also provide insight into the molecular basis of Ly49A to distinguish MHC class I polymorphism.     

Structural basis of the differential stability and receptor specificity of H-2Db in complex with murine versus human beta2-microglobulin

beta(2)-Microglobulin (beta(2)m) is non-covalently linked to the major histocompatibility complex (MHC) class I heavy chain and interacts with CD8 and Ly49 receptors. Murine MHC class I heavy chains can bind human beta(2)m (hbeta(2)m) and peptide, and such hybrid molecules are often used in structural and functional studies. The replacement of mouse beta(2)m (mbeta(2)m) with hbeta(2)m has several functional consequences for MHC class I complex stability and specificity, but the structural basis for this is presently unknown. To investigate the impact of species-specific beta(2)m subunits on MHC class I conformation, we provide a crystallographic comparison of H-2D(b) in complex with LCMV-derived gp33 peptide and either hbeta(2)m or mbeta(2)m. The conformation of the gp33 peptide is not affected by the beta(2)m species. Comparison of the interface between beta(2)m and the alpha(1)alpha(2) domains of the heavy chain in these two crystal structures reveals a marked increase in both polarity and number of hydrogen bonds between hbeta(2)m and the alpha(1)alpha(2) domains of H-2D(b). We propose that the positioning of two hydrogen bond rich regions at the hbeta(2)m/alpha(1)alpha(2) interface plays a central role in the increased overall stability and peptide exchange capacity in the H-2D(b)/hbeta(2)m complex. These two regions act as bridges, holding and stabilizing the underside of the alpha(1) and alpha(2) helices, enabling a prolonged peptide-receptive conformation of the peptide binding cleft. Furthermore, analysis of H-2D(b) in complex with either mbeta(2)m or hbeta(2)m provides a structural explanation for the differential binding of H-2D(b)/hbeta(2)m to both Ly49A and Ly49C. Our comparative structural study emphasizes the importance of beta(2)m residues at positions 3, 6 and 29 for binding to Ly49A and suggests that sterical hindrance by residue K6 on hbeta(2)m impairs the recognition of Ly49C by H-2D(b)/gp33/hbeta(2)m. Finally, comparison of the two H-2D(b) crystal structures implies that the beta(2)m species may affect the strength of TCR recognition by affecting CD8 binding.     

Binding of the natural killer cell inhibitory receptor Ly49A to its major histocompatibility complex class I ligand. Crucial contacts include both H-2Dd AND beta 2-microglobulin.

Ly49A, an inhibitory C-type lectin-like mouse natural killer cell receptor, functions through interaction with the major histocompatibility complex class I molecule, H-2D(d). The x-ray crystal structure of the Ly49A.H-2D(d) complex revealed that homodimeric Ly49A interacts at two distinct sites of H-2D(d): Site 1, spanning one side of the alpha1 and alpha2 helices, and Site 2, involving the alpha1, alpha2, alpha3, and beta(2)m domains. Mutants of Ly49A, H-2D(d), and beta(2)-microglobulin at intermolecular contacts and the Ly49A dimer interface were examined for binding affinity and kinetics. Although mutations at Site 1 had little affect, several at Site 2 and at the dimer interface hampered the Ly49A.H-2D(d) interaction, with no effect on gross structure or T cell receptor interaction. The region surrounding the most critical residues (in H-2D(d), Asp(122); in Ly49A, Asp(229), Ser(236), Thr(238), Arg(239), and Asp(241); and in beta(2)-microglobulin, Gln(29) and Lys(58)) of the Ly49A.H-2D(d) interface at Site 2 includes a network of water molecules, suggesting a molecular basis for allelic specificity in natural killer cell recognition.     

The MHC class I molecule H-2Dp inhibits murine NK cells via the inhibitory receptor Ly49A.

