Genomic organization and evolutionary analysis of <Emphasis Type="Italic">Ly49</Emphasis> genes encoding the rodent natural killer cell receptors: rapid evolution by repeated gene duplication

Ly49 genes regulate the cytotoxic activity of natural killer (NK) cells in rodents and provide important protection against virus-infected or tumor cells. About 15 Ly49 genes have been identified in mice, but only a few genes have been reported to date in rats. Here we studied all Ly49 genes in the entire rat genome sequence and identified 17 putative functional and 16 putative non-functional genes together with their genomic locations in a 1.8-Mb region of chromosome 4. Phylogenetic analysis of these genes indicated that the Ly49 gene family expanded rapidly in recent years, and this expansion was mediated by both tandem and genomic block duplication. The joint phylogenetic analysis of mouse and rat genes suggested that the most recent common ancestor of the two species had at least several Ly49 genes, but that the majority of current duplicate genes were generated after divergence of the two species. In both species Ly49 genes are apparently subject to birth-and-death evolution, but the birth and death rates of Ly49 genes are higher in rats than in mice. The rate of gene expansion in the Ly49 gene family in rats is one of the highest among all mammalian multigene families so far studied. The biochemical function of Ly49 genes is essentially the same as that of KIR genes in primates, but the molecular structures of the two groups of NK cell receptors are very different. A hypothesis was presented to explain the origin of the differential use of Ly49 and KIR genes in rodents and primates.     

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.     

<Emphasis Type="Italic">Ly49</Emphasis> genes in non-rodent mammals

The Ly49 family of natural killer (NK) receptors is encoded by a highly polymorphic multigene family in the mouse and is also present in multiple copies in the rat. However, this gene exists as a single copy in primates and is mutated to non-function in humans. We recently showed that the cow also likely has only one Ly49 gene, but it is unclear what the Ly49 gene content is for other mammals. We have now isolated Ly49 cDNAs from the domestic cat, dog and pig and show that the corresponding gene appears to be single copy in these three species. The open reading frame is intact in all the genes and the putative proteins contain an immune tyrosine-based inhibition motif (ITIM), suggesting a role as an inhibitory receptor. In contrast to the other mammals, several Ly49-like genes appear to exist in the horse, indicating that amplification of this locus has occurred in a non-rodent lineage. Finally, phylogenetic analysis suggests that the rodent Ly49 genes have evolved more rapidly than their counterparts in mammals where the gene has remained as a single copy.     

The genomic organization and evolution of the natural killer immunoglobulin-like receptor (KIR) gene cluster

Natural killer (NK) immunoglobulin-like receptors (KIRs) are a family of polymorphic receptors which interact with specific motifs on HLA class I molecules and modulate NK cytolytic activity. In this study, we analyzed a recently sequenced subgenomic region on chromosome 19q13.4 containing eight members of the KIR receptor repertoire. Six members are clustered within a 100-kb continuous sequence. These genes include a previously unpublished member of the KIR gene family 2DS6, as well as 2DL1, 2DL4, 3DL1, 2DS4, 3DL2, from centromere to telomere. Two additional KIR genes, KIRCI and 2DL3, which may be located centromeric of this cluster were also analyzed. We show that the KIR genes have undergone repeated gene duplications. Diversification between the genes has occurred postduplication primarily as a result of retroelement indels and gene truncation. Using pre- and postduplication Alu sequences identified within these genes as evolutionary molecular clocks, the evolution and duplication of this gene cluster is estimated to have occurred 30–45 million years ago, during primate evolution. A proposed model of the duplication history of the KIR gene family leading to their present organization is presented. Received: 25 November 1999 / Revised: 10 January 2000     

KIR Gene Content in Amerindians Indicates Influence of Demographic Factors

Although the KIR gene content polymorphism has been studied worldwide, only a few isolated or Amerindian populations have been analyzed. This extremely diverse gene family codifies receptors that are expressed mainly in NK cells and bind HLA class I molecules. KIR-HLA combinations have been associated to several diseases and population studies are important to comprehend their evolution and their role in immunity. Here we analyzed, by PCR-SSP (specific sequencing priming), 327 individuals from four isolated groups of two of the most important Brazilian Amerindian populations: Kaingang and Guarani. The pattern of KIR diversity among these and other ten Amerindian populations disclosed a wide range of variation for both KIR haplotypes and gene frequencies, indicating that demographic factors, such as bottleneck and founder effects, were the most important evolutionary factors in shaping the KIR polymorphism in these populations.     

