The evolution of natural killer cell receptors

Natural killer (NK) cells are immune cells that play a crucial role against viral infections and tumors. To be tolerant against healthy tissue and simultaneously attack infected cells, the activity of NK cells is tightly regulated by a sophisticated array of germline-encoded activating and inhibiting receptors. The best characterized mechanism of NK cell activation is “missing self” detection, i.e., the recognition of virally infected or transformed cells that reduce their MHC expression to evade cytotoxic T cells. To monitor the expression of MHC-I on target cells, NK cells have monomorphic inhibitory receptors which interact with conserved MHC molecules. However, there are other NK cell receptors (NKRs) encoded by gene families showing a remarkable genetic diversity. Thus, NKR haplotypes contain several genes encoding for receptors with activating and inhibiting signaling, and that vary in gene content and allelic polymorphism. But if missing-self detection can be achieved by a monomorphic NKR system why have these polygenic and polymorphic receptors evolved? Here, we review the expansion of NKR receptor families in different mammal species, and we discuss several hypotheses that possibly underlie the diversification of the NK cell receptor complex, including the evolution of viral decoys, peptide sensitivity, and selective MHC-downregulation.     

Human-specific evolution and adaptation led to major qualitative differences in the variable receptors of human and chimpanzee natural killer cells

Natural killer (NK) cells serve essential functions in immunity and reproduction. Diversifying these functions within individuals and populations are rapidly-evolving interactions between highly polymorphic major histocompatibility complex (MHC) class I ligands and variable NK cell receptors. Specific to simian primates is the family of Killer cell Immunoglobulin-like Receptors (KIR), which recognize MHC class I and associate with a range of human diseases. Because KIR have considerable species-specificity and are lacking from common animal models, we performed extensive comparison of the systems of KIR and MHC class I interaction in humans and chimpanzees. Although of similar complexity, they differ in genomic organization, gene content, and diversification mechanisms, mainly because of human-specific specialization in the KIR that recognizes the C1 and C2 epitopes of MHC-B and -C. Humans uniquely focused KIR recognition on MHC-C, while losing C1-bearing MHC-B. Reversing this trend, C1-bearing HLA-B46 was recently driven to unprecedented high frequency in Southeast Asia. Chimpanzees have a variety of ancient, avid, and predominantly inhibitory receptors, whereas human receptors are fewer, recently evolved, and combine avid inhibitory receptors with attenuated activating receptors. These differences accompany human-specific evolution of the A and B haplotypes that are under balancing selection and differentially function in defense and reproduction. Our study shows how the qualitative differences that distinguish the human and chimpanzee systems of KIR and MHC class I predominantly derive from adaptations on the human line in response to selective pressures placed on human NK cells by the competing needs of defense and reproduction.     

Virus Encoded MHC-Like Decoys Diversify the Inhibitory KIR Repertoire

Natural killer (NK) cells are circulating lymphocytes that play an important role in the control of viral infections and tumors. Their functions are regulated by several activating and inhibitory receptors. A subset of these receptors in human NK cells are the killer immunoglobulin-like receptors (KIRs), which interact with the highly polymorphic MHC class I molecules. One important function of NK cells is to detect cells that have down-regulated MHC expression (missing-self). Because MHC molecules have non polymorphic regions, their expression could have been monitored with a limited set of monomorphic receptors. Surprisingly, the KIR family has a remarkable genetic diversity, the function of which remains poorly understood. The mouse cytomegalovirus (MCMV) is able to evade NK cell responses by coding “decoy” molecules that mimic MHC class I. This interaction was suggested to have driven the evolution of novel NK cell receptors. Inspired by the MCMV system, we develop an agent-based model of a host population infected with viruses that are able to evolve MHC down-regulation and decoy molecules. Our simulations show that specific recognition of MHC class I molecules by inhibitory KIRs provides excellent protection against viruses evolving decoys, and that the diversity of inhibitory KIRs will subsequently evolve as a result of the required discrimination between host MHC molecules and decoy molecules.     