MHC class I molecules strongly influence the phenotype and function of mouse NK cells. NK cell-mediated lysis is prevented through the interaction of Ly49 receptors on the effector cell with appropriate MHC class I ligands on the target cell. In addition, host MHC class I molecules have been shown to modulate the in vivo expression of Ly49 receptors. We have previously reported that H-2Dd and H-2Dp MHC class I molecules are able to protect (at the target cell level) from NK cell-mediated lysis and alter the NK cell specificity (at the host level) in a similar manner, although the mechanism behind this was not clear. In this study, we demonstrate that the expression of both H-2Dd and H-2Dp class I molecules in target cells leads to inhibition of B6 (H-2b)-derived Ly49A+ NK cells. This inhibition could in both cases be reversed by anti-Ly49A Abs. Cellular conjugate assays showed that Ly49A-expressing cells indeed bind to cells expressing H-2Dp. The expression of Ly49A and Ly49G2 receptors on NK cells was down-regulated in H-2Dp-transgenic (B6DP) mice compared with nontransgenic B6 mice. However, B6DP mice expressed significantly higher levels of Ly49A compared with H-2Dd-transgenic (D8) mice. We propose that both H-2Dd and H-2Dp MHC class I molecules can act as ligands for Ly49A.     

Distinctive interactions at multiple site 2 subsites by allele-specific rat and mouse ly49 determine functional binding and class I MHC specificity

Rodent Ly49 exhibit allele-specific MHC I recognition, yet the interaction site, site 2, encompassing the area below the MHC peptide-binding groove, the alpha3 domain, and associated beta(2) microglobulin, is highly conserved among rat and mouse MHC I alleles. We previously demonstrated that allele-specific Ly49 recognition can be affected by polymorphisms specifically in the peptide anchor-binding and supertype-defining B pocket of MHC I, possibly through differential conformations assumed by solvent-exposed interaction residues when articulating with this pocket. Through mutagenesis of RT1-A1(c) and H-2D(d), we map for the first time the interaction site(s) on rat MHC I mediating rat Ly49i2 recognition and the previously unexamined Ly49G(BALB/c) interaction with H-2D(d). We demonstrate that rat Ly49i2 and mouse Ly49G use both unique and common interactions at three MHC I H chain subsites to mediate functional binding and allele-specific recognition. We find that the F subsite, formed by solvent-exposed residues below the more conserved C-terminal anchor residue-binding F pocket, acts as an anchoring location for both Ly49i2 and Ly49G, whereas these receptors exhibit distinctive reliance on solvent-exposed residues articulating with the polymorphic anchor-binding and supertype-defining pocket(s) at subsite B, as well as on interaction residues at subsite C in the MHC I alpha3 domain. Our findings, combined with previous Ly49A/H-2D(d) and Ly49C/H-2K(b) cocrystal data, suggest how allele-specific MHC I conformations and Ly49 polymorphisms may affect Ly49 placement on MHC I ligands and residue usage at site 2, thereby mediating allele-specific recognition at the highly conserved MHC I interface.     

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.     

MHC class I-Ly49 interactions shape the Ly49 repertoire on murine NK cells

This study aims to determine how the interaction of Ly49 receptors with MHC class I molecules shapes the development of the Ly49 repertoire. We have examined the percentage of NK cells that expressed Ly49A, Ly49G2, and Ly49D in single and double Ly49A/C-transgenic mice on four different MHC backgrounds, H-2(b), H-2(d), H-2(b/d), and beta(2)-microglobulin(-/-). The results show that the total numbers of NK cells were not different among the strains. The prior expression of a Ly49 receptor capable of binding to self MHC class I altered the percentage of NK cells expressing endogenous Ly49A, Ly49G2, and Ly49D even in mice in which no MHC ligand was present for the latter receptors. The NK cells in the Ly49-transgenic mice expressed the same level of endogenous Ly49 receptors as wild-type mice of a similar MHC background. In contrast, the number of NK T cells was reduced in mice in which the Ly49 transgene could bind to a MHC class I molecule. The onset of Ly49 receptor expression on NK cells during ontogeny was not altered in the presence of transgenic Ly49 receptors. These data support a sequential model and argue against a selection model for Ly49 repertoire development on NK cells.     