Localization of five new Ly49 genes, including three closely related to Ly49c

 Nine genes belonging to the mouse Ly49 multigene family of natural killer cell receptors have been identified to date. Two of these genes, Ly49h and i, are very closely related to the well characterized Ly49c gene in the carbohydrate recognition domain. Here we show by Southern blotting that at least two additional new sequences exist in C57BL/6 mice that are also closely related to Ly49c in the carbohydrate recognition domain. Furthermore, in contrast to Ly49a, extensive variation in the arrangement and number of Ly49c–related genes in different mouse strains was observed. To characterize and localize the new Ly49c–related genes in C57BL/6 mice, we isolated and mapped genomic P1 clones hybridizing to an Ly49C exon 7 probe. Locations and the relative order of all Ly49 genes found within the clones was determined. We also used polymerase chain reaction to sequence exons 2, 4, and 7 from all genes. In this manner, we identified five new potential Ly49 genes which have been tentatively termed Ly49j-n. Ly49j, k, and n belong to the Ly49c–related subfamily, whereas Ly49l and Ly49m are most similar to Ly49d and g, respectively. Interestingly, the members of the Ly49c–related subfamily are not clustered as a unit but are interspersed among other Ly49 genes. These results illustrate the complex nature of the Ly49 gene family and should aid in the understanding of functions, such as the mediation of hybrid resistance, in which Ly49c–related genes play a role. Received: 10 December 1997 · Revised: 28 February 1998     

Characterization of rhesus macaque KIR genotypes and haplotypes

Certain combinations of the killer immunoglobulin-like receptors (KIR) and major histocompatibility complex class I ligands in humans predispose carriers to a variety of diseases, requiring sophisticated genotyping of the highly polymorphic and diverse KIR and HLA genes. Particularly, KIR genotyping is challenging due to polymorphisms (allelic substitutions), genomic diversity (presence/absence of genes), and frequent duplications. Rhesus macaques are often used as important animal models of human diseases such as, e.g. AIDS. However, typing of rhesus macaque KIR genes has not been described so far. In this study, we report the identification of additional novel rhesus macaque KIR cDNA sequences and a sequence-specific KIR genotyping assay. From a cohort of four rhesus macaque families with a total of 70 individuals, we identified 25 distinct KIR genotypes. Segregation analyses of KIR genes and of two polymorphic microsatellite markers allowed the identification of 21 distinct KIR haplotypes in these families, with five to 11 segregating KIR genes per haplotype. Our analyses confirmed and extended knowledge on differential gene KIR gene content in macaques and indicate that rhesus macaque and human KIR haplotypes show a comparable level of diversity and complexity.     

Ly49Q, an ITIM-bearing NK receptor, positively regulates osteoclast differentiation

Osteoclasts, multinucleated cells that resorb bone, play a key role in bone remodeling. Although immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling is critical for osteoclast differentiation, the significance of immunoreceptor tyrosine-based inhibitory motif (ITIM) has not been well understood. Here we report the function of Ly49Q, an Ly49 family member possessing an ITIM motif, in osteoclastogenesis. Ly49Q is selectively induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) stimulation in bone marrow-derived monocyte/macrophage precursor cells (BMMs) among the Ly49 family of NK receptors. The knockdown of Ly49Q resulted in a significant reduction in the RANKL-induced formation of tartrate-resistance acid phosphatase (TRAP)-positive multinucleated cells, accompanied by a decreased expression of osteoclast-specific genes such as Nfatc1, Tm7sf4, Oscar, Ctsk, and Acp5. Osteoclastogenesis was also significantly impaired in Ly49Q-deficient cells in vitro. The inhibitory effect of Ly49Q-deficiency may be explained by the finding that Ly49Q competed for the association of Src-homology domain-2 phosphatase-1 (SHP-1) with paired immunoglobulin-like receptor-B (PIR-B), an ITIM-bearing receptor which negatively regulates osteoclast differentiation. Unexpectedly, Ly49Q deficiency did not lead to impaired osteoclast formation in vivo, suggesting the existence of a compensatory mechanism. This study provides an example in which an ITIM-bearing receptor functions as a positive regulator of osteoclast differentiation.     