MHC class I genes of birds of prey: isolation, polymorphism and diversifying selection

The threat of emerging infectious diseases encourages the investigation of functional loci related to host resilience, such as those belonging to the major histocompatibility complex (MHC). Through careful primer design targeting to conserved regions of MHC class I sequences in birds, we successfully amplified a genomic fragment spanning exons 2–4 in three birds of prey. The identification of a highly conserved region within intron 2 allowed cross-amplifying complete exon 3 sequences in diurnal raptors, owls and New World vultures. We found evidence through PCR and cloning for 1–2 polymorphic class I loci, although this is almost certainly an underestimate. Inferences of diversifying selection in the kestrel MHC revealed that the two major regions of exon 3 exhibiting positive selection mostly agree with those described for the human HLA-A2 molecule. In contrast to passerines, where a high incidence of gene duplications and pseudogenes has been commonly documented, birds of prey emerge as nice model species for the investigation of the evolutionary significance and conservation implications of MHC diversity in vertebrates.     

Positive impact of inhibitory Ly49 receptor-MHC class I interaction on NK cell development

NK cells can kill MHC-different or MHC-deficient but not syngeneic MHC-expressing target cells. This MHC class I-specific tolerance is acquired during NK cell development. MHC recognition by murine NK cells largely depends on clonally distributed Ly49 family receptors, which inhibit NK cell function upon ligand engagement. We investigated whether these receptors play a role for the development of NK cells and provide evidence that the expression of a Ly49 receptor transgene on developing NK cells endowed these cells with a significant developmental advantage over NK cells lacking such a receptor, but only if the relevant MHC ligand was present in the environment. The data suggest that the transgenic Ly49 receptor accelerates and/or rescues the development of NK cells which would otherwise fail to acquire sufficient numbers of self-MHC-specific receptors. Interestingly, the positive effect on NK cell development is most prominent when the MHC ligand is simultaneously present on both hemopoietic and nonhemopoietic cells. These findings correlate with functional data showing that MHC class I ligand on all cells is required to generate functionally mature NK cells capable of reacting to cells lacking the respective MHC ligand. We conclude that the engagement of inhibitory MHC receptors during NK cell development provides signals that are important for further NK cell differentiation and/or maturation.     

Primate‐specific regulation of natural killer cells

Natural killer (NK) cells are circulating lymphocytes that function in innate immunity and placental reproduction. Regulating both development and function of NK cells is an array of variable and conserved receptors that interact with major histocompatibility complex (MHC) class I molecules. Families of lectin‐like and immunoglobulin‐like receptors are determined by genes in the natural killer complex (NKC) and leukocyte receptor complex (LRC), respectively. As a consequence of the strong, varying pressures on the immune and reproductive systems, NK cell receptors and their MHC class I ligands evolve rapidly, are highly diverse and exhibit dramatic species‐specific differences. The variable, polymorphic family of killer cell immunoglobulin‐like receptors (KIR) that regulate human NK cell development and function arose recently, from a single‐copy gene during the evolution of simian primates. Our studies of KIR and MHC class I genes in representative species show how these two unlinked but functionally intertwined genetic complexes have co‐evolved. In humans, combinations of KIR and HLA class I factors are associated with infectious diseases, including HIV/AIDS, autoimmunity, reproductive success and the outcome of therapeutic transplantation. The extraordinary, and unanticipated, divergence of human NK cell receptors and MHC class I ligands from their mouse counterparts can in part explain the difficulties experienced in finding informative mouse models for human diseases. Non‐human primate models have far greater potential, but to realize their promise will first require more complete definition of the genetics and function of KIR and MHC variation in non‐human primate species, at a level comparable to that achieved for the human species.     