Mapping the ligand of the NK inhibitory receptor Ly49A on living cells

We have used a recombinant, biotinylated form of the mouse NK cell inhibitory receptor, Ly49A, to visualize the expression of MHC class I (MHC-I) ligands on living lymphoid cells. A panel of murine strains, including MHC congenic lines, was examined. We detected binding of Ly49A to cells expressing H-2D(d), H-2D(k), and H-2D(p) but not to those expressing other MHC molecules. Cells of the MHC-recombinant strain B10.PL (H-2(u)) not only bound Ly49A but also inhibited cytolysis by Ly49A(+) effector cells, consistent with the correlation of in vitro binding and NK cell function. Binding of Ly49A to H-2D(d)-bearing cells of different lymphoid tissues was proportional to the level of H-2D(d) expression and was not related to the lineage of the cells examined. These binding results, interpreted in the context of amino acid sequence comparisons and the recently determined three-dimensional structure of the Ly49A/H-2D(d) complex, suggest a role for amino acid residues at the amino-terminal end of the alpha1 helix of the MHC-I molecule for Ly49A interaction. This view is supported by a marked decrease in affinity of an H-2D(d) mutant, I52 M, for Ly49A. Thus, allelic variation of MHC-I molecules controls measurable affinity for the NK inhibitory receptor Ly49A and explains differences in functional recognition in different mouse strains.     

Critical residues at the Ly49 natural killer receptor's homodimer interface determine functional recognition of m157, a mouse cytomegalovirus MHC class I-like protein

NK cell function is regulated by Ly49 receptors in mice and killer cell Ig-like receptors in humans. Although inhibitory Ly49 and killer cell Ig-like receptors predominantly ligate classical MHC class I molecules, recent studies suggest that their activating counterparts recognize infection. The quintessential example is resistance to the mouse CMV in C57BL/6 mice, which depends on the functional recognition of m157, a mouse CMV-encoded MHC class I-like molecule, by Ly49H, an activating NK cell receptor. We have taken advantage of the natural variation in closely related members of the Ly49C-like receptors and the availability of Ly49 crystal structures to understand the molecular determinants of the Ly49H-m157 interaction and to identify amino acid residues discriminating between m157 binding and nonbinding receptors. Using a site-directed mutagenesis approach, we have targeted residues conserved in receptors binding to m157 (Ly49H and Ly49I(129)) but different from receptors lacking m157 recognition (Ly49C, Ly49I(B6), and Ly49U). Wild-type and mutant receptors were transfected into reporter cells, and physical binding as well as functional activation by m157 was studied. Our findings suggested that the Ly49 MHC class I contact "site 2," I226, may not be involved in m157 binding. In contrast, residue Y146 and G151, mapping at the receptor homodimer interface, are likely critical for functional recognition of the m157 glycoprotein. Our combined functional and three-dimensional modeling approach suggested that the architecture of the Ly49H dimer is crucial to accessing m157, but not MHC class I. These results link Ly49 homodimerization variability to the direct recognition of pathogen products.     

Altered donor and recipient Ly49+ NK cell subsets in allogeneic H-2d --> H-2b and H-2b --> H-2d bone marrow chimeras

NK cells reject non-self hematopoietic bone marrow (BM) grafts via Ly49 receptor-mediated MHC class I-specific recognition and calibration of receptor expression levels. In this paper we investigated how Ly49+ subset frequencies were regulated dependent on MHC class I expression. The development of donor and host Ly49A+ (recognizes H-2Dd and H-2Dk ligands) and Ly49C/I+ (Ly49CBALB/c recognizes H-2Kb, H-2Kd, and H-2Dd, and Ly49CB6 recognizes only H-2Kb) NK cell frequencies were monitored for 120 days in murine-mixed allogeneic BM chimeras. C57BL/6 (H-2b) BM was transplanted into BALB/c (H-2d) mice and vice versa. Peripheral NK cell populations were examined every 5 days. Chimerism was found to be stable with 80-90% donor NK cells. In contrast to syngeneic controls reexpressing pretransplant patterns, donor and host NK cells revealed new and mainly reduced subset frequencies 55 days after allogeneic transplantation. Recipient NK cells acquired these later than donor NK cells. In H-2d --> H-2b chimeras Ly49A+, Ly49C/I+, and Ly49A+/Ly49C/I+ proportions were mainly diminished upon interaction with cognate ligands. Also in H-2b --> H-2d chimeras, Ly49A+ and Ly49A+/Ly49C/I+ subsets were reduced, but there was a transient normalization of Ly49C/I+ proportions in the noncognate host. After 120 days all subsets were reduced. Therefore, down-regulation of developing Ly49A+ and Ly49C/I+ chimeric NK cell frequencies by cognate ligands within 7-8 wk after BM transplantation may be important for successful engraftment.     

NK cell inhibitory receptor Ly-49C residues involved in MHC class I binding.

