Rapid expansion of killer cell immunoglobulin-like receptor genes in primates and their coevolution with MHC Class I genes

The gene family of killer cell immunoglobulin-like receptors (KIRs) in primates provides the first line of defense against virus infection and tumor transformation. Interacting with MHC class I molecules, KIRs can regulate the cytotoxic activity of natural killer (NK) cells and distinguish the tumor and virus infected cells from normal body cells. Phylogenetic analysis and comparison of domain structures identified three major groups of KIR genes (group I, II, and III genes). These groups of KIR genes, generated by a series of gene duplications, have acquired different MHC-binding specificity. Inference of ancestral KIR sequences suggested that the functional divergence of group I genes from group II genes occurred by positive selection at the MHC-binding sites after duplication. Our evolutionary study has shown that group I genes diverged from group II genes about 17 million years ago (Mya) apparently after separation of hominoids from Old World (OW) monkeys. Around the same time, gene duplication generating the class I MHC-C locus appears to have occurred. These findings suggest that KIR and MHC class I genes have coevolved as an interacting system. The KIR gene family has experienced a rapid expansion in primate species. The rate of expansion of this gene family seems to be one of the highest among all hominoid gene families. The KIR gene family is also subject to birth-and-death evolution.     

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.     

Complexity in cattle <Emphasis Type="Italic">KIR</Emphasis> genes: transcription and genome analysis

Killer immunoglobulin (Ig)-like receptors (KIRs) are the major functional natural killer (NK) cell receptors in human. The presence of KIR genes has only recently been demonstrated in other (non-primate) species, and their expression, genomic arrangement, and function in these species have yet to be investigated. In this study, we describe the KIR gene family in cattle. KIR sequences were amplified from cDNA derived from four animals. Seventeen new sequences were identified in total. Some are alleles of two previously described genes, and the remainder are representative of at least four additional genes. These cDNA data, together with analysis of the cattle genome sequence, confirm that, as in humans, cattle have multiple inhibitory and activating KIR genes, with variable haplotype composition, and putative framework genes. In contrast to human, the majority of the cattle KIR genes encode three Ig-domain KIRs; most of the inhibitory genes encode only one immunoreceptor tyrosine-based inhibitory motif (ITIM), and the activating genes encode molecules with arginine rather than the more usual lysine in the transmembrane domain. A divergent gene, 2DL1, encodes a two Ig-domain KIR with an unusual D0-D2 structure, and a distinct signaling domain with two ITIMs. Similarity to pig and human two Ig-domain (D0-D2) KIRs suggest these may be more related to an ancestral gene than the other cattle KIR genes. Cattle have multiple NKG2A-related genes and at least one Ly49 gene; thus, the data presented here suggest that they have the potential to express more major histocompatibility complex-binding NK receptors than other species.     

Domain shuffling has been the main mechanism forming new hominoid killer cell Ig-like receptors

The killer cell Ig-like receptor (KIR) gene family encodes MHC class I-specific receptors, which regulate NK cell responses and are also expressed on subpopulations of T cells. KIR haplotypes vary in gene content, which, in combination with allelic polymorphism, extensively diversifies the KIR genotype both within and between human populations. Species comparison indicates that formation of new KIR genes and loss of old ones are frequent events, so that few genes are conserved even between closely related species. In this regard, the hominoids define a time frame that is particularly informative for understanding the processes of KIR evolution and its potential impact on killer cell biology. KIR cDNA were characterized from PBMC of three gorillas, and genomic DNA were characterized for six additional individuals. Eleven gorilla KIR genes were defined. With attainment of these data, a set of 75 KIR sequences representing five hominoid species was assembled, which also included rhesus monkey, cattle, and rodent KIR. Searching this data set for recombination events, and phylogenetic analysis using Bayesian methods, demonstrated that new KIR were usually the result of recombination between loci in which complete protein domains were shuffled. Further phylogenetic analysis of the KIR sequences after removal of confounding recombined segments showed that only two KIR genes, KIR2DL4 and KIR2DL5, have been preserved throughout hominoid evolution, and one of them, KIR2DL4, is also common to rhesus monkey and hominoids. Other KIR genes represent recombinant forms present in a minority of species, often only one, as exemplified by 8 of the 11 gorilla KIR genes.     

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.     