Impact of KIR/HLA ligand combinations on immune responses in malignant melanoma

Tumor growth and dissemination depend partly on the reactivity of natural killer (NK) cells and T cells expressing NK-associated receptors. Their effector functions are regulated by an array of activating and inhibitory cell surface receptors with MHC class I ligand specificity, such as the killer immunoglobulin-like receptors (KIRs). Given the extensive genomic diversity of KIRs and their HLA ligands, it is reasonable to speculate that HLA, KIR gene variations and specific KIR-ligand combinations will have an impact on disease susceptibility and/or progression. Here, we discuss how KIR genotypes and KIR/HLA immunogenetic profiles may be involved in tumorigenesis, especially in malignant melanoma (MM). A hypothetical model of the impact of KIR/ligand combinations on immune responses in MM is proposed.     

Reciprocal translocation of small numbers of inbred individuals rescues immunogenetic diversity

Genetic rescue can reduce inbreeding depression and increase fitness of small populations, even when the donor populations are highly inbred. In a recent experiment involving two inbred island populations of the New Zealand South Island robin, Petroica australis, reciprocal translocations improved microsatellite diversity and individual fitness. While microsatellite loci may reflect patterns of genome‐wide diversity, they generally do not indicate the specific genetic regions responsible for increased fitness. We tested the effectiveness of this reciprocal translocation for rescuing diversity of two immunogenetic regions: Toll‐like receptor (TLR) and major histocompatibility complex (MHC) genes. We found that the relatively small number of migrants (seven and ten per island) effectively brought the characteristic TLR gene diversity of each source population into the recipient population. However, when migrants transmitted TLR alleles that were already present at high frequency in the recipient population, it was possible for offspring of mixed heritage to have decreased gene diversity compared to recipient population diversity prior to translocation. In contrast to TLRs, we did not observe substantial changes in MHC allelic diversity following translocation, with limited evidence of a decrease in differentiation, perhaps because most MHC alleles were observed at both sites prior to the translocation. Overall, we conclude that small numbers of migrants may successfully restore the diversity of immunogenetic loci with few alleles, but that translocating larger numbers of animals would provide additional opportunity for the genetic rescue of highly polymorphic immunity regions, such as the MHC, even when the source population is inbred.     

A ligand for the murine NK activation receptor Ly-49D: activation of tolerized NK cells from beta 2-microglobulin-deficient mice

Mouse NK cells express inhibitory NK receptors that recognize target cell MHC class I molecules and activation receptors that are less well defined. The Ly-49D activation receptor on C57BL/6 NK cells recognizes Chinese hamster ovary cells and triggers natural killing. In this study, we demonstrate that a Chinese hamster classical MHC class I molecule is the ligand for Ly-49D in a reporter gene assay system as well as in NK cell killing assays. Ly-49D recognizes the Chinese hamster class I molecule better when it is expressed with Chinese hamster beta(2)-microglobulin (beta(2)m) than murine beta(2)m. However, it is still controversial that Ly-49D recognizes H-2D(d), as we were unable to demonstrate the specificity previously reported. Using this one ligand-one receptor recognition system, function of an NK activation receptor was, for the first time, investigated in NK cells that are tolerized in beta(2)m-deficient mice. Surprisingly, Ly-49D-killing activity against ligand-expressing targets was observed with beta(2)m-deficient mouse NK cells, albeit reduced, even though "tolerized" function of Ly-49D was expected. These results indicate that Ly-49D specifically recognizes the Chinese hamster MHC class I molecule associated with Chinese hamster beta(2)m, and indicate that the Ly-49D NK cell activation receptor is not tolerized in beta(2)m deficiency.     