Investigation of transferability of BovineSNP50 BeadChip from cattle to water buffalo for genome wide association study

Cattle and water buffalo belong to the same subfamily Bovinae and share chromosome banding and gene order homology. In this study, we used genome-wide Illumina BovineSNP50 BeadChip to analyze 91 DNA samples from three breeds of water buffalo (Nili-Ravi, Murrah and their crossbred with local GuangXi buffalos in China), to demonstrate the genetic divergence between cattle and water buffalo through a large single nucleotide polymorphism (SNP) transferability study at the whole genome level, and performed association analysis of functional traits in water buffalo as well. A total of 40,766 (75.5 %) bovine SNPs were found in the water buffalo genome, but 49,936 (92.5 %) were with only one allele, and finally 935 were identified to be polymorphic and useful for association analysis in water buffalo. Therefore, the genome sequences of water buffalo and cattle shared a high level of homology but the polymorphic status of the bovine SNPs varied between these two species. The different patterns of mutations between species may associate with their phenotypic divergence due to genome evolution. Among 935 bovine SNPs, we identified a total of 9 and 7 SNPs significantly associated to fertility and milk production traits in water buffalo, respectively. However, more works in larger sample size are needed in future to verify these candidate SNPs for water buffalo.     

Distinct diversity of KIR genes in three southern Indian populations: comparison with world populations revealed a link between KIR gene content and pre-historic human migrations

By interacting with polymorphic HLA class I molecules, the killer cell immunoglobulin-like receptors (KIR) influence the innate and adaptive immune response to infection. The KIR family varies in gene content and sequence polymorphism, thereby, distinguishing individuals and populations. To investigate KIR diversity in the earliest settlers of India, we have characterized the KIR gene content in three Dravidian-speaking populations (Mollukurumba, Kanikar, and Paravar) from the state of Tamil Nadu, southern India. The activating KIR genes and putative group-B KIR haplotypes were frequent in Paravar and Kanikar, a scenario analogous to those seen previously in other populations of Indian origin, indicating that predominance of group-B KIR haplotypes is the characteristic feature of Indian populations. In contrast, the KIR gene profile of Mollukurumba was more related to Caucasian type. It is not clear whether a local-specific selection or a recent admixture from Iran is responsible for such discrete profile in Mollukurumba. Each southern Indian population had distinct KIR genotype profile. Comparative analyses with world populations revealed that group-B KIR haplotypes were frequent in the natives of India, Australia, and America, the populations associated with those involved in extensive prehistoric human migrations. Whether or not natural selection has acted to enrich group-B KIR haplotypes in these migratory descendants is an issue that requires objective testing. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of the Ly49I promoter

 Fourteen potential Ly49 genes have been identified in the C57Bl/6 mouse strain, and cDNAs containing a complete coding region have been isolated for 10 members of this gene family. Ly49 proteins are primarily expressed in natural killer (NK) cells. Although the sequence of the Ly49a promoter region has been published, no study of the cell-specific activity of the promoter has been reported. A 12-kb genomic fragment of the Ly49I gene was isolated and characterized by DNA sequencing. Approximately 5 kb of DNA sequence upstream of the first Ly49I exon was determined and this region was used to perform promoter analysis using luciferase reporter plasmid constructs. A core promoter was identified that was preferentially transcribed in a Ly49-expressing cell line, EL-4. Electrophoretic mobility shift assays using oligonucleotide probes from the core Ly49i promoter and comparable regions from the Ly49a promoter demonstrated the importance of TATA-related elements in generating EL-4 and NK cell-specific DNA/protein complexes. Received: 15 October 1999 / Revised: 26 November 1999     

Remarkably Low KIR and HLA Diversity in Amerindians Reveals Signatures of Strong Purifying Selection Shaping the Centromeric KIR Region

The killer-cell immunoglobulin-like receptors (KIR) recognize human leukocyte antigen (HLA) molecules to regulate the cytotoxic and inflammatory responses of natural killer cells. KIR genes are encoded by a rapidly evolving gene family on chromosome 19 and present an unusual variation of presence and absence of genes and high allelic diversity. Although many studies have associated KIR polymorphism with susceptibility to several diseases over the last decades, the high-resolution allele-level haplotypes have only recently started to be described in populations. Here, we use a highly innovative custom next-generation sequencing method that provides a state-of-art characterization of KIR and HLA diversity in 706 individuals from eight unique South American populations: five Amerindian populations from Brazil (three Guarani and two Kaingang); one Amerindian population from Paraguay (Aché); and two urban populations from Southern Brazil (European and Japanese descendants from Curitiba). For the first time, we describe complete high-resolution KIR haplotypes in South American populations, exploring copy number, linkage disequilibrium, and KIR–HLA interactions. We show that all Amerindians analyzed to date exhibit the lowest numbers of KIR–HLA interactions among all described worldwide populations, and that 83–97% of their KIR–HLA interactions rely on a few HLA-C molecules. Using multiple approaches, we found signatures of strong purifying selection on the KIR centromeric region, which codes for the strongest NK cell educator receptors, possibly driven by the limited HLA diversity in these populations. Our study expands the current knowledge of KIR genetic diversity in populations to understand KIR–HLA coevolution and its impact on human health and survival.     