Differential association of gene content polymorphisms of killer cell immunoglobulin-like receptors with placental malaria in HIV- and HIV+ mothers

Pregnant women have abundant natural killer (NK) cells in their placenta, and NK cell function is regulated by polymorphisms of killer cell immunoglobulin-like receptors (KIRs). Previous studies report different roles of NK cells in the immune responses to placental malaria (PM) and human immunodeficiency virus (HIV-1) infections. Given these references, the aim of this study was to determine the association between KIR gene content polymorphism and PM infection in pregnant women of known HIV-1 status. Sixteen genes in the KIR family were analyzed in 688 pregnant Kenyan women. Gene content polymorphisms were assessed in relation to PM in HIV-1 negative and HIV-1 positive women, respectively. Results showed that in HIV-1 negative women, the presence of the individual genes KIR2DL1 and KIR2DL3 increased the odds of having PM, and the KIR2DL2/KIR2DL2 homozygotes were associated with protection from PM. However, the reverse relationship was observed in HIV-1 positive women, where the presence of individual KIR2DL3 was associated with protection from PM, and KIR2DL2/KIR2DL2 homozygotes increased the odds for susceptibility to PM. Further analysis of the HIV-1 positive women stratified by CD4 counts showed that this reverse association between KIR genes and PM remained only in the individuals with high CD4 cell counts but not in those with low CD4 cell counts. Collectively, these results suggest that inhibitory KIR2DL2 and KIR2DL3, which are alleles of the same locus, play a role in the inverse effects on PM and PM/HIV co-infection and the effect of KIR genes on PM in HIV positive women is dependent on high CD4 cell counts. In addition, analysis of linkage disequilibrium (LD) of the PM relevant KIR genes showed strong LD in women without PM regardless of their HIV status while LD was broken in those with PM, indicating possible selection pressure by malaria infection on the KIR genes.     

Receptor-ligand analyses define minimal killer cell Ig-like receptor (KIR) in humans

Interactions between inhibitory killer cell immunoglobulin-like receptors (iKIR) and human leukocyte antigen (HLA) class I molecules regulate natural killer (NK) cell responses to eliminate infected and transformed cells while maintaining tolerance to healthy cells. Unlinked polymorphic gene families encode KIR receptors and HLA class I ligands and their independent segregation results in a variable number and type of iKIR + HLA pairs inherited in individuals. The diversity in the co-inheritance of iKIR + HLA pairs and activating KIR (aKIR) genes in 759 unrelated individuals from four ethnic populations was analyzed. Every individual studied inherited a minimum of one iKIR + HLA pair; suggesting that major histocompatibility complex class I-dependent inhibitory KIR signaling is essential for human NK cell function. In contrast, 13.4% of the study group lacked all aKIR genes. Twenty percent of the study group carried only one of the four iKIR + HLA pairs. Interestingly, 3% of the study group carrying only KIR2DL3 + HLA-C1 as an iKIR + HLA pair lacked aKIR genes. These data suggest that a single iKIR can constitute the minimal KIR repertoire for human NK cells. Genotypes carrying an equal number of iKIR + HLA pairs and aKIR genes represented 20% of the study group. The remaining individuals had either a dominant inhibitory KIR genotype (iKIR + HLA > aKIR) or a dominant activating KIR genotype (iKIR + HLA < aKIR). Genotypes encoding these imbalanced inhibitory and activating interactions may contribute to susceptibility or resistance to human diseases.     

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.     

Comparison of chimpanzee and human leukocyte Ig-like receptor genes reveals framework and rapidly evolving genes.

The leukocyte receptor complex (LRC) on human chromosome 19 contains related Ig superfamily killer cell Ig-like receptor (KIR) and leukocyte Ig-like receptor (LIR) genes. Previously, we discovered much difference in the KIR genes between humans and chimpanzees, primate species estimated to have approximately 98.8% genomic sequence similarity. Here, the common chimpanzee LIR genes are identified, characterized, and compared with their human counterparts. From screening a chimpanzee splenocyte cDNA library, clones corresponding to nine different chimpanzee LIRs were isolated and sequenced. Analysis of genomic DNA from 48 unrelated chimpanzees showed 42 to have all nine LIR genes, and six animals to lack just one of the genes. In structural diversity and functional type, the chimpanzee LIRs cover the range of human LIRs. Although both species have the same number of inhibitory LIRs, humans have more activating receptors, a trend also seen for KIRs. Four chimpanzee LIRs are clearly orthologs of human LIRs. Five other chimpanzee LIRs have paralogous relationships with clusters of human LIRs and have undergone much recombination. Like the human genes, chimpanzee LIR genes appear to be organized into two duplicated blocks, each block containing two orthologous genes. This organization provides a conserved framework within which there are clusters of faster evolving genes. Human and chimpanzee KIR genes have an analogous arrangement. Whereas both KIR and LIR genes can exhibit greater interspecies differences than the genome average, within each species the LIR gene family is more conserved than the KIR gene family.     