Polymorphic Alu insertions within the Major Histocompatibility Complex class I genomic region: a brief review

Most polymorphic Alu insertions (POALINs) belong to a subgroup of the Alu multicopy retrotransposon family of short interspersed nucleotide elements (SINEs) that are categorized as AluYb8 and AluYa5. The number of AluYb8/AluYa5 members (approximately 4,492 copies) is significantly less than the approximately one million fixed Alu copies per human genome. We have studied the presence of POALINs within the Major Histocompatibility Complex (MHC) class I region on the short arm of chromosome 6 (6p21.3) because this region has a high gene density, many genes with immune system functions, large sequence variations and diversity, duplications and redundancy, and a strong association with more than 100 different diseases. Since little is known about POALINs within the MHC genomic region, we undertook to identify some of the members of the AluYb8/AluYa5 subfamily and to study their frequency of distribution and genetic characteristics in different populations. As a result of our comparative genomic analyses, we identified the insertion sites for five POALINs distributed within the MHC class I region. This brief review outlines the locations of the insertions and sequence features of the five MHC POALINs, their single site and haplotype frequencies in different geographic populations, and their association with different HLA class I genes and disease. We show that the MHC POALINs have a potential value as lineage and linkage markers for the study of human population genetics, disease associations, genomic diversity and evolution.     

Distinct effects of interferon-gamma and MHC class I surface antigen levels on resistance of the K562 tumor cell line to natural killer-mediated lysis

Various investigators have examined the relationship between tumor cell susceptibility to natural killer (NK) cell lysis and the expression of HLA class I antigens on the tumor cell. There is controversy as to whether or not an inverse relationship exists, and if so, the basis of the relationship between these two phenomena remains undefined. To address these questions, the genomic clones for two HLA antigens were transfected into the erythroleukemia cell line K562, a cell line that is used as the standard to assess human NK and major histocompatibility complex (MHC) nonrestricted cytolysis. Susceptibility to NK lysis was not affected by the de novo expression of HLA antigens on the K562 after DNA mediated gene transfer. Interferon-gamma (IFN-gamma) treatment of K562 induced levels of MHC class I antigen surface expression comparable to those found on the transfected cells; however, the IFN-gamma-treated cells were resistant to NK lysis. When very high levels of surface HLA antigens were induced on the transfectants, a potential effect of class I MHC expression on K562 lysis could be discerned that was distinct from the resistance to NK lysis induced by IFN-gamma-treatment.     

A possible basis for major histocompatibility complex-restricted T-cell recognition

Four distinct T-cell antigen-receptor gene loci have now been identified and partly characterized: alpha, beta, gamma and delta. All of these loci can rearrange in an immunoglobulin-like fashion and express polypeptides that contribute to either alpha:beta or gamma:delta T-cell receptor-CD3 complexes. Surprisingly, the T-cell receptor (TCR) delta coding regions are located entirely, or almost entirely, within the TCR alpha locus and share at least some of the V region gene segments, thus at least partly linking the two different types of receptor heterodimers. Analysis of potential T-cell receptor diversity, particularly that of the delta chain, indicates a striking concentration of somatic polymorphism in the V-J junctional region of the two heterodimers, four to six orders of magnitude higher than similar calculations for immunoglobulin light- and heavy-chain combinations. In contrast, the number of possible V region combinations in T-cell receptors is one hundredth to one thousandth that of immunoglobulins. TCR alpha: beta heterodimers are known to recognize many possible fragments of antigens embedded in the peptide-binding clefts of a relatively small number of major histocompatibility complex (MHC) molecules. Thus it is attractive to speculate that the V-J junctional portions of both types of T-cell receptor contact peptide antigens, whereas the remaining diversity regions contact the MHC. This contention is supported by molecular modelling studies and has interesting implications for the evolution of antigen-receptor genes.     