Mouse NK cells express Ly-49 receptors specific for classical MHC class I molecules. Several of the Ly-49 receptors have been characterized in terms of function and ligand specificity. However, the only Ly-49 receptor-ligand interaction previously described in detail is that between Ly-49A and H-2D(d), as studied by point mutations in the ligand and the crystal structure of the co-complex of these molecules. It is not known whether other Ly-49 receptors bind MHC class I in a similar manner as Ly-49A. Here we have studied the effect of mutations in Ly-49C on binding to the MHC class I molecules H-2K(b), H-2D(b), and H-2D(d). The MHC class I molecules were used as soluble tetramers to stain transiently transfected 293T cells expressing the mutated Ly-49C receptors. Three of nine mutations in Ly-49C led to loss of MHC class I binding. The three Ly-49C mutations that affected MHC binding correspond to Ly-49A residues that are in contact or close to H-2D(d) in the co-crystal, demonstrating that MHC class I binding by Ly-49C is dependent on residues in the same area as that used by Ly-49A for ligand contacts.     

Recognition of class I MHC by NK receptor Ly-49C: identification of critical residues.

The Ly-49 family of inhibitory receptors plays a major role in regulating mouse NK cell cytotoxicity. Two of its members, Ly-49C and I, are recognized by the mAb 5E6, which also defines a subset of NK cells involved in the hybrid resistance phenomenon. Previous studies have shown that Ly-49C binds to a broad spectrum of class I MHC molecules, while Ly-49I apparently does not bind to any class I MHC molecules tested. In the present investigation we have defined the amino acid residues of Ly-49C that are critical for determining its ligand specificities. First, using quantitative COS cell adhesion assays, we demonstrated that Ly-49CB6 bound to Dd, Db, Kb, or Kk as well as to murine leukemic cell lines GM979 (H-2s) and IC-21 (H-2b). In contrast, COS cells expressing Ly-49IB6 did not significantly bind to any of the class I MHC tested. To determine which amino acid residues of Ly-49C are critical for their specific binding to class I MHC, a series of chimeric and mutant Ly-49C and I were generated and tested. Exchanging the critical residues between Ly-49C and I significantly affected their binding specificities. Finally, we identified the epitopes on Ly-49C recognized by mAbs 5E6 and 4LO3311 that functionally inhibit Ly-49C recognition of its ligands. These results further define the class I specificities of Ly-49C and provide insight into the structural basis for how class I MHC is recognized by the Ly-49 family of NK receptors.     

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.     

Broad recognition of cytotoxic T cell epitopes from the HIV-1 envelope protein with multiple class I histocompatibility molecules.

A few cases have been described of antigenic determinants that are broadly presented by multiple class II MHC molecules, especially murine I-E or human DR, in which polymorphism is limited to the beta chain, and the alpha chain is conserved. However, no similar cases have been studied for presentation by class I MHC molecules. Because both domains of the MHC peptide binding site are polymorphic in class I molecules, exploring permissiveness in class I presentation would be of interest, and also such broadly presented antigenic determinants would clearly be useful for vaccine development. We had defined an immunodominant determinant, P18, of the HIV-1 gp160 envelope protein recognized by human and murine CTL. To determine the range of class I MHC molecules that could present this peptide and to determine whether two HIV-1 gp160 Th cell determinants, T1 and HP53, could also be presented by class I MHC molecules, we attempted to generate CTL specific for these three peptides in 10 strains of B10 congenic mice, representing 10 MHC types, and BALB/c mice. P18 was presented by at least four different class I MHC molecules from independent haplotypes (H-2d, p, u, and q to CD8+ CTL. In H-2d and H-2q the presentation was mapped to the D-end class I molecule, and for Dd, a requirement for both the alpha 1 and alpha 2 domains of Dd, not Ld, was found. HP53 was also presented by the same four different class I MHC molecules to CD8+ CTL although at higher concentrations. T1 was presented by class I molecules in three different strains of distinct MHC types (B10.M, H-2f; B10.A, H-2a; and B10, H-2b) to CTL. The CTL specific for P18 and HP53 were shown to be CD8+ and CD4- and to kill targets expressing endogenously synthesized whole gp160 as well as targets pulsed with the corresponding peptide. To compare the site within each peptide presented by the different class I molecules, we used overlapping and substituted peptides and found that the critical regions of each peptide are the similar for all four MHC molecules. Thus, antigenic sites are broadly or permissively presented by class I MHC molecules even without a nonpolymorphic domain as found in DR and I-E, and these sequences may be of broad usefulness in a synthetic vaccine.     