Bimodal evolution of the killer cell Ig-like receptor (KIR) family in New World primates

The immunoglobulin-like receptor (KIR) gene family in New World primates (Platyrrhini) has been characterized only in the owl monkey (Aotus sp.). To gain a better understanding of the KIR system in Platyrrhini, we analyzed a KIR haplotype in Ateles geoffroyi, and sequenced KIR complementary DNAs (cDNAs) from other three Atelidae species, Ateles hybridus, Ateles belzebuth, and Lagothrix lagotricha. Atelidae expressed a variable set of activating and inhibitory KIRs that diversified independently from their Catarrhini counterparts. They had a unique mechanism to generate activating receptors from inhibitory ones, involving a single nucleotide deletion in exon 7 and a change in the donor splice site of intron 7. The A. geoffroyi haplotype contained at least six gene models including a pseudogene, two coding inhibitory receptors, and three coding activating receptors. The centromeric region was in a tail-to-tail orientation with respect to the telomeric region. The owl monkey KIR haplotype shared this organization, and in phylogenetic trees, the centromeric genes clustered together with those of A. geoffroyi, whereas their telomeric genes clustered independently. KIR cDNAs from the other Atelidae species conformed to this pattern. Signatures of positive selection were found in residues predicted to interact with the major histocompatibility complex. Such signatures, however, primarily explained variability between paralogous genes but not between alleles in a locus. Atelidae, therefore, has expanded the KIR family in a bimodal fashion, where an inverted centromeric region has remained relatively conserved and the telomeric region has diversified by a rapid process of gene duplication and divergence, likely favored by positive selection for ligand binding.     

Natural Killer Cell Tolerance Persists Despite Significant Reduction of Self MHC Class I on Normal Target Cells in Mice

Background

A major group of murine inhibitory receptors on Natural Killer (NK) cells belong to the Ly49 receptor family and recognize MHC class I molecules. Infected or transformed target cells frequently downmodulate MHC class I molecules and can thus avoid CD8⁺ T cell attack, but may at the same time develop NK cell sensitivity, due to failure to express inhibitory ligands for Ly49 receptors. The extent of MHC class I downregulation needed on normal cells to trigger NK cell effector functions is not known.

Methodology/Principal Findings

In this study, we show that cells expressing MHC class I to levels well below half of the host level are tolerated in an in vivo assay in mice. Hemizygous expression (expression from only one allele) of MHC class I was sufficient to induce Ly49 receptor downmodulation on NK cells to a similar degree as homozygous expression, despite a strongly reduced cell surface level of MHC class I. Co-expression of weaker MHC class I ligands in the host did not have any further effect on the degree of Ly49 downmodulation. Furthermore, a single MHC class I allele could downmodulate up to three Ly49 receptors on individual NK cells. Only when NK cells simultaneously expressed several Ly49 receptors and hemizygous MHC class I levels, a putative threshold for Ly49 downmodulation was reached.

Conclusion

Collectively, our findings suggest that in interactions between NK cells and normal untransformed cells, MHC class I molecules are in most cases expressed in excess compared to what is functionally needed to ensure self tolerance and to induce maximal Ly49 downmodulation. We speculate that the reason for this is to maintain a safety margin for otherwise normal, autologous cells over a range of MHC class I expression levels, in order to ensure robustness in NK cell tolerance.     

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.     

The novel inhibitory NKR-P1C receptor and Ly49s3 identify two complementary, functionally distinct NK cell subsets in rats

The proximal region of the NK gene complex encodes the NKR-P1 family of killer cell lectin-like receptors which in mice bind members of the genetically linked C-type lectin-related family, while the distal region encodes Ly49 receptors for polymorphic MHC class I molecules. Although certain members of the NKR-P1 family are expressed by all NK cells, we have identified a novel inhibitory rat NKR-P1 molecule termed NKR-P1C that is selectively expressed by a Ly49-negative NK subset with unique functional characteristics. NKR-P1C(+) NK cells efficiently lyse certain tumor target cells, secrete cytokines upon stimulation, and functionally recognize a nonpolymorphic ligand on Con A-activated lymphoblasts. However, they specifically fail to kill MHC-mismatched lymphoblast target cells. The NKR-P1C(+) NK cell subset also appears earlier during development and shows a tissue distribution distinct from its complementary Ly49s3(+) subset, which expresses a wide range of Ly49 receptors. These data suggest the existence of two major, functionally distinct populations of rat NK cells possessing very different killer cell lectin-like receptor repertoires.