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.

External and internal calibration of the MHC class I-specific receptor Ly49A on murine natural killer cells.

Expression of the H-2Dd-specific inhibitory receptor Ly49A on murine NK cells is subject to MHC class I-dependent modulation in vivo. As a result, NK cells in H-2Dd-transgenic mice express low cell surface levels of Ly49A, whereas NK cells from nontransgenic C57BL/6 (B6) mice express high levels. The purpose of this study was to assess the role of MHC class I molecules on the NK cell itself vs those on surrounding cells in this calibration and to test whether the Ly49A levels are subject to regulation in mature NK cells also. Analysis of transgenic mice with mosaic expression of an H-2Dd/Ld transgene showed that MHC class I molecules on surrounding cells (external ligands) and on the NK cell itself (internal ligands) played distinct roles in the determination of Ly49A levels. External ligands were involved in down-regulation of Ly49A levels in vivo, whereas internal ligands kept the down-regulated levels of Ly49A low upon NK cell activation in vitro. Furthermore, in an experimental system based on adoptive transfer of spleen cells, receptor down-regulation of Ly49A occurred as a rapid adaptation process in mature NK cells after interaction with the H-2Dd ligand in vivo. This suggests that Ly49 levels are not fixed but can be changed in mature NK cells when they are exposed to a changed MHC class I environment.

Impaired MHC class I (H-2Dd)-mediated protection against Ly-49A+ NK cells after amino acid substitutions in the antigen binding cleft.

The MHC class I molecule H-2Dd (Dd) acts as a ligand for the inhibitory NK cell receptor Ly-49A. We have constructed altered Dd molecules by site-directed mutagenesis, replacing residues with the corresponding amino acids from the Db molecule, which fails to inhibit via Ly-49A. Mutations at positions 73 and 156 (DdS73WD156Y) impaired the protective effect of the Dd molecule, as evaluated by testing lymphoma cells transfected with the mutant gene for sensitivity to killing by Ly-49A+ NK cells in vitro and rejection by NK cells in vivo. The altered residues form a hydrophobic ridge across the floor of the antigen binding cleft. A mutation in the alpha helix of the alpha2 domain, facing the solvent and without direct contact with the peptide (DdA150S) had no effect. Dd recognition by Ly-49A+ NK cells is considered to be peptide dependent, but not peptide specific. Our results indicate that alterations of residues buried in the antigen binding cleft can induce changes in peptide binding patterns and/or conformational changes in the Dd molecule that make the trimolecular complex less permissive for inhibition of Ly-49A+ NK cells.

NK cell receptor calibration: effects of MHC class I induction on killing by Ly49Ahigh and Ly49Alow NK cells.

NK cells from C57BL/6 (B6) mice and H-2Dd transgenic B6 (D8) mice express different levels of the inhibitory receptor Ly49A, and they also differ in their target cell specificity. Here, we examined this differential specificity with respect to the role of the Ly49A receptor expression on effector cells and levels of H-2Dd inhibitory ligands on target cells. NK cells from D8 mice express low levels of Ly49A receptor (Ly49Alow), and are able to kill SP2/0 tumor cells in spite of their expression of H-2Dd. H-2Dd is expressed at reduced levels on SP2/0 cells; when these were increased three- to fivefold after IFN-gamma treatment, the killing by Ly49Alow NK cells from D8 mice was markedly reduced. Efficient killing was restored when the effectors were preincubated with anti-Ly49A F(ab')2 Abs. A separate experimental system was based on D8 TAP1-deficient Con A blasts exogenously loaded with H-2Dd-specific peptides. In this system, higher levels of cell surface H-2Dd had to be induced by peptide to inhibit D8 Ly49Alow NK cells to an extent similar to that of B6 Ly49Ahigh NK cells. Ly49A receptors on NK cells from H-2Dd transgenic mice are thus functional, although they require high levels of ligand to inhibit progression of the NK-target cell interaction. The data are in favor of the "receptor-calibration" model, which suggests that down-regulation of inhibitory receptors on NK cells may be useful in order for NK cells to discriminate between normal and reduced levels of MHC class I molecules.

Altered expression of Ly49 inhibitory receptors on natural killer cells from MHC class I-deficient mice.

MHC class I molecules in the host affect the specificity of NK cells. Previous work has suggested that this specificity is conferred by the expression of products encoded by the Ly49 gene family. This gene family encodes receptors that upon specific recognition of MHC class I ligands mediate an inhibitory signal that prevents killing by NK cells. The pattern of expression of the Ly49 MHC class I binding inhibitory receptors on NK cells is thought to be adapted to the host to ensure the generation of a self-tolerant, yet functional, NK cell repertoire. In the present study we have examined the expression of inhibitory receptors (Ly49A, Ly49C, and Ly49G2) on NK1.1+ cells from B6 (H-2b) and D8 (B6 mice transgenic for H-2Dd) mice as well as corresponding TAP1 -/-, beta2m -/-, and TAP1/beta2m -/- mutants of these mice. We demonstrate that receptor expression on NK1.1+ cells can be specifically modulated by host MHC class I molecules in at least two different ways: alteration of numbers of cells expressing a given receptor and modulation of the levels of expression of a given receptor at the cell surface. The degree of this modulation varies significantly among the various receptors studied and may depend upon the nature of their MHC class I ligands. The results are discussed in relation to the influence of MHC class I molecules on the development of an NK cell repertoire.