Cell surface expression of the amino-terminal domain of A kappa alpha. Recognition of an isolated MHC antigenic structure by allospecific T cells but not alloantibodies

To evaluate the relationship between structure and function of the alpha 1 and beta 1 domains of class II MHC molecules, a strategy has been developed whereby isolated alpha 1 or beta 1 domains can be expressed on the surface of transfected mouse L cells as a part of a hybrid class II/class I molecule. The construction and expression of a chimeric class II/class I gene encoding the first two exons of A kappa alpha linked to the C2, TM and cytoplasmic exons of H-2Dd (A kappa alpha 1/DdC2) are described. High levels of A kappa alpha 1/DdC2 protein were detected on transfected L cells with a DdC2-specific mAb. An anti-Ia xenoantiserum specifically bound to A kappa alpha 1/DdC2-transfected L cells, although 12 different mAb reactive against I-A kappa alpha and an anti-Ia kappa alloantiserum did not bind these cells. However, alloreactive Ia kappa-specific CTL lines were able specifically to lyse cells expressing the hybrid A kappa alpha 1/DdC2 molecule. This indicates that the isolated A kappa alpha 1 domain preserves some of the structure of the native molecule and demonstrates the recognition by T cells of domain-specific A alpha allodeterminants.     

Impaired anti-viral T cell responses due to expression of the Ly49A inhibitory receptor.

Inhibitory receptors specific for alleles of MHC class I proteins play an important role in determining the reactivity and specificity of NK cells. To determine whether these receptors are also able to regulate T cell functions, we have studied anti-viral immune responses in mice transgenic for a class I-specific inhibitory receptor, Ly49A. Although nontransgenic mice express Ly49A primarily on NK cells and some T cells, the Ly49A transgenic mice express Ly49A on all lymphocytes, including T cells. We have assessed the activation, expansion, cytokine production, and cytotoxic activity of CD8 T cells in both transgenic and nontransgenic mice following infection with lymphocytic choriomeningitis virus. As expected, nontransgenic mice made a potent virus-specific CD8 T cell response following virus infection. However, as measured in cytolysis assays and by cytokine production, virus-specific CD8 T cell activity was reduced in Ly49A transgenic mice. This inhibition was largely, but not always exclusively, dependent upon the presence, either in vivo or in vitro, of the Ly49A ligand, H-2Dd. Strikingly Ly49A transgenic mice have reduced capacity to control infection with the virulent lymphocytic choriomeningitis virus variant clone 13. Overall, these studies demonstrate that expression of killer inhibitory receptors can modulate anti-viral T cell responses in vivo and in vitro.     

Ly49-dependent NK cell licensing and effector inhibition involve the same interaction site on MHC ligands

NK cells become functionally competent to be triggered by their activation receptors through the interaction of NK cell inhibitory receptors with their cognate self-MHC ligands, an MHC-dependent educational process termed "licensing." For example, Ly49A(+) NK cells become licensed by the interaction of the Ly49A inhibitory receptor with its MHC class I ligand, H2D(d), whereas Ly49C(+) NK cells are licensed by H2K(b). Structural studies indicate that the Ly49A inhibitory receptor may interact with two sites, termed site 1 and site 2, on its H2D(d) ligand. Site 2 encompasses the α1/α2/α3 domains of the H2D(d) H chain and β(2)-microglobulin (β2m) and is the functional binding site for Ly49A in effector inhibition. Ly49C functionally interacts with a similar site in H2K(b). However, it is currently unknown whether this same site is involved in Ly49A- or Ly49C-dependent licensing. In this study, we produced transgenic C57BL/6 mice expressing wild-type or site 2 mutant H2D(d) molecules and studied whether Ly49A(+) NK cells are licensed. We also investigated Ly49A- and Ly49C-dependent NK licensing in murine β2m-deficient mice that are transgenic for human β2m, which has species-specific amino acid substitutions in β2m. Our data from these transgenic mice indicate that site 2 on self-MHC is critical for Ly49A- and Ly49C-dependent NK cell licensing. Thus, NK cell licensing through Ly49 involves specific interactions with its MHC ligand that are similar to those involved in effector inhibition.     