MHC class I-independent recognition of NK-activating receptor KIR2DS4

Natural killer cells are capable of killing tumor and virus-infected cells. This killing is mediated primarily via the natural cytotoxicity receptors, including NKp46, NKp44, NKp30, and by the NKG2D receptor. Killer cell Ig-like receptors (KIRs) are mainly involved in inhibiting NK killing (inhibitory KIRs) via interaction with MHC class I molecules. Some KIRs, however, have been found to enhance NK killing when interacting with MHC class I molecules (activating KIRs). We have previously demonstrated that KIR2DS4, an activating KIR, recognizes the HLA-Cw4 protein. The interaction observed was weak and highly restricted to HLA-Cw4 only. These findings prompted us to check whether KIR2DS4 might have additional ligand(s). In this study, we show that KIR2DS4 is able to also interact with a non-class I MHC protein expressed on melanoma cell lines and on a primary melanoma. This interaction is shown to be both specific and functional. Importantly, site-directed mutagenesis analysis reveals that the amino acid residues involved in the recognition of this novel ligand are different from those interacting with HLA-Cw4. These results may shed new light on the function of activating KIRs and their relevance in NK biology.     

Killer cell Ig-like receptor and leukocyte Ig-like receptor transgenic mice exhibit tissue- and cell-specific transgene expression

To generate an experimental model for exploring the function, expression pattern, and developmental regulation of human Ig-like activating and inhibitory receptors, we have generated transgenic mice using two human genomic clones: 52N12 (a 150-Kb clone encompassing the leukocyte Ig-like receptor (LILR)B1 (ILT2), LILRB4 (ILT3), and LILRA1 (LIR6) genes) and 1060P11 (a 160-Kb clone that contains ten killer cell Ig-like receptor (KIR) genes). Both the KIR and LILR families are encoded within the leukocyte receptor complex, and are involved in immune modulation. We have also produced a novel mAb to LILRA1 to facilitate expression studies. The LILR transgenes were expressed in a similar, but not identical, pattern to that observed in humans: LILRB1 was expressed in B cells, most NK cells, and a small number of T cells; LILRB4 was expressed in a B cell subset; and LILRA1 was found on a ring of cells surrounding B cell areas on spleen sections, consistent with other data showing monocyte/macrophage expression. KIR transgenic mice showed KIR2DL2 expression on a subset of NK cells and T cells, similar to the pattern seen in humans, and expression of KIR2DL4, KIR3DS1, and KIR2DL5 by splenic NK cells. These observations indicate that linked regulatory elements within the genomic clones are sufficient to allow appropriate expression of KIRs in mice, and illustrate that the presence of the natural ligands for these receptors, in the form of human MHC class I proteins, is not necessary for the expression of the KIRs observed in these mice.     

Recruitment of activation receptors at inhibitory NK cell immune synapses

Natural killer (NK) cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR) family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses.     

Extensive Variation in Gene Copy Number at the Killer Immunoglobulin-Like Receptor Locus in Humans

Killer immunoglobulin-like receptors (KIRs) are involved in the regulation of natural killer cell cytotoxicity. Within the human genome seventeen KIR genes are present, which all contain a large number of allelic variants. The high level of homology among KIR genes has hampered KIR genotyping in larger cohorts, and determination of gene copy number variation (CNV) has been difficult. We have designed a multiplex ligation-dependent probe amplification (MLPA) technique for genotyping and CNV determination in one single assay and validated the results by next-generation sequencing and with a KIR gene-specific short tandem repeat assay. In this way, we demonstrate in a cohort of 120 individuals a high level of CNV for all KIR genes except for the framework genes KIR3DL3 and KIR3DL2. Application of our MLPA assay in segregation analyses of families from the Centre d’Etude du Polymorphisme Humaine, previously KIR-genotyped by classical techniques, confirmed an earlier reported duplication and resulted in the identification of a novel duplication event in one of these families. In summary, our KIR MLPA assay allows rapid and accurate KIR genotyping and CNV detection, thus rendering improved transplantation programs and oncology treatment feasible, and enables more detailed studies on the role of KIRs in human (auto)immunity and infectious disease.