Host MHC class I gene control of NK-cell specificity in the mouse.

The missing self model predicts that NK cells adapt somatically to the type as well as levels of MHC class I products expressed by their host. Transgenic and gene knock-out mice have provided conclusive evidence that MHC class I genes control specificity and tolerance of NK cells. The article describes this control and discusses the possible mechanisms behind it, starting from a genetic model to study how natural resistance to tumors is influenced by MHC class I expression in the host as well as in the target cells. Data on host gene regulation of NK-cell functional specificity as well as Ly49 receptor expression are reviewed, leading up to the central question: how does the system develop and maintain "useful" NK cells, while avoiding "harmful" and "useless" ones? The available data can be fitted within each of two mutually none-exclusive models: cellular adaptation and clonal selection. Recent studies supporting cellular adaptation bring the focus on different possibilities within this general mechanism, such as anergy, receptor calibration and, most importantly, whether the specificity of each NK cell is permanently fixed or subject to continuous regulation.

Lack of F1 anti-parental resistance in H-2b/d F1 hybrids devoid of beta2-microglobulin.

F1 hybrid mice often reject parental hematopoietic grafts, a phenomenon known as hybrid resistance. Hybrid resistance is mediated by natural killer (NK) cells and although the molecular interactions responsible for this phenomenon are largely unknown, one hypothesis suggests that parental cells are rejected because they fail to express a complete set of host major histocompatibility complex (MHC) class I molecules. Inherent in this theory is that NK cells in the F1 hybrid are instructed by self MHC class I molecules to form an NK cell repertoire capable of reacting against cells lacking these self MHC class I molecules. Here, we show that C57BL/6 x DBA/2 mice (H-2b/d) devoid of beta2-microglobulin (beta2m) are incapable of rejecting beta2m-/- parental C57BL/6 cells (H-2b) both in vivo and in vitro. From this, we conclude that the development of an NK cell repertoire, at least in F1 mice of the H-2b/d haplotype, requires expression of MHC class I molecules complexed with beta2m.

Influence of glycosylphosphatidylinositol-linked H-2Dd molecules on target cell protection and natural killer cell specificity in transgenic mice.

The expression of certain major histocompatibility complex (MHC) class I ligands on target cells is one important determinate of their susceptibility to lysis by natural killer (NK) cells. NK cells express receptor molecules that bind to MHC class I. Upon binding to their MHC class I ligand, the NK cell is presumed to receive a signal through its receptor that inhibits lysis. It is unclear what role the MHC class I molecules of the effector and target cells play in signaling to the NK cell. We have investigated the role of the cytoplasmic and transmembrane domains of MHC class I molecules by producing a glycosylphosphatidylinositol (GPI)-linked H-2Dd molecule. The GPI-linked H-2Dd molecule is recognized by H-2Dd-specific antibodies and cytotoxic T lymphocytes. Expression of the GPI-linked H-2Dd molecule on H-2b tumor cells resulted in protection of the tumor cells after transplantation into D8 mice (H-2b, H-2Dd) from rejection by NK cells. In addition, NK cells from mice expressing the GPI-linked H-2Dd molecule as a transgene were able to kill nontransgenic H-2b lymphoblast target cells. The GPI-linked MHC class I molecule was able to alter NK cell specificity at the target and effector cell levels. Thus, the expression of the cytoplasmic and transmembrane domains of MHC class I molecules are not necessary for protection and alteration of NK cell specificity.

H-2Dp transgene alters natural killer cell specificity at the target and effector cell levels. Comparison with an H-2Dd transgene.

The expression of MHC class I molecules is an important determinate of natural killer (NK) cell specificity. The missing self hypothesis proposes that NK cells express receptors for self-MHC class I molecules so that target cells that share MHC class I alleles with the NK cells are not killed by those NK cells. However, some effector cells fail to kill some allogeneic target cells suggesting that shared motifs between different MHC class I alleles can interact with the effector cell class I receptors and prevent lysis. We have used transgenic mice to critically assess whether different MHC class I alleles can exert common influences on NK cell specificity at the host/effector and target cell levels. The specificity of NK cells have been compared between C57BL/6 (H-2b) mice and B6DP (H-2b, H-2Dp) and D8 (H-2b, H-2Dd) transgenic mice. The data indicate that H-2Dp and H-2Dd confer similar protection and specific lysis, such that NK cells from either of the H-2Dp or H-2Dd transgenic mice kill nontransgenic target cells yet they do not kill either of the transgenic target cells. The expression of an H-2Dp transgene also provides protection for C57BL/6 lymphoblasts from allogeneic BALB/c (H-2d) NK cells. Furthermore, H-2Dp and H-2Dd transgenic target cells are lysed to a similar extent by H-2k effector cells. These data suggest that H-2Dp and H-2Dd may be able to inhibit the same NK cell population. This may occur through a shared motif recognized by the same receptor, or different motifs recognized by different, but co-expressed receptors.