Ly-49P activates NK-mediated lysis by recognizing H-2Dd

Little is known regarding the ligand specificity of Ly-49 activating receptor subfamily members expressed by NK cells. A new Ly-49 activating receptor related to Ly-49A in its extracellular domain, designated Ly-49P, was recently cloned from 129 strain mice. We independently cloned an apparent allele of Ly-49P expressed by nonobese diabetic and nonobese diabetes-resistant mouse strain NK cells. We found it to be reactive with the A1 Ab thought to recognize a polymorphic epitope expressed only by the Ly-49A inhibitory receptor of the C57BL/6 strain. Rat RNK-16 cells transfected with Ly-49P mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49P can activate NK-mediated lysis. We determined that RNK-16 lysis of Con A blasts induced by Ly-49P was MHC dependent, resulting in efficient lysis of H-2Dd-bearing targets. We found that the Dd alpha1/alpha2 domain is required for Ly-49P-mediated RNK-16 activation, as determined by exon shuffling and transfection. Thus, Ly-49P is the second activating Ly-49 receptor demonstrated to induce NK cytotoxicity by recognizing a class I MHC molecule.     

Ly49C-Dependent Control of MCMV Infection by NK Cells Is Cis-Regulated by MHC Class I Molecules

Natural Killer (NK) cells are crucial in early resistance to murine cytomegalovirus (MCMV) infection. In B6 mice, the activating Ly49H receptor recognizes the viral m157 glycoprotein on infected cells. We previously identified a mutant strain (MCMV^G1F) whose variant m157 also binds the inhibitory Ly49C receptor. Here we show that simultaneous binding of m157 to the two receptors hampers Ly49H-dependent NK cell activation as Ly49C-mediated inhibition destabilizes NK cell conjugation with their targets and prevents the cytoskeleton reorganization that precedes killing. In B6 mice, as most Ly49H⁺ NK cells do not co-express Ly49C, the overall NK cell response remains able to control MCMVm157^G1F infection. However, in B6 Ly49C transgenic mice where all NK cells express the inhibitory receptor, MCMV infection results in altered NK cell activation associated with increased viral replication. Ly49C-mediated inhibition also regulates Ly49H-independent NK cell activation. Most interestingly, MHC class I regulates Ly49C function through cis-interactions that mask the receptor and restricts m157 binding. B6 Ly49C Tg, β2m ko mice, whose Ly49C receptors are unmasked due to MHC class I deficient expression, are highly susceptible to MCMVm157^G1F and are unable to control a low-dose infection. Our study provides novel insights into the mechanisms that regulate NK cell activation during viral infection.     

The complementarity-determining region-like loops of CD8 alpha interact differently with beta 2-microglobulin of the class I molecules H-2Kb and thymic leukemia antigen, while similarly with their alpha 3 domains

The murine CD8 glycoprotein interacts with both classical MHC class I molecules and some nonclassical molecules, including the thymic leukemia Ag (TL). TL binds preferentially to CD8alphaalpha homodimers with a 10-fold higher affinity than H-2K(b) class I molecules. To understand the molecular basis for this difference, we created a panel of CD8alpha mutants and tested the ability of the CD8alphaalpha homodimers to bind to H-2K(b) tetramers and TL tetramers. Mutations in three CD8 residues located on the complementarity-determining region-like loops contacting the negatively charged loop in the alpha3 domain of MHC class I greatly reduced binding to both tetramers. Because TL and H-2K(b) class I sequences are highly conserved in the alpha3 domain of MHC class I, this suggests that CD8 contacts the alpha3 domain of TL and H-2K(b) in a similar manner. In contrast, mutations in residues on the A and B beta strands of CD8 that are involved in contact with beta(2)-microglobulin affected interaction with the H-2K(b) tetramer, but not the TL tetramer. Therefore, the orientation of interaction of TL with CD8 appears to be different from that of H-2K(b). The unique high affinity binding of TL with CD8alphaalpha is most likely a result of amino acid differences in the alpha3 domain between TL and H-2K(b), particularly at positions 198 (K to D) and 228 (M to T), which are contact residues in the CD8alphaalpha-H-2K(b) cocrystal.