Cmv4, a new locus linked to the NK cell gene complex, controls innate resistance to cytomegalovirus in wild-derived mice

CMV can cause life-threatening disease in immunodeficient hosts. Experimental infection in mice has revealed that the genetically determined natural resistance to murine CMV (MCMV) may be mediated either by direct recognition between the NK receptor Ly49H and the pathogen-encoded glycoprotein m157 or by epistatic interaction between Ly49P and the host MHC H-2D(k). Using stocks of wild-derived inbred mice as a source of genetic diversity, we found that PWK/Pas (PWK) mice were naturally resistant to MCMV. Depletion of NK cells subverted the resistance. Analysis of backcrosses to susceptible BALB/c mice revealed that the phenotype was controlled by a major dominant locus effect linked to the NK gene complex. Haplotype analysis of 41 polymorphic markers in the Ly49h region suggested that PWK mice may share a common ancestral origin with C57BL/6 mice; in the latter, MCMV resistance is dependent on Ly49H-m157 interactions. Nevertheless, PWK mice retained viral resistance against m157-defective mutant MCMV. These results demonstrate the presence of yet another NK cell-dependent viral resistance mechanism, named Cmv4, which most likely encodes for a new NK activating receptor. Identification of Cmv4 will expand our understanding of the specificity of the innate recognition of infection by NK cells.     

KIR3DL1 polymorphisms that affect NK cell inhibition by HLA-Bw4 ligand

The killer cell Ig-like receptor (KIR) gene family encodes MHC class I receptors expressed by NK cells and several T cell subpopulations. Factors contributing to human KIR haplotype diversity are differences in gene number, gene content, and allelic polymorphism. Whereas functional and clinical consequences of the first two factors are established, knowledge of the effects of KIR gene polymorphism is limited to special cases in which signaling function is reversed or cell surface expression lost. In this study we use retrovirally transduced human cell lines to show that 3DL1*002 is a stronger inhibitory receptor for HLA-Bw4 ligands than 3DL1*007. Analysis of mutant 3DL1*002 and 3DL1*007 molecules demonstrates that residue 238 in the D2 domain and 320 in the transmembrane region contribute to the difference in receptor strength. Neither position 238 nor 320 is predicted to interact directly with HLA-Bw4 ligand. This study also revealed that KIR3DL1 and LILRB1 both contribute to developing an inhibitory response to HLA-Bw4 ligands.     

Successful control of viruses by NK cells--a balance of opposing forces?

Natural killer (NK) cells play a crucial role in limiting the severity of diseases caused by a range of viruses. Recent data have shown that the effector functions of NK cells can be specifically stimulated when NK cell activation receptors engage cellular major histocompatibility complex (MHC) class I-like ligands induced after infection or by specific viral gene products. However, to counter this NK cell response viruses have evolved an array of strategies to subvert efficient NK cell activation. These data indicate that the balance of host NK cell responses and viral NK cell escape mechanisms can be strategically poised as each strives for survival.     

Characterization of a novel killer cell lectin-like receptor (KLRH1) expressed by alloreactive rat NK cells

NK cells have the ability to recognize and kill MHC-mismatched hemopoietic cells. In the present study, strain-specific differences in the rat NK allorecognition repertoire were exploited to generate Abs against receptors that may be involved in allogeneic responses. A mAb termed STOK9 was selected, and it reacted with subsets of NK cells and NKR-P1(+) T cells from certain rat strains possessing highly alloreactive NK cells. The STOK9(+) NK subset was broadly alloreactive and lysed Con A lymphoblast targets from a range of MHC-mismatched strains. The mAb STOK9 precipitated a 75-kDa dimeric glycoprotein from NK lysates. Expression cloning revealed that each monomer consisted of 231 aa with limited homology to other previously characterized killer cell lectin-like receptors (KLRs). This glycoprotein therefore constitutes a novel KLR branch, and it has been termed KLRH1. A gene in the central region of the natural killer gene complex on rat chromosome 4 encodes KLRH1. A mouse homolog appears to be present as deduced from analyses of genomic trace sequences. The function of KLRH1 is unknown, but it contains an immunoreceptor tyrosine-based inhibitory motif, suggesting an inhibitory function. The MHC haplotype of the host appears to influence KLRH1 expression, suggesting that it may function as an MHC-binding receptor on subsets of NK cells and T lymphocytes.